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1 pages, 126 KiB

Open AccessAbstract

In Situ Capture RT-qPCR Method for Detection of Human Norovirus in Food and Environmental Samples

by Peng Tian

Proceedings 2020, 50(1), 1; https://doi.org/10.3390/proceedings2020050001 - 1 Jun 2020

Viewed by 1591

Abstract

Human noroviruses (HuNoVs) are the major cause of non-bacterial acute gastroenteritis worldwide. RT-qPCR is a widely used method to detect HuNoVs. However, the method is unable to extract a virus from environmental samples and to discriminate between infectious and non-infectious viruses. In this [...] Read more.

Human noroviruses (HuNoVs) are the major cause of non-bacterial acute gastroenteritis worldwide. RT-qPCR is a widely used method to detect HuNoVs. However, the method is unable to extract a virus from environmental samples and to discriminate between infectious and non-infectious viruses. In this study, we explored a new in situ capture RT-qPCR (ISC-RT-qPCR) methodology to estimate the infectivity of HuNoV in environmental and food samples. This assay was based on capturing encapsidated HuNoV by viral receptors, followed by in situ amplification of the captured viral genomes by RT-qPCR. We demonstrated that ISC-RT-qPCR did not capture and enable signal amplification of the heat-denatured Tulane virus (TV) and HuNoVs. Therefore, ISC-RT-qPCR provides better estimates for infectivity of HuNoV than RT-qPCT. We then utilized the ISC-RT-qPCR to detect HuNoV in environmental water samples and food samples, as compared to a conventional RT-qPCR procedure. The presence of HuNoV was examined in 36 oyster samples from retail markets using by both assays for detection. The detection rates of HuNoV in gill, digestive glands, and other tissues were 33.3%, 25%, and 19.4%, respectively, by ISC-RT-qPCR; and were 5.6%, 11.1%, and 11.1%, respectively, by RT-qPCR. ISC-RTqPCR is more sensitive than RT-qPCR for the detection of HuNoV in oysters. By contrast, the HuNoV detection rate by ISC-RTqPCR is lower for environmental samples. Of the 72 water samples that tested positive for HuNoV by RT-qPCR, only 20 (27.8%) of these tested positive by ISC-RT-qPCR, suggesting that 72.2% of RT-qPCR-positive samples were unlikely to be infectious. A better detection rate by ISC-RT-qPCR in oyster samples indicates the likelihood of infectious HuNoV that accumulated in oysters, and a lower detection rate of HuNoV in environmental water by ISC-RT-qPCR, indicating that the majority of RT-qPCR-positive samples were from non-infectious viral RNA. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 146 KiB

Open AccessAbstract

Identification of Aquarius and Senataxin as Restriction Host Factors for Hepatitis B Virus Infection

by Christina Whitten-Bauer, Andoni Gómez-Moreno and Urtzi Garaigorta

Proceedings 2020, 50(1), 2; https://doi.org/10.3390/proceedings2020050002 - 1 Jun 2020

Cited by 1 | Viewed by 1653

Abstract

Hepatitis B virus (HBV) represents an important human pathogen causing acute and chronic hepatitis. Over 240 million people are chronically infected, many of whom will die due to complications such as liver cirrhosis and hepatocellular carcinoma. Currently approved therapies are very effective in [...] Read more.

Hepatitis B virus (HBV) represents an important human pathogen causing acute and chronic hepatitis. Over 240 million people are chronically infected, many of whom will die due to complications such as liver cirrhosis and hepatocellular carcinoma. Currently approved therapies are very effective in suppressing virus replication and viremia, but they are not curative, because they do not completely eliminate the nuclear episomal DNA form of HBV (cccDNA) that re-establishes infection upon interruption of therapy. Despite our understanding of many aspects of the HBV lifecycle, details of the HBV cccDNA biology remain poorly understood. Our group is pursuing a loss-of-function genetic screening approach, to identify cellular factors regulating HBV infection. A lentivirus-delivered short hairpin RNA (shRNA) library, composed of 384 shRNAs, was used to interrogate the function of 80 DNA damage repair pathway proteins in the establishment of HBV infection. The primary screening identified 10 cellular factors that regulate the HBV infection both positively or negatively. Two of those proteins, aquarius (AQR) and senataxin (SETX), were subsequently validated as factors restricting the HBV infection in independent experiments. Silencing of AQR and SETX led to an increased infection efficiency that was characterized by higher intracellular levels of HBV cccDNA, HBV mRNA, and core protein, and increased HBV e antigen (HBeAg) accumulation in the supernatants of infected cells. The expression level, glycosylation pattern, and localization of the HBV receptor, sodium taurocholate cotransporting polypeptide (NTCP), in AQR- and SETX-downregulated cells was equivalent to that of the control cells. Collectively, our results are compatible with AQR and SETX restricting early steps in the HBV lifecycle and downstream HBV entry, that affect the establishment of the HBV cccDNA pool. Experiments to unravel the function of these proteins in the context of HBV infection are currently underway. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 120 KiB

Open AccessAbstract

Imaging Viral Factories

by Cristina Risco Ortiz

Proceedings 2020, 50(1), 3; https://doi.org/10.3390/proceedings2020050003 - 1 Jun 2020

Viewed by 1531

Abstract

Viruses remodel cellular compartments to build their replication factories. Remarkably, viruses are also able to induce new membranes and new organelles. As a result of recent advances in light and transmission electron microscopy (TEM), we are starting to become aware of the variety [...] Read more.

Viruses remodel cellular compartments to build their replication factories. Remarkably, viruses are also able to induce new membranes and new organelles. As a result of recent advances in light and transmission electron microscopy (TEM), we are starting to become aware of the variety of structures that viruses assemble inside cells. Viral factories are intracellular compartments harboring replication organelles that contain viral replication complexes and the sites of virus particle assembly. This lecture will revise the most relevant imaging technologies for studying the biogenesis of viral replication factories. Live cell microscopy, correlative light and electron microscopy, cryo-TEM, and three-dimensional imaging methods are unveiling how viruses manipulate cell organization. In particular, methods for molecular mapping in situ, in two and three dimensions, are revealing how macromolecular complexes build functional replication complexes inside infected cells. The combination of all these imaging approaches is uncovering the viral lifecycle events with a detail never seen before. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 154 KiB

Open AccessAbstract

First Report on Detection and Genetic Diversity of Picobirnaviruses in the Small Indian Mongoose (Herpestes auropunctatus)

by Alyssa Kleymann, Anne A.M.J. Becker and Souvik Ghosh

Proceedings 2020, 50(1), 4; https://doi.org/10.3390/proceedings2020050004 - 1 Jun 2020

Viewed by 1331

Abstract

Picobirnaviruses (PBVs), family Picobirnaviridae, are bi-segmented, double-stranded RNA viruses. PBVs are considered opportunistic enteric pathogens. Gene segment-1 of PBV encodes the capsid protein, whilst gene segment-2 codes for RNA-dependent RNA polymerase (RdRp). Based on differences in gene segment-2, PBVs are classified into [...] Read more.

Picobirnaviruses (PBVs), family Picobirnaviridae, are bi-segmented, double-stranded RNA viruses. PBVs are considered opportunistic enteric pathogens. Gene segment-1 of PBV encodes the capsid protein, whilst gene segment-2 codes for RNA-dependent RNA polymerase (RdRp). Based on differences in gene segment-2, PBVs are classified into genogroup-I (GI) and GII. Although PBVs have been detected in a wide variety of host species, there are no reports on PBVs from mongoose so far. We report here high rates of detection (35.36%, 29/82) of GI PBVs in fecal samples from the small Indian mongoose (Herpestes auropunctatus) on the Caribbean island of St. Kitts. Applying a combination of a non-specific primer-based amplification method and conventional RT-PCR using a newly designed primer targeting the 3′-untranslated region (UTR), we could amplify and sequence the complete/nearly complete gene segment-2 of eight mongoose PBV strains. Except for a single strain, the gene segment-2 of the remaining mongoose PBV strains contained the putative open reading frame encoding the RdRp. The gene segment-2/putative RdRps of the mongoose PBV strains retained various features that are conserved in other PBVs (5′- and 3′-terminal nucleotide sequences, bacterial ribosomal binding site sequence in 5′-UTR, and the three domains in putative RdRps). On the other hand, phylogenetic analysis and sequence identities of the putative RdRps revealed high genetic diversity among the mongoose PBV strains and with those of PBVs from other host species. To our knowledge, this is the first report on detection and genetic diversity of PBVs from the mongoose, expanding the host range of PBVs and providing vital insights into the various features and evolution of putative RdRps of PBVs in a new host species. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 168 KiB

Open AccessAbstract

FDA Approved Drugs Efavirenz, Tipranavir, and Dasabuvir Inhibit Replication of Multiple Flaviviruses In Vitro

by Michal Stefanik, Fortunatus C Ezebuo, Jan Haviernik, Ikemefuna C. Uzochukwu, Martina Fojtikova, Jiri Salat, Ludek Eyer and Daniel Ruzek

Proceedings 2020, 50(1), 6; https://doi.org/10.3390/proceedings2020050006 - 2 Jun 2020

Viewed by 1633

Abstract

Arthropod-borne flaviviruses such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Zika virus (ZIKV), Dengue virus (DENV), and yellow fever virus (YFV) cause several serious life-threatening syndromes (encephalitis, miscarriages, paralysis, etc.). No effective antiviral therapy against these viruses has been approved yet. [...] Read more.

Arthropod-borne flaviviruses such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Zika virus (ZIKV), Dengue virus (DENV), and yellow fever virus (YFV) cause several serious life-threatening syndromes (encephalitis, miscarriages, paralysis, etc.). No effective antiviral therapy against these viruses has been approved yet. We selected, via in silico modeling, 12 U.S. Food and Drug Administration (FDA)-approved antiviral drugs (paritaprevir, dolutegravir, raltegravir, efavirenz, elvitegravir, tipranavir, saquinavir, dasabuvir, delavirdine, maraviroc, trifluridine, and tauroursodeoxycholic acid) for their interaction with ZIKV proteins (NS3 helicase and protease, non-structural protein 5 (NS5) RNA-dependent RNA polymerase, and methyltransferase). Only three of them were active against ZIKV, namely, dasabuvir (ABT-333), efavirenz, and tipranavir. These compounds inhibit virus replication of ZIKV (MR-766 and Paraiba_01) in Vero cells; therefore, we tested these compounds against other medically important flaviviruses WNV (13-104 and Eg101) and TBEV (Hypr). Dasabuvir was originally developed as an antiviral drug against hepatitis C virus (HCV); tipranavir and efavirenz are used for treating human immunodeficiency virus (HIV) infection. The antiviral effects of efavirenz, tipranavir, and dasabuvir were tested for ZIKV in HUH-7, astrocytes (HBCA), and UKF-NB-4 cells, where we also identified a significant inhibition effect of these compounds. For Vero cells, efavirenz inhibited all investigated viruses with EC₅₀ ranging from 9.70 to 29.26 µM; the tipranavir inhibition effect was from 16.19 (WNV 13-104) to 27.47 µM (TBEV), while the strongest and most robust antiviral effect was demonstrated in the case of dasabuvir (EC₅₀ values ranging from 9.09 (TBEV) to 10.85 µM (WNV 13-104)). These results warrant further research of these drugs, either individually or in combination, as possible pan-flavivirus inhibitors. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 153 KiB

Open AccessAbstract

Lyophilized Matrix Containing Ready-to-Use Primers and Probe Solutions for Standardization of Real-Time PCR and RT-qPCR Diagnostics

by Remi N Charrel and Laurence Thirion

Proceedings 2020, 50(1), 7; https://doi.org/10.3390/proceedings2020050007 - 2 Jun 2020

Viewed by 1672

Abstract

Real-time molecular techniques have become the reference methods for the direct diagnosis of pathogens. The reduction of steps is a key factor in order to decrease the risk of human errors resulting in invalid series and delayed results. We describe here a process [...] Read more.

Real-time molecular techniques have become the reference methods for the direct diagnosis of pathogens. The reduction of steps is a key factor in order to decrease the risk of human errors resulting in invalid series and delayed results. We describe here a process involving the preparation of oligonucleotide primers and a hydrolysis probe in a single tube at predefined optimized concentrations that are stabilized via lyophilization (Lyoph-P&P). Lyoph-P&P was compared to the classic protocol using extemporaneously prepared liquid reagents, assaying (i) sensitivity, (ii) long-term stability at 4 °C, and (iii) long-term stability at 37 °C, mimicking transportation without a cold chain. Two previously published molecular assays were selected for this study. They target two emerging viruses that are listed on the blueprint of the WHO to be considered for preparedness and response actions: chikungunya virus (CHIKV) and Rift Valley fever phlebovirus (RVFV). The results of our study demonstrate that (i) Lyoph-P&P is stable for at least four days at 37 °C, supporting shipping without the need of a cold chain, (ii) Lyoph-P&P rehydrated solution is stable at 4 °C for at least two weeks, (iii) the sensitivity observed with Lyoph-P&P is at least equal to, and often better than, that observed with liquid formulation, and (iv) the validation of results observed with low-copy specimens is rendered easier by higher fluorescence levels. In conclusion, Lyoph-P&P holds several advantages over extemporaneously prepared liquid formulations and merits consideration as a novel real-time molecular assay for implementation into a laboratory with routine diagnostic activity. Since the meeting, this concept has been applied to the COVID-19 situation: two diagnostic assays (E gene and RdRp) have been developed and can be ordered on the European Virus Archive catalog (https://www.european-virus-archive.com/detection-kit/lyophilized-primers-and-probe-rt-pcr-2019-ncov-e-gene; https://www.european-virus-archive.com/detection-kit/lyophilized-primers-and-probe-rt-pcr-sars-cov-2-rdrp-gene). Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 130 KiB

Open AccessAbstract

Mapping the Interface between New World Hantaviruses and Their Receptor, PCDH1

by Megan M. Slough, Andrew S. Herbert, Ana I. Kuehne, John M. Dye, Kartik Chandran and Rohit K. Jangra

Proceedings 2020, 50(1), 8; https://doi.org/10.3390/proceedings2020050008 - 3 Jun 2020

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Abstract

Hantaviruses are found throughout the world and can cause deadly diseases in humans, specifically, hantavirus cardiopulmonary syndrome (HCPS) in the New World and hemorrhagic fever with renal syndrome (HFRS) in the Old World. Currently, no FDA-approved, specific antiviral drugs or vaccines are available. [...] Read more.

Hantaviruses are found throughout the world and can cause deadly diseases in humans, specifically, hantavirus cardiopulmonary syndrome (HCPS) in the New World and hemorrhagic fever with renal syndrome (HFRS) in the Old World. Currently, no FDA-approved, specific antiviral drugs or vaccines are available. Recently, we showed that New World hantaviruses utilize protocadherin-1 (PCDH1) for endothelial cell entry and infection by directly engaging its first extracellular cadherin repeat (EC1) domain. The knockout of PCDH1 also greatly reduced pulmonary infection and was highly protective in a Syrian hamster model of lethal challenge with Andes virus (ANDV). To further understand PCDH1’s role in hantavirus entry, we sought to map the binding interface between hantavirus Gn/Gc and PCDH1-EC1. Accordingly, we screened a panel of EC1 proteins, bearing point mutations in solvent-exposed residues, for their capacity to recognize Gn/Gc and block viral entry. EC1 mutations defective in Gn/Gc binding were engineered individually and in combinations into full-length PCDH1, expressed in PCDH1-knockout cells, and evaluated for their capacity to complement viral infection. We identified a surface in the PCDH1-EC1 domain, comprising contiguous residues, which was required for virus PCDH1 recognition and PCDH1-dependent viral entry. However, this region does not overlap with the EC1–EC4 heterodimer interface recently described by Modak and Sotomayor. In addition, through the use of recombinant vesicular stomatitis viruses bearing chimeric hantavirus Gn/Gc glycoproteins, we were able to pinpoint the importance of the N-terminal domain of the Gn subunit for PCDH1-mediated entry. With these taken together, identifying the location of the interface could provide a direction for the development of host-directed antiviral drugs that do not interfere with PCDH1’s endogenous function, as well as help to map an antigen target on Gn/Gc for antiviral antibodies. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 131 KiB

Open AccessAbstract

Expression of Two Foreign Genes from the Optimal Insertion Sites of Newcastle Disease Virus Vector for Use as a Multivalent Vaccine and Gene Therapy Vector

by Lei He, Zhenyu Zhang and Qingzhong Yu

Proceedings 2020, 50(1), 9; https://doi.org/10.3390/proceedings2020050009 - 3 Jun 2020

Viewed by 1608

Abstract

Many Newcastle disease virus (NDV) strains have been developed as vectors to express a foreign gene (FG) for vaccine and gene therapy purposes. A majority of these NDV vectors express only a single FG or two FGs from suboptimal insertion sites in the [...] Read more.

Many Newcastle disease virus (NDV) strains have been developed as vectors to express a foreign gene (FG) for vaccine and gene therapy purposes. A majority of these NDV vectors express only a single FG or two FGs from suboptimal insertion sites in the NDV genome, obtaining various levels of FG expression. To improve the FG expression, we generated NDV LaSota vaccine strain-based recombinant viruses to express two FGs, green fluorescent protein (GFP) and red fluorescent protein (RFP) genes, from the identified optimal insertion sites, through a combination of the independent transcription unit (ITU) and the internal ribosomal entry site (IRES) dependent expression approaches. Biological assessments showed that these recombinants expressing two FGs were slightly attenuated with approximately one order of magnitude lower in virus titers than those containing a single FG. The FG expression efficiencies from two-FG viruses were also lower than those from the single-FG viruses. However, the expression of two FGs from the optimal insertion sites was significantly (p < 0.05) higher than those from the suboptimal insertion sites. The expression of FGs through the ITU approach was approximately 4-fold more efficient than that through the IRES-dependent approach. These results suggest that the NDV LaSota vector could efficiently express two FGs from the identified optimal insertions sites. The ITU strategy could be used for the expression of a higher amount of FG products, whereas the IRES tactic might be useful when a lower amount of FG products are needed. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 157 KiB

Open AccessAbstract

Bacteriophage Biocontrol of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch

by Aryan Rahimi-Midani and Tae-Jin Choi

Proceedings 2020, 50(1), 10; https://doi.org/10.3390/proceedings2020050010 - 3 Jun 2020

Cited by 2 | Viewed by 1809

Abstract

Bacterial fruit blotch caused by Acidovorax citrulli is known to be the major threat to cucurbit crop production worldwide. The pathogen can penetrate into seed coat and cause disease symptoms at any stage of plant growth, which results in fruit loss. Two main [...] Read more.

Bacterial fruit blotch caused by Acidovorax citrulli is known to be the major threat to cucurbit crop production worldwide. The pathogen can penetrate into seed coat and cause disease symptoms at any stage of plant growth, which results in fruit loss. Two main genotypes (genotype I and II) are reported in A. citrulli, in which genotype II is the main cause of Bacterial Fruit Blotch (BFB) in watermelon and group I is known to be a causal agent of BFB in melon. To date, there are no commercially available cultivars resistant to BFB, and available strategies are not able to completely manage the disease. In this study, we aim to isolate bacteriophages to control BFB. Samples collected from watermelon, melon, and pumpkin were used to isolate bacteriophages. All isolated bacteriophages were tested against 42 strains of A. citrulli, among which two phages with the ability to lyse a greater number of hosts were selected and characterized. Bacteriophage ACP17 from the Myoviridae family, with a head size of 100 ± 5 nm and tail of 150 ± 5 nm, infected 29 strains of A. citrulli mostly belonging to genotype group I, whereas the second isolated bacteriophage, ACPWH from Siphoviridae, with a head size of 60 ± 5 nm and tail of 180 ± 5 nm, infected 39 A. citrulli strains. Genome analysis of both bacteriophages using Next generation Sequencing (NGS) showed that ACP17 and ACPWH have double-stranded DNA with sizes of 156,972 kb and 424,299 kb, respectively. Watermelon seeds coated with ACPWH showed a germination rate of up to 90% in the presence of A. citrulli in contrast to untreated seed, which showed no germination or germinated juveniles with BFB symptoms in the presence of A. citrulli. The results of this study show that the use of bacteriophages of A. citrulli represents a potential biocontrol method for controlling BFB. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 143 KiB

Open AccessAbstract

Antioxidant Effect of A. chilensis on the Production of Infectious Viral Particles of ISAv and Its Consequences on the SUMOylation of NP Protein

by Fernanda Fredericksen, Gardenia Payne, Nicolás Maldonado, Melina Villalba and Víctor Olavarría

Proceedings 2020, 50(1), 11; https://doi.org/10.3390/proceedings2020050011 - 3 Jun 2020

Viewed by 1424

Abstract

Infectious salmon anemia virus (ISAv) is a pathogen of high economic importance worldwide; it produces a highly fatal clinical symptomatology called infectious salmon anemia (ISA), which is one of the main causes of economic loss in Chilean aquaculture, specifically in Chilean salmon, being [...] Read more.

Infectious salmon anemia virus (ISAv) is a pathogen of high economic importance worldwide; it produces a highly fatal clinical symptomatology called infectious salmon anemia (ISA), which is one of the main causes of economic loss in Chilean aquaculture, specifically in Chilean salmon, being responsible for a mortality rate greater than 80% when outbreaks of this pathogen occur in fish farms. ISAv dramatically increases levels of reactive oxygen species (ROS) by increasing the activity of the p38MAPK protein, which activates p47phox, by phosphorylation, allowing its binding to the membrane subunits of the NADPH oxidase complex, which is an important positive regulator of ROS levels in cells. Further, it is known that oxidative stress is able to regulate the SUMOylation machinery, producing an increase in SUMOylated proteins. Together with this background and various bioinformatic analyses, it was found that the ISAv nucleoprotein (NP) has a highly conserved capacity for SUMOylation, and this protein alone is capable of causing strong oxidative stress in transfected cells and is therefore able to regulate the SUMOylation machinery. Immunoprecipitation assays confirmed the bioinformatic analyses, where NP was seen to be SUMOylated, and this signal decreased considerably when cells were treated with a p38MAPK inhibitor. Together with this, the number of copies of NP and the viability in cells infected with ISAv were also evaluated, where it was observed that there was a strong increase in the number of copies of NP and a marked decrease in cell viability, this being in contrast to when, in addition to the infection, the cells were treated with a natural product “maqui” (A. chilensis), which, due to its high content of polyphenolic compounds, has been shown to have a high antioxidant capacity, greatly reducing the number of copies of NP and the percentage of mortality compared to cells that are only infected with ISAv. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 173 KiB

Open AccessAbstract

An E460D Substitution in the NS5 Protein of Tick-Borne Encephalitis Virus Confers Resistance to the Inhibitor Galidesivir (BCX4430) and Also Attenuates the Virus in Mice

by Jan Haviernik, Ludek Eyer, Antoine Nougairède, Marie Uhlířová, Jean-Sélim Driouich, Darina Zouharová, James Jason Valdés, Ernest Gould, Erik De Clercq, Xavier de Lamballerie and Daniel Ruzek

Proceedings 2020, 50(1), 12; https://doi.org/10.3390/proceedings2020050012 - 4 Jun 2020

Viewed by 1811

Abstract

Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia for which there is currently no specific therapy. The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics [...] Read more.

Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia for which there is currently no specific therapy. The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of TBEV and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. In our study, we performed serial in vitro passaging of TBEV in the presence of increasing concentrations of galidesivir (up to 50 μM), which resulted in the generation of two drug-resistant TBEV mutants. The first TBEV mutant was characterized by a single amino acid change, E460D. The other carried two amino acid changes, E460D and Y453H. Both mutations mapped to the active site of the viral RNA-dependent RNA polymerase (RdRp). Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2′-C-methyladenosine, 2′-C-methyladenosine, and 4′-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. By contrast, infection with wild-type virus resulted in fatal infections for all animals. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 157 KiB

Open AccessAbstract

Application of SORTS, a Novel Gene-Edited Cell Selection Method for HIV Study and Therapy

by Dmitriy Mazurov, Alexandra Maslennikova, Dmitriy Komkov and Anastasia Zotova

Proceedings 2020, 50(1), 13; https://doi.org/10.3390/proceedings2020050013 - 4 Jun 2020

Viewed by 1451

Abstract

We have recently developed surface oligopeptide knock-in for rapid target selection (SORTS), a novel method to isolate mammalian cells with gene modifications using FACS-sorting. It relies on CRISPR/Cas9-mediated targeted knock-in of a very short promoterless expression construct (250 bp) comprising a Flag or [...] Read more.

We have recently developed surface oligopeptide knock-in for rapid target selection (SORTS), a novel method to isolate mammalian cells with gene modifications using FACS-sorting. It relies on CRISPR/Cas9-mediated targeted knock-in of a very short promoterless expression construct (250 bp) comprising a Flag or HA epitope embedded into the smallest GPI-protein CD52, and a polyA signal from the beta-globin. CD52 efficiently delivers the epitope to the cell surface, where it serves as a marker for selection, while polyA terminates transcription and silences target gene expression. Primarily, SORTS was developed to knock out genes encoding intracellular or secreted proteins, which cannot be used as markers for selection of live cells. Using in-frame modification of SORTS, we demonstrated the possibility of HIV-1 provirus inactivation via sorting of GPI-tag positive cells. In order to make the “cured” cells resistant to a subsequent HIV-1 infection, the epitope tag in the CD52 molecule was substituted by one of the fusion inhibitor peptides from the CHR-domain of gp41. We selected a series of cell-surface-expressed, GPI-anchored, C34-based peptides that confer a strong cellular resistance to HIV-1 infection mediated by NL4-3, JRFL, or ZM153 Env. These findings together with a monoclonal antibody raised against the C34 peptide provide an opportunity to generate and select HIV-resistant lymphocytes for a therapeutic goal. SORTS was also adapted to engineer transgenic HIV-1 effector Т cells and to study cell-to-cell transmission. To facilitate transgenesis, we developed a knock-in strategy to express GPI-tag from the intronic region of the human PPP1R12C gene (AAVS1 locus) and delivered FRT sites of recombination into both alleles. In summary, SORTS is a novel instrument to isolate rare cells with precise genomic modifications with broad applications, including HIV biology. This work was supported by the Russian Science Foundation (grant 18-14-00333) and the Russian Foundation for Basic Research (grants 18-29-07052, 18-04-01016). Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 138 KiB

Open AccessAbstract

Activation and Antagonism of the OAS–RNase L Pathway

by Susan R. Weiss

Proceedings 2020, 50(1), 14; https://doi.org/10.3390/proceedings2020050014 - 4 Jun 2020

Cited by 1 | Viewed by 2045

Abstract

The oligoadenylate synthetase–ribonuclease L (OAS–RNase L) system is a potent antiviral pathway that severely limits the pathogenesis of many viruses. Upon sensing dsRNA, OASs produce 2′,5′-oligoadenylates (2-5A) that activate RNase L to cleave both host and viral single-stranded RNA, thereby limiting protein production, [...] Read more.

The oligoadenylate synthetase–ribonuclease L (OAS–RNase L) system is a potent antiviral pathway that severely limits the pathogenesis of many viruses. Upon sensing dsRNA, OASs produce 2′,5′-oligoadenylates (2-5A) that activate RNase L to cleave both host and viral single-stranded RNA, thereby limiting protein production, virus replication and spread, leading to apoptotic cell death. Endogenous host dsRNA, which accumulates in the absence of adenosine deaminase acting on RNA (ADAR)1, can also activate RNase L and lead to apoptotic cell death. RNase L activation and antiviral activity during infections with several types of viruses in human and bat cells is dependent on OAS3 but independent of virus-induced interferon (IFN) and, thus, RNase L can be activated even in the presence of IFN antagonists. Differently from other human viruses examined, Zika virus is resistant to the antiviral activity of RNase L and instead utilizes RNase L to enhance its replication factories to produce more infectious virus. Some betacoronaviruses antagonize RNase L activation by expressing 2′,5′-phosphodiesterases (PDEs) that cleave 2-5A and thereby antagonize activation of RNase L. The best characterized of these PDEs is the murine coronavirus (MHV) NS2 accessory protein. Enzymatically active NS2 is required for replication in myeloid cells and in the liver. Interestingly, while wild type mice clear MHV from the liver by 7–10 days post-infection, RNase L knockout mice fail to effectively clear MHV, probably due to diminished apoptotic death of infected cells. We suggest that RNase L antiviral activity stems from direct cleavage of viral genomes and cessation of protein synthesis as well as through promoting death of infected cells, limiting the spread of virus. Importantly, OASs are pattern recognition receptors and the OAS–RNase L pathway is a primary innate response pathway to viruses, capable of early response, coming into play before IFN is induced or when the virus shuts down IFN signaling. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 146 KiB

Open AccessAbstract

Identification of a Novel Yellow Fever Virus Cellular Restriction Factor

by Jinhua Xiang, James H. McLinden, Qing Chang, Thomas M. Kaufman, Jennifer L. Welch, Nirjal Bhattarai and Jack T. Stapleton

Proceedings 2020, 50(1), 15; https://doi.org/10.3390/proceedings2020050015 - 4 Jun 2020

Viewed by 1399

Abstract

Background: We previously identified a yellow fever virus (YFV) genome-derived, short noncoding RNA (vsRNA) generated during infection that targets the 3’ untranslated region (UTR) of a Src-kinase regulatory phosphatase (PTPRE), reducing PTPRE translation. By contrast, a related flavivirus (Zika) did not regulate PTPRE. [...] Read more.

Background: We previously identified a yellow fever virus (YFV) genome-derived, short noncoding RNA (vsRNA) generated during infection that targets the 3’ untranslated region (UTR) of a Src-kinase regulatory phosphatase (PTPRE), reducing PTPRE translation. By contrast, a related flavivirus (Zika) did not regulate PTPRE. We assessed the role of PTPRE in YFV and Zika replication. Methods: Human PTPRE with a β -Globulin 3’UTR to remove the 3’UTR sequence targeted by the YFV vsRNA was stably expressed in Jurkat and Huh7D cells using a tetracycline-regulated promoter. YFV (17D strain) and Zika (PR strain) were used to infect the different cell lines, and viral replication was measured by viral RNA and infectivity. Attempts to knock out PTPRE by CRISPR were unsuccessful, suggesting that PTPRE may be required for cell survival. Results: The expression of PTPRE with nontargeted 3′UTR led to a 100-fold reduction in infectious virus released from Jurkat and Huh7D cells by infectivity and a 3–10-fold reduction in viral RNA levels. Infectivity was restored in cells grown in doxycycline. By contrast, nontargeted PTPRE expression had no effect on the release of infectious Zika virus. PTPRE expression did not alter YFV attachment or entry. Despite reduced infectivity of the supernatant virus produced by PTPRE-nontargeted cells, YFV envelope content in culture supernatants was similar to that released by cells not expressing nontargeted PTPRE. Electron microscopy demonstrated large, empty-appearing viral particles in YFV-infected cells expressing nontargeted PTPRE that were not present in cells not expressing nontargeted PTPRE. Conclusions: PTPRE is a novel YFV restriction factor that appears to increase the proportion of defective YFV particles released into cell culture media, potentially by preventing YFV RNA encapsidation. PTPRE’s effects on viral replication are not conserved among flaviviruses, as Zika replication was not altered by PTPRE overexpression. Understanding how PTPRE inhibits YFV may provide insight into YFV assembly and release and potentially help to identify novel drug targets. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 150 KiB

Open AccessAbstract

Honey Bee Viruses, Colony Health, and Antiviral Defense

by Katie F. Daughenbaugh, Alex J. McMenamin, Laura M. Brutscher, Fenali Parekh and Michelle L. Flenniken

Proceedings 2020, 50(1), 16; https://doi.org/10.3390/proceedings2020050016 - 4 Jun 2020

Viewed by 1433

Abstract

Honey bee colony losses are influenced by multiple abiotic and biotic factors, including viruses. To investigate the effects of RNA viruses on honey bees, we infected bees with a model virus (Sindbis-GFP) in the presence or absence of double-stranded RNA (dsRNA). In honey [...] Read more.

Honey bee colony losses are influenced by multiple abiotic and biotic factors, including viruses. To investigate the effects of RNA viruses on honey bees, we infected bees with a model virus (Sindbis-GFP) in the presence or absence of double-stranded RNA (dsRNA). In honey bees, dsRNA is the substrate for sequence-specific RNA interference (RNAi)-mediated antiviral defense and is a trigger of sequence-independent\antiviral responses. Transcriptome sequencing identified more than 200 differentially expressed genes, including genes in the RNAi, Toll, Imd, JAK-STAT, and heat shock response pathways, and many uncharacterized genes. To confirm the virus limiting role of two genes (i.e., dicer and mf116383) in honey bees, we utilized RNAi to reduce their expression in vivo and determined that the virus abundance increased. To evaluate the role of the heat shock stress response in antiviral defense, bees were heat stressed post-virus infection and the virus abundance and gene expression were assessed. Heat-stressed bees had reduced virus levels and a greater expression of several heat shock protein encoding genes (hsps) compared to the controls. To determine if these genes are universally associated with antiviral defense, bees were infected with another model virus, Flock House virus (FHV), or deformed wing virus and the gene expression was assessed. The expression of dicer was greater in bees infected with either FHV or Sindbis-GFP compared to the mock-infected bees, but not in the deformed wing virus-infected bees. To further investigate honey bee antiviral defense mechanisms and elucidate the function of key genes (dicer, ago-2, mf116383, and hsps) at the cellular level, primary honey bee larval hemocytes were transfected with dsRNA or infected with the Lake Sinai virus 2 (LSV2). These studies indicate that mf116383 and hsps mediate dsRNA detection and that MF116383 is involved in limiting LSV2 infection. Together, these results further our understanding of honey bee antiviral defense, particularly dsRNA-mediated antiviral responses, at both the individual bee and cellular levels. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 131 KiB

Open AccessAbstract

HIV-1 Envelope Glycoprotein Trafficking and Viral Transmission

by Melissa Victoria Fernandez, Lwar N Naing, David A Scheiblin, Sherimay D Ablan, Jennifer A Simmons and Eric O Freed

Proceedings 2020, 50(1), 17; https://doi.org/10.3390/proceedings2020050017 - 4 Jun 2020

Viewed by 1502

Abstract

HIV-1 encodes an envelope glycoprotein complex (Env) containing a long cytoplasmic tail (CT) harboring trafficking motifs implicated in Env incorporation into virions. Although the requirement for the Env CT in viral transmission is known, the precise mechanism by which Env is incorporated into [...] Read more.

HIV-1 encodes an envelope glycoprotein complex (Env) containing a long cytoplasmic tail (CT) harboring trafficking motifs implicated in Env incorporation into virions. Although the requirement for the Env CT in viral transmission is known, the precise mechanism by which Env is incorporated into nascent virions and localizes to the virological synapse remains poorly defined. To further elucidate the mechanism of Env trafficking, we examined three HIV-1 strains: the lab-adapted clade B strain, NL4-3, and a transmitted/founder (T/F) clade C virus, K3016, and a T/F clade B virus, CH077. The HIV-1 Env CT contains two invariant trafficking motifs: tyrosine endocytosis motif, Y⁷¹²SPL, and C-terminal dileucine motif, LL⁸⁵⁵. Virion Env incorporation analysis revealed that Y⁷¹²SPL is necessary for efficient Env incorporation, while LL⁸⁵⁵ is dispensable. Spreading infection kinetics were analyzed in various T-cell lines and primary human PBMCs; the results indicated that both endocytic motifs contribute to efficient viral spread in culture. Analysis of Env localization to the T-cell uropod, the portion of the plasma membrane that forms a virological synapse with uninfected cells, was found to be dependent on the Env CT and the Y⁷¹²SPL motif. Cell-to-cell and cell-free transmission assays using T cells infected with HIV-1 bearing Y₇₁₂A or LL₈₅₅AA Env CT mutations are ongoing to establish a role for these motifs in both modes of viral transmission. These studies will significantly enhance our understanding of Env trafficking and viral transmission, providing insights into viral Env–host interactions in physiologically relevant cells. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 148 KiB

Open AccessAbstract

Exploitation of Host Factors and Cellular Pathways by Tombusviruses for the Biogenesis of the Viral Replication Organelles

by Peter D. Nagy

Proceedings 2020, 50(1), 18; https://doi.org/10.3390/proceedings2020050018 - 4 Jun 2020

Viewed by 1254

Abstract

Plus-stranded RNA viruses recruit cellular vesicles and co-opt cellular proteins involved in cellular metabolism and lipid biosynthesis to build viral replicase complexes (VRCs) within the large viral replication compartments. We use tombusviruses (TBSV), which are small (+)RNA viruses, as model plant viruses to [...] Read more.

Plus-stranded RNA viruses recruit cellular vesicles and co-opt cellular proteins involved in cellular metabolism and lipid biosynthesis to build viral replicase complexes (VRCs) within the large viral replication compartments. We use tombusviruses (TBSV), which are small (+)RNA viruses, as model plant viruses to study virus replication, recombination, and virus–host interactions using yeast (Saccharomyces cerevisiae) as a surrogate host. Several systematic genome-wide screens and global proteomic and lipidomic approaches have led to the identification of ~500 host proteins/genes that are implicated in TBSV replication. We characterized the role of two-dozen co-opted host proteins, sterols, and phosphatidylethanolamine in tombusvirus VRC assembly and viral RNA synthesis. We provide evidence on the critical roles of phosphoinositides and co-opted membrane-shaping proteins in VRC formation. We also present data that tombusviruses hijack the glycolytic and fermentation pathways to obtain ATP, which is required for the biogenesis of the replication compartment. Finally, we show evidence that TBSV usurps COPII and endosomal vesicles to form a unique microenvironment involving peroxisomes and endoplasmic reticulum (ER) to support viral replication. These new insights highlight the amazingly complex nature of virus-host interactions. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 143 KiB

Open AccessAbstract

Expression Profiles of NOD-Like Receptors in Salmonid Cells after Infection with Infectious Pancreatic Necrosis Virus (IPNV)

by Gardenia Payne, Fernanda Fredericksen, Nicolás Maldonado, Melina Villalba and Victor Olavarría

Proceedings 2020, 50(1), 19; https://doi.org/10.3390/proceedings2020050019 - 5 Jun 2020

Viewed by 1585

Abstract

Infectious pancreatic necrosis virus (IPNv) is a worldwide etiologic agent of one disease that causes severe economic losses in several species of fish, mainly in young salmonids. Its genome consists of two linear double-stranded RNA segments that encode five viral proteins. Teleost fish [...] Read more.

Infectious pancreatic necrosis virus (IPNv) is a worldwide etiologic agent of one disease that causes severe economic losses in several species of fish, mainly in young salmonids. Its genome consists of two linear double-stranded RNA segments that encode five viral proteins. Teleost fish respond to infectious agents, mainly through the components of innate immunity. This response to viral infections is initiated, conducted, and coordinated by pathogen recognition receptors (PRRs), which can detect the presence of microorganisms through the identification of molecular patterns associated with pathogens (PAMPs). Heterologous PRR molecules have been found in salmonids, even in teleost fish. NOD-like receptors (NLRs) are a multigenic family of cytoplasmic molecules involved in immunity and apoptosis; these receptors have been little studied in fish. However, they have recently been linked to antiviral defense. There is no information that relates the expression of NOD-like receptors with IPNv infection. Thus, the objective of this study was to analyze the gene expression of several members of subfamily A of the NLRs (NOD1, NOD2, NLR-C3, NLR-C5, and NLR-X1) in response to IPNv infection by real-time quantitative PCR (RT-qPCR) and cellular models used in vitro and ex vivo. The expression analysis revealed that CHSE-214 cells, infected with IPNv, show a positive regulation of the NLRs, with the NLRX1 gene being the one with the highest expression. A similar result was obtained when primary cultures of head kidney of rainbow trout were infected with IPNv, but in this case, the most stimulated receptor was found to be NLR-C5. Overall, the results suggest that NLRs could play a key role in the regulation of defense mechanisms of salmonids against viral pathogens and justify the exploration of the precise molecular mechanism related to the immune system of the NLRs in these fish. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 144 KiB

Open AccessAbstract

Determining the Role of Bacteriophages in the Virulence of Streptococcus agalactiae

by Melody N. Neely, Caitlin Sar Wiafe-Kwakye and Sally D. Molloy

Proceedings 2020, 50(1), 20; https://doi.org/10.3390/proceedings2020050020 - 8 Jun 2020

Viewed by 1861

Abstract

Bacteriophages (phage) are viruses that specifically infect bacteria and contribute to their evolution. Nearly all bacterial pathogens contain integrated phage, or prophage, within their genome, and many phage genes are associated with bacterial virulence and/or bacterial fitness. Prophages that are associated with the [...] Read more.

Bacteriophages (phage) are viruses that specifically infect bacteria and contribute to their evolution. Nearly all bacterial pathogens contain integrated phage, or prophage, within their genome, and many phage genes are associated with bacterial virulence and/or bacterial fitness. Prophages that are associated with the virulence of human pathogens include those found in Vibrio cholerae, Salmonella sp., Staphylococcus aureus, and Streptococcus pyogenes. Streptococcus agalactiae (Group B streptococcus or GBS) also contain prophages and, in many cases, multiple prophages in a single strain. GBS is an opportunistic pathogen that is found associated with the mucus membranes of the genitourinary tract in about 25% of the adult population. However, if GBS colonizes an immunocompromised individual (such as a neonate or pregnant mother), it can invade and survive in the bloodstream, resulting in serious morbidity and mortality. Unfortunately, treatment of neonates with antibiotics results in disruption of their normal gut microflora and has major effects on the development of their immune system. In order to address the threat of GBS to newborns, we need to fully understand the mechanisms by which this pathogen causes disease. We hypothesize that phage integration into the S. agalactiae genome may enhance pathogenicity and play a role in neonatal-associated infection. Through analysis of the genome sequences of seven S. agalactiae strains, we detected intact phage sequences in three out of the seven strains, which are A909, CNCTC 10/84, and 2603V/R. We have successfully cured the prophage, Callidus, from the CNCTC10/84 genome by overexpression of the excise gene. Comparative analyses between the WT lysogen and the cured strain show differences in chain length, cell morphology and virulence using a well-established zebrafish infectious disease model. Understanding GBS virulence mechanisms is key to developing novel, effective treatments and vaccines for GBS. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 134 KiB

Open AccessAbstract

Does the Cytoplasmic Tail Matter? Mechanism of Viral Envelope Glycoprotein Targeting by Membrane-Associated-RING-CH (MARCH) Proteins

by Cheng man Lun, Abdul A. Waheed and Eric O. Freed

Proceedings 2020, 50(1), 21; https://doi.org/10.3390/proceedings2020050021 - 8 Jun 2020

Viewed by 1351

Abstract

The MARCH family of RING-finger E3 ubiquitin ligases comprise 11 members that have been reported to play a variety of roles in the downregulation of cell-surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to ubiquitinate the cytoplasmic [...] Read more.

The MARCH family of RING-finger E3 ubiquitin ligases comprise 11 members that have been reported to play a variety of roles in the downregulation of cell-surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to ubiquitinate the cytoplasmic tails (CTs) of target proteins, leading to protein degradation through either lysosomal or proteasomal pathways. Three MARCH proteins (MARCH1, 2, and 8) have recently been reported to target the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G glycoprotein (VSV-G), thereby impairing the infectivity of HIV-1 virions bearing HIV-1 Env or VSV-G. However, the mechanism of antiviral activity remains poorly defined. Our data show that MARCH proteins antagonize the full-length forms of HIV-1 Env, VSV-G, and Ebola glycoprotein (GP), and impair the infectivity of HIV-1 virions bearing these viral glycoproteins. This Env-targeting activity of the MARCH proteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that the MARCH protein targeting of VSV-G is, to a large extent, CT-dependent. In striking contrast, the MARCH-protein targeting of HIV-1 Env and Ebola GP does not require the CT. Confocal microscopy data demonstrate that MARCH proteins are able to trap the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in T-cell lines and PBMCs is inducible by type I interferons (a and b) and is also upregulated by HIV-1 infection. Current studies are aimed at identifying the cellular target for MARCH-mediated ubiquitination in the context of their antiviral activity. These results will clarify the mechanism by which MARCH proteins antagonize viral glycoproteins and provide insights into the antiviral role of cellular inhibitory factors in Env biogenesis, trafficking, and virion incorporation. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 139 KiB

Open AccessAbstract

Divergent Traits and Ligand-Binding Features of the Cytomegalovirus CD48 Gene Family

by Pablo Martinez-Vicente, Domènec Farré, Elena Gracia-Latorre, Pablo Engel and Ana Angulo

Proceedings 2020, 50(1), 23; https://doi.org/10.3390/proceedings2020050023 - 9 Jun 2020

Viewed by 1418

Abstract

The genesis of gene families through the capture of host genes and their subsequent duplication is a crucial process in the evolution of large DNA viruses. CD48 is a cell surface protein with an ectodomain composed of two immunoglobulin (Ig) domains. Via its [...] Read more.

The genesis of gene families through the capture of host genes and their subsequent duplication is a crucial process in the evolution of large DNA viruses. CD48 is a cell surface protein with an ectodomain composed of two immunoglobulin (Ig) domains. Via its N-terminal Ig domain, CD48 interacts with the cell surface receptor 2B4, triggering signal transduction events that regulate leukocyte cytotoxicity. We previously reported the presence of five CD48 homologs (vCD48s) in two related cytomegaloviruses, derived from a common host CD48 ancestor gene acquired by retrotranscription. Recently, we examined one member of this family, A43, showing that it acts as a functional viral decoy receptor, binding with high affinity and stability to 2B4 and impairing NK-cell cytotoxicity. Here, we have characterized the rest of the vCD48s. We show that they are highly glycosylated type I transmembrane proteins that display remarkably distinct features: dissimilar structures (e.g., different size stalks and cytoplasmic tails), biochemical properties, locations (cell surface/soluble), and temporal kinetic classes. We found that, in contrast to A43, none of them interacts with 2B4. Consistent with this, the molecular modeling of the N-terminal Ig domains of these vCD48s evidences significant changes in the numbers and lengths of their β-strands and inter-sheet loops that participate in the interaction of CD48 with 2B4. This suggests that these vCD48s have diverged to perform new 2B4-independent functions. Interestingly, we determined that one of them, S30, tightly binds CD2, a T- and NK-cell adhesion and costimulatory molecule whose primary ligand is CD58. Thus, altogether, our results show how a key host immune receptor captured by a virus can be subsequently remodeled during viral evolution to yield new molecules with improved affinities to their cognate receptors or with shifted binding specificities to additional immune targets, expanding the repertoire of viral immunoevasins. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 153 KiB

Open AccessAbstract

Elucidating the Role of HIV-2 Viral Protein X

by Mohamed Mahdi, Tamás Richárd Linkner, Zsófia Ilona Szojka and József Tőzsér

Proceedings 2020, 50(1), 24; https://doi.org/10.3390/proceedings2020050024 - 9 Jun 2020

Viewed by 1480

Abstract

Human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) are the causative agents of the acquired immunodeficiency syndrome (AIDS). While both viruses share a similar structural and genomic organization, a difference in replication dynamics and the clinical course of infection is evident [...] Read more.

Human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) are the causative agents of the acquired immunodeficiency syndrome (AIDS). While both viruses share a similar structural and genomic organization, a difference in replication dynamics and the clinical course of infection is evident between the two. Patients dually infected were shown to have lower viral loads and generally a slower rate of progression to AIDS than those who are mono-infected. While the roles of the unique accessory proteins have been studied in detail for HIV-1, those of HIV-2, including viral protein X (Vpx), remain largely uncharacterized. In our previous experiments, Vpx of HIV-2 was found to be involved in decreasing the infectivity of HIV-1 in dual infection cell culture assays. We set out to elucidate the function of this accessory protein, identifying protein–protein interactions of HIV-2 Vpx with cellular and possibly HIV-1 proteins in dual infection, using in-vitro proteomics techniques and proximity ligation assays. Results showed that wild-type Vpx interacted with many cellular proteins involved in splicing, packaging of pre-mRNA, nuclear export, and translation. Of particular interest was the interaction between HIV-2 Vpx and the pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15, which is required for HIV-1 viral DNA synthesis, and the eukaryotic translation initiation factor 2 subunit 3 (EIF2S3), involved in the early steps of protein synthesis. Additionally, Vpx was found to interact directly with the cellular transcriptional repressor C-Terminal Binding Protein 2 (CTBP-2). Moreover, Vpx was shown to hinder the function of HIV-1 reverse transcriptase in in-vitro assays. These findings shed light on the functions of this accessory protein and add to our understanding of the replication dynamics of HIV-2 and its role in dual infection. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 118 KiB

Open AccessAbstract

BKTyper—Web Application for VP1 and NCCR Polyoma BK Typing

by Joan Martí-Carreras and Piet Maes

Proceedings 2020, 50(1), 25; https://doi.org/10.3390/proceedings2020050025 - 9 Jun 2020

Viewed by 1302

Abstract

Human polyoma BK virus (BKV) prevalence has been increasing due to the introduction of more potent immunosuppressive agents, mostly in immunocompromised patients. BKV has been linked mostly to polyomavirus-associated hemorrhagic cystitis, and polyomavirus-associated nephropathy. BKV is a circular double stranded DNA virus (cdsDNA) [...] Read more.

Human polyoma BK virus (BKV) prevalence has been increasing due to the introduction of more potent immunosuppressive agents, mostly in immunocompromised patients. BKV has been linked mostly to polyomavirus-associated hemorrhagic cystitis, and polyomavirus-associated nephropathy. BKV is a circular double stranded DNA virus (cdsDNA) with an average genome size of 5100 bp and an average GC content of 40%. Its genome codifies for five proteins: VP1, VP2, VP3, Angio gene, and the antigen T (which includes an event of alternative splicing, yielding a short and a large antigen T transcript). Additionally, it contains the non-coding control region (NCCR), known to be highly repetitive and to vary in number, length, and location of the repeats. Subtyping of BKV has been mainly studied in VP1 and the NCCR. Subtyping and subgrouping of BKV is conducted routinely in diagnostic assays and in epidemiological studies. Recently, Morel et al. published (Journal of Clinical Microbiology 2017; 55, 4) a strategy to subtype BKV through 100 bp VP1 amplicon. NCCR diversity is more complex than VP1, as it is configured by five repeat blocks (O, P, Q, R, and S). NCCR blocks can vary in number and length, resulting in a gradient of infectivity and replication. Rearranged NCCR have been linked to diverse patient etiologies, although any specific arrangement has failed to correlate with disease outcome or to have any predictive value. Due to the high abundance of BKV individuals and the clinical implications for human health that may represent BKV typing, a reliable, automatic, and free typing tool would be of great interest. Here, BKTyper is presented, a whole genome genotyper for polyoma BKV, based on a VP1 typing by Morel’s algorithm and NCCR block identification. BKTyper can accept both whole BKV genome or regions of interest in fasta format to generate the typing profile and phylogenetic analysis. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 133 KiB

Open AccessAbstract

Deciphering the RNA Silencing Suppressor Function in the Potyvirus SPV2

by Ornela Chase, Giannina Bambaren and Juan José López-Moya

Proceedings 2020, 50(1), 26; https://doi.org/10.3390/proceedings2020050026 - 9 Jun 2020

Viewed by 1327

Abstract

In most eukaryotes, RNA silencing is a key element in the regulation of gene expression and defense against pathogens. Plants have developed a defensive barrier against exogenous microorganisms, such as plant-infecting viruses, by specifically targeting and degrading the viral RNAs and thus limiting [...] Read more.

In most eukaryotes, RNA silencing is a key element in the regulation of gene expression and defense against pathogens. Plants have developed a defensive barrier against exogenous microorganisms, such as plant-infecting viruses, by specifically targeting and degrading the viral RNAs and thus limiting the negative effects of the diseases caused by them. On the other hand, plant viruses encode for suppressor proteins that repress the host-silencing machinery, hence allowing viral replication and infection establishment. Our current project focuses on the characterization of gene products contributing to the RNA silencing suppressor (RSS) function of Sweet potato virus 2 (SPV2), genus Potyvirus, family Potyviridae. SPV2 infects sweet potatoes (Ipomoea batatas, family Convolvulaceae), one of the most important staple food crops worldwide. Infections by potyvirids result in the high yield losses of sweet potatoes, especially from coinfection with unrelated viruses, and our final goal is to develop efficient control strategies. Our preliminary results analyzing the P1 and HCPro proteases of SPV2, transiently expressed in N. benthamiana together with a reporter GFP construct, revealed that HCPro constitutes a strong RSS. This is a novel finding, and we are currently characterizing the functions of other gene products. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 128 KiB

Open AccessAbstract

Development of a T7-Independent MARV Minigenome System

by Bert Vanmechelen, Joren Stroobants, Kurt Vermeire and Piet Maes

Proceedings 2020, 50(1), 27; https://doi.org/10.3390/proceedings2020050027 - 9 Jun 2020

Viewed by 1326

Abstract

Marburg virus (MARV) is the only known pathogenic filovirus that does not belong to the genus Ebolavirus. It causes a severe hemorrhagic fever that is associated with a high mortality rate (>80%). The potential for filoviruses to cause devastating outbreaks, in combination [...] Read more.

Marburg virus (MARV) is the only known pathogenic filovirus that does not belong to the genus Ebolavirus. It causes a severe hemorrhagic fever that is associated with a high mortality rate (>80%). The potential for filoviruses to cause devastating outbreaks, in combination with the lack of licensed therapeutics and vaccines for Marburg virus disease, illustrates the need for more MARV research. However, research involving MARV is hindered by its dependency on access to high-containment laboratories. Virus alternatives such as minigenomes have proven to be a useful tool to study virus replication and transcription at lower biosafety levels, and can be used for antiviral compound screening. All currently available MARV minigenomes are dependent on the addition of an ectopic T7 RNA polymerase that can drive minigenome expression. While this allows for high expression levels, the ectopic expression of a T7 polymerase is not feasible in all cell types, and acts as a confounding factor in compound screening assays. We have developed an alternative MARV minigenome system that is controlled by an RNA polymerase II promoter, which is natively expressed in most mammalian cell types. We show here that this novel minigenome can be used in a wide range of cell types, and can be easily amended to a 96-well format to be used for high-throughput compound screening, thereby providing a valuable alternative to previously developed MARV minigenomes. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 163 KiB

Open AccessAbstract

Development of a Tubular Bacteriophage-Based Vaccine Platform that Induces an Immune Response in Mice

by Aliona Špakova, Indrė Dalgėdienė, Aurelija Žvirblienė and Rasa Petraitytė-Burneikienė

Proceedings 2020, 50(1), 28; https://doi.org/10.3390/proceedings2020050028 - 9 Jun 2020

Viewed by 1417

Abstract

Current vaccines against infectious diseases have primarily relied on attenuated or inactivated pathogens. However, self-assembled, virus-based nanoparticles (VNPs) are noninfectious multiprotein structures regarded as safe vaccine platforms for an efficient foreign antigen display within a host immune system. Currently, there is a low [...] Read more.

Current vaccines against infectious diseases have primarily relied on attenuated or inactivated pathogens. However, self-assembled, virus-based nanoparticles (VNPs) are noninfectious multiprotein structures regarded as safe vaccine platforms for an efficient foreign antigen display within a host immune system. Currently, there is a low diversity of self-assembled, rod-shaped VNPs. Additionally, there is no information regarding the generation of tailed-bacteriophage nanotubes in yeast and their immunogenicity in mice. Here, we developed a novel tubular VNP-based vaccine platform utilizing a yeast-synthesized recombinant tail tube gp39 protein from bacteriophage vB_EcoS_NBD2 (NBD2). The diameter of these extremely flexible polytubes was ~12 nm, while the length varied from 0.1 µm to >3.95 µm. In this study, the immunogenicity of polytubes formed by the recombinant gp39 protein and the elicited antibody response were tested. The tubular structures formed by the recombinant gp39 protein were immunogenic in mice, although the addition of Freund’s adjuvant enhanced the anti-gp39 antibody response compared to the use of tubular structures alone. To further examine the applicability of novel polytubes as a carrier for foreign epitopes, the carboxy-terminal region within the gp39 protein was identified, allowing insertion of six histidine residues with no effect on the recombinant protein synthesis or structure self-assembly. This genetic insertion of a foreign epitope within the surface-exposed gp39 domains resulted in a repetitive display of the insert on the surface of NBD2 tail tube-originated polytubes. The combination of a repetitive, highly ordered display of foreign epitopes as well as tubular shape of nanoparticles can greatly enhance the immune response. Although more studies are needed, the flexible and extremely long polytubes formed by the recombinant tail tube gp39 protein represent a new potential platform for presenting target sequences on the exterior surface of the nanotubes. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 154 KiB

Open AccessAbstract

Atypical Porcine Pestivirus Molecular Evolution within a Persistently Infected Swine Farm

by Alba Folgueiras-González, Robin van den Braak, Bartjan Simmelink, Martin Deijs, Ad de Groof and Lia van der Hoek

Proceedings 2020, 50(1), 29; https://doi.org/10.3390/proceedings2020050029 - 9 Jun 2020

Viewed by 1146

Abstract

Atypical porcine pestivirus (APPV) is a single stranded RNA virus from the family Flaviviridae. [...] Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 122 KiB

Open AccessAbstract

Differential Roles of Lipin1 and Lipin2 in the Hepatitis C Virus Replication Cycle

by Victoria Castro, Gema Calvo, Ginés Ávila-Pérez, Marlène Dreux and Pablo Gastaminza

Proceedings 2020, 50(1), 30; https://doi.org/10.3390/proceedings2020050030 - 9 Jun 2020

Viewed by 1234

Abstract

Although their origin, nature and structure are not identical, a common feature of positive-strand RNA viruses is their ability to subvert host lipids and intracellular membranes to generate replication and assembly complexes. Recently, lipin1, a cellular enzyme that converts phosphatidate into diacylglycerol, has [...] Read more.

Although their origin, nature and structure are not identical, a common feature of positive-strand RNA viruses is their ability to subvert host lipids and intracellular membranes to generate replication and assembly complexes. Recently, lipin1, a cellular enzyme that converts phosphatidate into diacylglycerol, has been involved in the formation of the membranous web that hosts hepatitis C virus (HCV) replicase. In the liver, lipin1 cooperates with lipin2 to maintain glycerolipid homeostasis. We extended our previous study of the lipin family in HCV infection by determining the impact of the lipin2 silencing on viral replication. In contrast to the specific impact of lipin1 silencing on HCV replication, our data suggest a broader function of lipin2 not only in HCV infection, but also for the replication of other RNA viruses. Moreover, uninfected lipin2- but not lipin1-deficient cells display alterations in mitochondrial and Golgi morphology, suggesting that lipin2 contributes to the maintenance of the overall organelle architecture. Coinciding with Golgi fragmentation, our data reveal that lipin2 silencing mainly interferes with HCV virion secretion at late stages of the infection without significantly affecting viral replication or assembly. Overall, this study reveals distinctive functions of lipin1 and lipin2 in cells of hepatic origin, a context in which they are often considered functionally redundant. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 170 KiB

Open AccessAbstract

ER-Shaping Atlastin Proteins Act as Central Hubs to Promote Flavivirus Replication and Virion Assembly

by Christopher J Neufeldt, Mirko Cortese, Pietro Scaturro, Berati Cerikan, Jeremy Wideman, Keisuke Tabata, Thais Morase, Olga Oleksiuk, Andreas Pichlmair and Ralf Bartenschlager

Proceedings 2020, 50(1), 31; https://doi.org/10.3390/proceedings2020050031 - 10 Jun 2020

Viewed by 1558

Abstract

Members of the Flavivirus genus rely extensively on the host cell endomembrane network to generate complex membranous replication organelles (ROs) that facilitate viral genome replication and the production of virus particles. For dengue virus and Zika virus, these ROs included vesicles which are [...] Read more.

Members of the Flavivirus genus rely extensively on the host cell endomembrane network to generate complex membranous replication organelles (ROs) that facilitate viral genome replication and the production of virus particles. For dengue virus and Zika virus, these ROs included vesicles which are formed through membrane invagination into the endoplasmic reticulum (ER) lumen, termed invaginated vesicles or vesicle packets (VPs), as well as large areas of bundled smooth ER, termed convoluted membranes. Though the morphology of these virus-induced membrane structures has been well characterized, the viral and host constituents that make up flaviviral ROs are still poorly understood. Here, we identified a subset of ER resident proteins (atlastins), normally required for maintaining ER tubule networks, as critical host factors for flavivirus infection. Specific changes in atlastin (ATL) levels had dichotomous effects on flaviviruses with ATL2 depletion, leading to replication organelle defects and ATL3 depletion to changes in viral assembly/release pathways. These different depletion phenotypes allowed us to exploit virus infection to characterize non-conserved functional domains between the three atlastin paralogues. Additionally, we established the ATL interactome and show how it is reprogrammed upon viral infection. Screening of specific ATL interactors confirmed non-redundant ATL functions and identified a role for ATL3 in vesicle trafficking. Our data demonstrate that ATLs are central host factors that coordinate the ER network and shape the ER during flavivirus infection. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 156 KiB

Open AccessAbstract

Impact of Capsid Anchor Length and Sequential Processing on the Assembly and Infectivity of Dengue Virus

by Oscar R. Burrone, José L. Slon Campos, Monica Poggianella and Jyoti Rana

Proceedings 2020, 50(1), 32; https://doi.org/10.3390/proceedings2020050032 - 10 Jun 2020

Viewed by 1531

Abstract

: The assembly and secretion of flaviviruses are part of an elegantly regulated process. During maturation, the viral polyprotein undergoes several co- and post-translational cleavage events mediated by both viral and host proteases. Among these, sequential cleavage at the N- and C-termini of [...] Read more.

: The assembly and secretion of flaviviruses are part of an elegantly regulated process. During maturation, the viral polyprotein undergoes several co- and post-translational cleavage events mediated by both viral and host proteases. Among these, sequential cleavage at the N- and C-termini of the hydrophobic capsid anchor (Ca) at the junction of C-PrM has been considered essential for the production of flaviviruses. Here, using a refined dengue pseudovirus production system, we show that Ca plays a key role in the processing efficiency of dengue virus type 2 (DENV2) structural proteins and the assembly of viral particles. The replacement of the relatively short DENV2 Ca with the homologous regions from West Nile or Zika viruses or, alternatively, the increase in its length, improved cleavage, and hence particle assembly. Furthermore, we show that the substitution of the Ca conserved proline residue (Pro-110), as alanine abolishes pseudovirus production, regardless of the Ca sequence length. Using two experimental approaches, we investigated the need for sequential cleavage (first on the cytosolic side, then on the luminal side) and found that, while cleavage at the Ca-Pr boundary is essential for the assembly of infective particles, the same is not true for cleavage at the C-Ca boundary. We show that both the mature (C) and unprocessed capsids (C-Ca) of DENV2 were equally efficient in packaging the viral RNA and in assembling the infective particles. This was further confirmed with mutants, in which cleavage at the luminal side, by the signal peptidase, occurred independently of cleavage at the cytosolic side, by the viral NS2B/NS3 protease. We thus demonstrate that, unlike other flaviviruses, DENV2 capsid does not require a cleavable Ca sequence and that sequential cleavage is not an obligatory requirement for the morphogenesis of infective particles. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 139 KiB

Open AccessAbstract

An Endogenous Retrovirus from Human Hookworm Encodes an Ancient Phlebovirus-Like Class II Envelope Fusion Protein

by Monique Merchant, Carlos P. Mata and Yorgo Modis

Proceedings 2020, 50(1), 33; https://doi.org/10.3390/proceedings2020050033 - 10 Jun 2020

Viewed by 1453

Abstract

Within the parasitic nematode Ancylostoma ceylanicum, a ~20 million-year-old Bel/Pao LTR retrotransposon encodes an ancient viral class II envelope fusion protein termed Atlas Gc. Typically, retroviruses and related degenerate retrotransposons encode a hemagglutinin-like class I envelope fusion protein. A subset of Bel/Pao [...] Read more.

Within the parasitic nematode Ancylostoma ceylanicum, a ~20 million-year-old Bel/Pao LTR retrotransposon encodes an ancient viral class II envelope fusion protein termed Atlas Gc. Typically, retroviruses and related degenerate retrotransposons encode a hemagglutinin-like class I envelope fusion protein. A subset of Bel/Pao LTR retrotransposons within the phylum Nematoda have acquired a phlebovirus-like envelope gene and utilized the encoded fusion machinery to escape the genome as intact exogenous retroviruses. This includes C. elegans retroelement 7 virus which was recently reclassified as a member of the genus Semotivirus. A 3.76 Å cryoEM reconstruction confirms Atlas Gc as a closely related phleboviral homologue and class II fusion protein in a novel case of gene exaptation. Preliminary biophysical and biochemical characterization indicate Atlas Gc functions under specific physiological conditions targeting late-endosomal membranes, much like modern viral class II envelope fusion proteins. Phylogenetic analyses support the reclassification of the Atlas endogenous retrovirus and five other A. ceylanicum ERVs as novel semotiviruses of Belpaoviridae of the new viral order of reverse-transcribing viruses Ortervirales. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 151 KiB

Open AccessAbstract

ADAR1 Function Regulates Innate Immune Activation and Susceptibility to Viral Infections

by Maria Pujantell, Eva Riveira-Muñoz, Edurne García-Vidal, Lucía Gutiérrez-Chamorro, Roger Badia, Bonaventura Clotet and Ester Ballana

Proceedings 2020, 50(1), 34; https://doi.org/10.3390/proceedings2020050034 - 10 Jun 2020

Viewed by 1410

Abstract

Viral infection induces innate intracellular antiviral defenses, aimed at restricting virus replication and spread. Therefore, understanding the role and function of innate immune modulators can help to establish novel strategies for viral control. Here, we explore the role of ADAR1 as a regulator [...] Read more.

Viral infection induces innate intracellular antiviral defenses, aimed at restricting virus replication and spread. Therefore, understanding the role and function of innate immune modulators can help to establish novel strategies for viral control. Here, we explore the role of ADAR1 as a regulator of the HIV, HCV, and HPV infections, both in vitro and in vivo, in a genetic association study. Depletion of ADAR1 induced innate immune activation, observed by a significant increase in IFNB1 mRNA and CXCL10 expression. Further characterization of ADAR1 knockdown also showed upregulation of the RNA sensors MDA5 and RIG-I, increased IRF7 expression, and phosphorylation of STAT1. ADAR1 deficiency had differential effects depending on the virus tested: siADAR1 cells showed a significant reduction in HIV-1 infection, whereas ADAR1 knockdown suggested a proviral role in HCV and HPV infections. In addition, genetic association studies were performed in a cohort of 155 HCV/HIV individuals with chronic coinfection, and a cohort of 173 HPV/HIV-infected individuals was followed for a median of six years (range 0.1–24). Polymorphisms within the ADAR1 gene were found to be significantly associated with poor clinical outcome of HCV therapy and advanced liver fibrosis in a cohort of HCV/HIV-1-coinfected patients. Moreover, we identified the low-frequency haplotype AACCAT to be significantly associated with recurrent HPV dysplasia, suggesting a role for ADAR1 in the outcome of HPV infection in HIV+ individuals. In conclusion, we show that ADAR1 regulates innate immune activation and plays a key role in susceptibility to viral infections by either limiting or enhancing viral replication. Overall, ADAR1 could be a potential target for designing immune-modulating therapeutic strategies. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 152 KiB

Open AccessAbstract

Ebola Virus Requires Phosphatidylserine Scrambling Activity for Efficient Budding and Optimal Infectivity

by Marissa Acciani, Maria Lay, Katherine E. Havranek, Avery Duncan, Hersha Iyer, Olivia Linn and Melinda Ann Brindley

Proceedings 2020, 50(1), 35; https://doi.org/10.3390/proceedings2020050035 - 11 Jun 2020

Cited by 2 | Viewed by 2046

Abstract

Ebola virus (EBOV) interacts with cells using multiple categories of cell-surface receptors, including C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically bind to apoptotic cell membrane PS and orchestrate the uptake and clearance of apoptotic bodies. Many viruses coated with PS-containing lipid [...] Read more.

Ebola virus (EBOV) interacts with cells using multiple categories of cell-surface receptors, including C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically bind to apoptotic cell membrane PS and orchestrate the uptake and clearance of apoptotic bodies. Many viruses coated with PS-containing lipid envelopes, acquired during budding from host cells, can also exploit these receptors for internalization. PS is restricted to the inner leaflet of the plasma membrane in homeostatic cells, an orientation that would be unfavorable for PS receptor-mediated uptake if conserved on the viral envelope. Therefore, it is theorized that viral infection induces host-cell PS externalization to the outer leaflet during replication. Cells have several membrane scramblase enzymes that enrich outer leaflet PS when activated. Here, we investigate the role of two scramblases, TMEM16F and XKR8, as possible mediators of cellular and viral envelope surface PS levels during recombinant vesicular stomatitis virus (VSV) in which the VSV glycoprotein was replaced with the Ebola glycoprotein (rVSV/EBOV-GP) replication and EBOV virus-like particle (VLP) production. We find that rVSV/EBOV-GP and EBOV VLPs produced in XKR8 knockout cells contain two- to threefold less PS in their outer leaflets. Consequently, rVSV/EBOV-GP produced in deltaXKR8 is 70% less efficient at infecting cells through apoptotic mimicry as compared to the viruses produced by parental cells. In addition, the budding efficiency of both recombinant VSV particles and VLPs was significantly reduced in cells lacking XKR8. Our data suggest that virion surface PS acquisition requires XKR8 activity, whereas the deletion of TMEM16F did not affect EBOV-GP-mediated entry of VLP production. Unexpectedly, we observed an additional role of XKR8 in rVSV/G, rVSV/EBOV-GP, and EBOV VLP budding. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 159 KiB

Open AccessAbstract

Influenza D Virus: A Potential Threat for Humans?

by Claudia Maria Trombetta, Serena Marchi, Ilaria Manini, Otfried Kistner, Feng Li, Pietro Piu, Alessandro Manenti, Fabrizio Biuso, Chithra Sreenivasan, Julian Druce and Emanuele Montomoli

Proceedings 2020, 50(1), 36; https://doi.org/10.3390/proceedings2020050036 - 11 Jun 2020

Cited by 2 | Viewed by 1780

Abstract

Influenza D virus (IDV) is a novel influenza virus first isolated from swine in 2011 in Oklahoma. Several studies have isolated IDV in cattle from multiple geographic areas, suggesting that cattle could be a possible primary natural reservoir for the virus. To date, [...] Read more.

Influenza D virus (IDV) is a novel influenza virus first isolated from swine in 2011 in Oklahoma. Several studies have isolated IDV in cattle from multiple geographic areas, suggesting that cattle could be a possible primary natural reservoir for the virus. To date, few studies have been performed on human samples and there is no conclusive evidence that IDV has the ability to infect humans. This serological study aimed to assess the prevalence of antibodies against IDV in the human population. The IDV used in the serological analysis was influenza D/bovine/Oklahoma/660/2013. The human serum samples, collected in Italy between 2005 and 2017, were randomly selected from the laboratory serum bank and tested by the haemagglutination inhibition (HI) assay. HI positivity has been confirmed using the virus neutralization (VN) assay. Based on HI positivity (HI titers ≥ 10), a low prevalence (5%–10%) was observed between 2005 and 2007. There has been a sharp increase since 2008, resulting in two main peaks in 2009–2010 and 2013–2014, a finding confirmed by the statistical trend analysis. The same pattern and trends can be seen with higher HI titers of >20 and ≥40. The prevalence of antibodies against IDV has increased in the human population in Italy from 2005 to 2017. Low prevalence values between 2005 and 2007 suggest that IDV most probably circulated before its detection in 2011, and perhaps even before 2005. In Italy, IDV has been shown to circulate among swine and bovine herds. It is, therefore, possible that prevalence peaks in humans follow the infection epidemics in animals and do not to persist in the population, resembling a spillover event from the animal reservoir and showing that the virus may not circulate consistently in the human population. However, IDV seemed to have the ability to elicit an immune response in humans. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 138 KiB

Open AccessAbstract

Cell Entry by Quasi-Enveloped and Naked Hepatoviruses

by Stanley M. Lemon

Proceedings 2020, 50(1), 37; https://doi.org/10.3390/proceedings2020050037 - 11 Jun 2020

Viewed by 1550

Abstract

Hepatoviruses are unusual picornaviruses, distinct genetically and structurally from other members of the Picornaviridae, exclusively hepatotropic, and released from infected cells without lysis in small membranous vesicles resembling exosomes. These quasi-enveloped virions (eHAV) are infectious and the only form of virus found [...] Read more.

Hepatoviruses are unusual picornaviruses, distinct genetically and structurally from other members of the Picornaviridae, exclusively hepatotropic, and released from infected cells without lysis in small membranous vesicles resembling exosomes. These quasi-enveloped virions (eHAV) are infectious and the only form of virus found circulating in blood during acute infection. By contrast, naked virions (nHAV) are shed in feces, having been stripped of membranes by bile salts during passage from the liver through the biliary system. nHAV is exceptionally stable, promoting efficient inter-host transmission through the environment, whereas the membranes cloaking quasi-enveloped eHAV virions protect the virus from neutralizing antibodies, facilitating stealthy spread of infection in newly infected hosts. Since quasi-enveloped eHAV lacks virus-encoded surface proteins, its mechanism of cell entry has been enigmatic. Previous studies in our laboratory have shown that both virion types are internalized primarily by clathrin- and dynamin-dependent endocytosis, facilitated by integrin β₁, followed by trafficking through early Rab-5A⁺ and late Rab-7a⁺ endosomes. eHAV undergoes further ALIX-dependent trafficking to LAMP1⁺ lysosomes where the quasi-envelope is enzymatically degraded. Although TIM1 (HAVCR) was reported many years ago to be a receptor for HAV, it is not essential for infection with either virion type and acts only to facilitate eHAV entry by binding phosphatidylserine on its surface. While late steps in entry remain uncertain, recent studies in our laboratory indicate that both virion types require a ganglioside within the late endolysosome to initiate transfer of the viral RNA to the cytoplasm to initiate replication. Ganglioside GD1a appears most active in facilitating cell entry, and binds to the capsid optimally at the low pH of endolysosomes Remarkably, neither virion type requires PLA2G16 for infection, although this phospholipase is essential for successful transfer of the RNA genome of many other picornaviruses to the cytoplasm. This, and other unusual features of HAV, including the fact that the assembly of capsid pentamers is driven by the C-terminal pX domain of VP1 rather than VP4, and the exceptional stability of the capsid, greatest at the low pH of endolysosomes, suggest an atypical mechanism for HAV uncoating and genome release. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 175 KiB

Open AccessAbstract

Characterization of DNA Polymerase from Thermus thermophilus MAT72 Phage Tt72

by Sebastian Dorawa, Magdalena Plotka, Anna-Karina Kaczorowska, Olafur H. Fridjonsson, Gudmundur O. Hreggvidsson, Arnthor Aevarsson and Tadeusz Kaczorowski

Proceedings 2020, 50(1), 38; https://doi.org/10.3390/proceedings2020050038 - 11 Jun 2020

Viewed by 1708

Abstract

Thermophilic phages are recognized as an untapped source of thermostable enzymes relevant in biotechnology; however, their biology is poorly explored. This has led us to start a project aimed at investigating thermophilic phages isolated from geothermal areas of Iceland. In this study, we [...] Read more.

Thermophilic phages are recognized as an untapped source of thermostable enzymes relevant in biotechnology; however, their biology is poorly explored. This has led us to start a project aimed at investigating thermophilic phages isolated from geothermal areas of Iceland. In this study, we present a structural and functional analysis of the DNA polymerase of phage Tt72, which infects thermophilic bacterium Thermus thermophilus MAT72. An in silico analysis of the Tt72 phage genome revealed the presence of a 2112-bp open reading frame (ORF) encoding protein homologous to the members of the A family of DNA polymerases. It contains a conserved nucleotidyltransferase domain and a 3′ → 5′ exonuclease domain but lacks the 5′ → 3′ exonuclease domain. The amino acid sequence of Tt72 DNA polymerase shows high similarity to two as yet uncharacterized DNA polymerases of T. thermophilus phages: ΦYS40 (91%) and ΦTMA (90%). The gene coding for Tt72 DNA polymerase was cloned and overexpressed in E. coli. The Tt72 polA gene is composed of 2112 nucleotides. The overall G+C content of this gene is 31.58%, which is lower than the G+C content of T. thermophilus genomic DNA (69.49%). The Tt72 polA gene codes for a 703-aa protein with a predicted molecular weight of 80,477. The enzyme was overproduced in E. coli, purified by heat treatment, followed by HiTrap TALON column and HiTrap Heparin HP column chromatography, then biochemically characterized. The optimum activity was found at 55 °C, pH 8.5, 25 mM KCl, and 0.5 mM Mg²⁺. Furthermore, the Tt72 DNA polymerase shows strong 3′ → 5′ exonucleolytic activity. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 129 KiB

Open AccessAbstract

Circulation of Toscana Virus in a Sample Population of Corsica, France

by Shirley Masse, Nazli Ayhan, Lisandru Capai, Rémi Charrel and Alessandra Falchi

Proceedings 2020, 50(1), 39; https://doi.org/10.3390/proceedings2020050039 - 11 Jun 2020

Cited by 1 | Viewed by 1192

Abstract

Sandfly-borne phleboviruses pathogenic to humans, such as Toscana virus (TOSV) and sandfly fever Sicilian virus (SFSV), are endemic in the Mediterranean region. In France, several autochthonous cases of TOSV infection have been described which cause either meningitis or encephalitis. The aim of the [...] Read more.

Sandfly-borne phleboviruses pathogenic to humans, such as Toscana virus (TOSV) and sandfly fever Sicilian virus (SFSV), are endemic in the Mediterranean region. In France, several autochthonous cases of TOSV infection have been described which cause either meningitis or encephalitis. The aim of the present study was to estimate the seroprevalence of TOSV and SFSV antibodies in a healthy population from Corsica. In this cross-sectional study, participants were enrolled from the medical staff at University of Corsica Pasquale Paoli (UCPP) and from general practitioners of the Corsican Sentinelles Network. The seroprevalence study was based on virus microneutralization (MN). A total of 240 patients were tested for TOSV and SFSV. Altogether, 54 serum samples were confirmed for TOSV infection (seroprevalence = 22.5%). None of the samples were positive for SFSV (0/240). The main place of residence was significantly associated with TOSV seropositivity (p-value = 0.005). The overall rate of TOSV antibody seroprevalence observed in our study suggests a more intense circulation of TOSV in Corsica, with a rate significantly higher than the 8.7% reported in Corsica in 2007 from blood donors. The absence of seropositivity to SFSV seems to confirm the low circulation of this virus in Corsica and in continental France. The increasing circulation of TOSV reported here should encourage the implementation of surveillance systems to control phlebovirus infection. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 135 KiB

Open AccessAbstract

Common Occurrence of Belerina Virus, a Novel Paramyxovirus Found in Belgian Hedgehogs

by Bert Vanmechelen, Valentijn Vergote, Michelle Merino and Piet Maes

Proceedings 2020, 50(1), 40; https://doi.org/10.3390/proceedings2020050040 - 11 Jun 2020

Cited by 1 | Viewed by 1324

Abstract

European hedgehogs (Erinaceus europaeus), also known as common hedgehogs, can be found all throughout Western Europe, including the Iberian Peninsula and Italy on the south side and Scandinavia in the north. Hedgehogs are known to carry a variety of bacterial and [...] Read more.

European hedgehogs (Erinaceus europaeus), also known as common hedgehogs, can be found all throughout Western Europe, including the Iberian Peninsula and Italy on the south side and Scandinavia in the north. Hedgehogs are known to carry a variety of bacterial and fungal pathogens, as well as viruses, the pathological and zoonotic potential of which is not yet fully elucidated. Here, we report the discovery of a novel paramyxovirus, named Belerina virus, in Belgian hedgehogs. Based on its divergence from other known paramyxovirus species, Belerina virus is thought to represent a new species in the family Paramyxoviridae. Phylogenetic analysis groups Belerina virus together with members of the genus Jeilongvirus, although its genome organization is most similar to that of several as yet unclassified bat viruses. Because of several dissimilarities with other Jeilongviruses, these bat viruses have been proposed to represent a new genus, tentatively called “Shaanvirus”. Out of 147 animals screened in this study, 57 tested positive for Belerina virus (39%), indicating the wide spread of this virus throughout the Belgian hedgehog population, although the virus’ pathogenic and zoonotic potential remains to be elucidated. In summary, we present here the complete genome sequence of Belerina virus, a putative new paramyxovirus species commonly found in Belgian hedgehogs. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 133 KiB

Open AccessAbstract

Independent Inhibition of the Polymerase and Deubiquitinase Activities of the Crimean–Congo Hemorrhagic Fever Virus Full-Length L-Protein

by Egor P. Tchesnokov, Ben A. Bailey-Elkin, Brian L. Mark and Matthias Götte

Proceedings 2020, 50(1), 41; https://doi.org/10.3390/proceedings2020050041 - 13 Jun 2020

Viewed by 1227

Abstract

The Crimean–Congo hemorrhagic fever virus (CCHFV) is a segmented negative-sense RNA virus that can cause severe human disease. The World Health Organization (WHO) has listed CCHFV as a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. We [...] Read more.

The Crimean–Congo hemorrhagic fever virus (CCHFV) is a segmented negative-sense RNA virus that can cause severe human disease. The World Health Organization (WHO) has listed CCHFV as a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. We report on the expression, characterization, and inhibition of the CCHFV full-length L-protein that provides an important tool in this regard. The requirements for high biosafety measures hamper drug discovery and development efforts with infectious CCHFV. Hence, we decided to adopt a biochemical approach that targets the viral RNA-dependent RNA polymerase (RdRp). The CCHFV RdRp activity is part of a multifunctional L protein that is unusually large, with a molecular weight of ~450 kDa. The CCHFV L-protein also contains an ovarian tumor (OTU) domain that exhibits deubiquitinating (DUB) activity. Previous studies have shown that DUB activity interferes with innate immune responses and viral replication. Here, we utilized the baculovirus expression system and generated a full-length CCHFV L protein. RdRp activity was seen in the presence of divalent metal ions, and inhibition of RNA synthesis was demonstrated with nucleotide analogues. The ubiquitin analogue CC.4 inhibits the CCHFV-associated DUB activity of the full-length L protein and the isolated DUB domain to a similar extent. We have finally shown that RdRp and DUB activities are functionally independent. The full-length CCHFV L-protein provides an important tool for the discovery of antiviral agents. High-throughput screening (HTS) campaigns are now feasible. The same enzyme preparations can be utilized to identify polymerase and DUB inhibitors. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 136 KiB

Open AccessAbstract

Interferon-Stimulated SAMHD1 Restricts Hepatitis C Virus Replication

by Ni An, Jianyuan Zhao and Shan Cen

Proceedings 2020, 50(1), 42; https://doi.org/10.3390/proceedings2020050042 - 13 Jun 2020

Viewed by 1293

Abstract

Human SAMHD1 is an IFN-induced dNTP triphosphatase that is able to restrict HIV-1 replication, whereas its role in innate immunity against virus infection remains largely unexplored. In this work, we provided evidence that SAMHD1 functions as an anti-HCV host factor. We found that [...] Read more.

Human SAMHD1 is an IFN-induced dNTP triphosphatase that is able to restrict HIV-1 replication, whereas its role in innate immunity against virus infection remains largely unexplored. In this work, we provided evidence that SAMHD1 functions as an anti-HCV host factor. We found that overexpression of SAMHD1 resulted in significant inhibition on the replication of HCV, but not other RNA viruses including influenza A virus and EV71. SAMHD1 knockdown partially relieved the inhibitory effect of IFN on HCV, suggesting its important role in the innate immune response against HCV. Mechanistic studies revealed that SAMHD1 targets viral RNA replication without impact on both protein translation and virus entry. Transcriptome analysis showed a broad inhibitory effect of SAMHD1 on host genes involved in cholesterol and fatty acid biosynthesis. In particular, SAMHD1 was shown to downregulate the mRNA abundance of SREBP1, a master transcriptional regulator of de novo lipid biosynthesis, impairing the formation of lipid droplets. Restoring intracellular lipid levels by either exogenous lipid addition or SREBP1 overexpression counteracted the restriction of HCV by SAMHD1, providing evidence that SAMHD1 inhibits the replication of HCV by suppressing host cholesterol and fatty acid biosynthesis. Together, these data unveil, for the first time, a novel antiviral mechanism of SAMHD1 and open new avenues for the development of novel anti-HCV therapeutics. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 129 KiB

Open AccessAbstract

Peroxisomes as Platforms for Cytomegalovirus’ Evasion from Cellular Antiviral Signaling

by Ana Rita Ferreira, Ana Gouveia, Mariana Marques and Daniela Ribeiro

Proceedings 2020, 50(1), 43; https://doi.org/10.3390/proceedings2020050043 - 13 Jun 2020

Viewed by 1255

Abstract

Peroxisomes, in concert with mitochondria, have been established as platforms for the establishment of a rapid and stable antiviral immune response, due to the presence of the mitochondrial antiviral signaling protein (MAVS) at their membranes. Upon intracellular recognition of viral RNA, retinoic acid [...] Read more.

Peroxisomes, in concert with mitochondria, have been established as platforms for the establishment of a rapid and stable antiviral immune response, due to the presence of the mitochondrial antiviral signaling protein (MAVS) at their membranes. Upon intracellular recognition of viral RNA, retinoic acid inducible gene-I (RIG-I)-like proteins interact with MAVS, inducing its oligomerization and the establishment of a signaling cascade that culminates with the production of direct antiviral effectors, preventing important steps in viral propagation. We and others have demonstrated that different viruses have developed specific mechanisms to counteract peroxisome-dependent antiviral signaling. We have shown that the human cytomegalovirus (HCMV) protein vMIA hijacks the peroxisome transport machinery to travel to the organelle, interact with MAVS, and inhibit the immune response. Here, we further unravel the mechanisms by which HCMV is able to evade peroxisome-dependent antiviral signaling. We demonstrate that vMIA localizes at the peroxisomes in a complex with MAVS and the stimulator of interferon genes (STING) protein. Furthermore, vMia interacts with mitochondrial fission factor (MFF) at the peroxisomal membrane, which we show to be essential for vMia-dependent inhibition of the antiviral immune response. Importantly, we demonstrate that vMIA’s interaction with MAVS impedes its oligomerization and the consequent activation of the downstream signaling cascade. Interestingly, our results underline important differences between vMIA’s mechanisms of action at the peroxisomes and the mitochondria. Our results unravel novel mechanisms involving the interplay between the HCMV and peroxisomes that may ultimately contribute to the discovery of novel targets for antiviral combat. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 129 KiB

Open AccessAbstract

Noncapped Genomic RNA Are Critical for Alphaviral Infection and Pathogenicity

by Autumn T. LaPointe and Kevin J Sokoloski

Proceedings 2020, 50(1), 44; https://doi.org/10.3390/proceedings2020050044 - 13 Jun 2020

Viewed by 1174

Abstract

Alphaviruses are positive-sense RNA arthropod-borne viruses that represent a significant threat to public health. During alphaviral replication, significant quantities of viral genomic RNAs that lack a canonical 5’ cap structure are produced and packaged into viral particles, despite the fact that the noncapped [...] Read more.

Alphaviruses are positive-sense RNA arthropod-borne viruses that represent a significant threat to public health. During alphaviral replication, significant quantities of viral genomic RNAs that lack a canonical 5’ cap structure are produced and packaged into viral particles, despite the fact that the noncapped genomes cannot be translated and are essentially noninfectious. Previously, we have reported that the capping efficiency of nsP1, the alphaviral capping enzyme, of Sindbis virus (SINV) could be modulated via point mutation. It was found that increasing RNA capping efficiency led to decreased viral growth kinetics via decreased particle production, despite increased innate immune evasion, whereas decreasing capping efficiency led to wild-type growth kinetics and particle production. This led to the conclusion that the noncapped viral RNAs meaningfully contribute to the biology of alphaviral infections at the molecular level. To determine the importance of the noncapped viral RNAs in vivo, we characterized the impact of altered capping efficiency in a murine model of infection utilizing a neurovirulent strain of SINV. Mice infected with the nsP1 mutant with decreased capping exhibited wild-type rates of mortality, weight loss, and neurological symptoms. Conversely, the mice infected with the increased capping nsP1 mutant showed significantly reduced mortality and morbidity compared to mice infected with the wild-type virus. Interestingly, viral titers in the ankle, serum, and brain were equivalent between the wild-type virus and the two mutant viruses. Importantly, examination of the brain tissue revealed that mice infected with the increased capping mutant had significantly reduced immune cell infiltration and expression of proinflammatory cytokines compared to the decreased capping mutant and wild-type virus. Collectively, these data indicate that the noncapped viral RNAs have important roles during the early and late stages of alphaviral infection and suggest a novel mechanism by which noncapped viral RNA aids in viral pathogenesis. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 137 KiB

Open AccessAbstract

Pioneering siRNA-Mediated Protection of Mammalian Cells against Zika Virus (MR-766) Infection

by Benedita K. L. Feron, Joachim J. Bugert and Simon C. W. Richardson

Proceedings 2020, 50(1), 45; https://doi.org/10.3390/proceedings2020050045 - 14 Jun 2020

Viewed by 1535

Abstract

Here, we present empirical data documenting the siRNA-mediated protection of cells after Zika virus (ZIKV) infection. siRNAs were designed to target well-conserved sequences across the ZIKV genome. Several delivery technologies were utilized. After the electroporation of 100 nM siRNA into human hepatocyte-derived carcinoma [...] Read more.

Here, we present empirical data documenting the siRNA-mediated protection of cells after Zika virus (ZIKV) infection. siRNAs were designed to target well-conserved sequences across the ZIKV genome. Several delivery technologies were utilized. After the electroporation of 100 nM siRNA into human hepatocyte-derived carcinoma (Huh7) cells, the Feron Zv-2 sequence (specific to the ZIKV NS3 gene) yielded a cell viability of 150.3% ± 7.4% (SEM: n = 4) (p = 0.0004) relative to the cells treated only with the virus (33.9% ± 12%, SEM: n = 4). Furthermore, 100 nM siRNA Feron Zv-4 (specific to ZIKV 3’UTR) resulted in 119.1% ± 11.2% cell viability (SEM: n = 4) relative to the control cells treated with ZIKV (p = 0.0021). The cells were electroporated with siRNA prior to ZIKV infection and viability was monitored four days after this. Additionally, two novel siRNA delivery systems were tested. The first utilized recombinant Bacillus anthracis PA83 (octomer-forming mutants), co-incubated with the N-terminal 255 amino acids of B. anthracis lethal factor (LFn) fused in-frame with the RNA binding domain for human protein kinase R (LFn-PKR) at a concentration of 50 µg/mL (each). Here, baby hamster kidney (BHK) cells, treated with 100 nM siRNA Feron Zv-1, yielded 79.0% ± 4.0% viability relative to the control (50.2% ± 1.7%, SEM: n = 3) three days after exposure to ZIKV (p = 0.0096). Finally, HeLa exosomes loaded with siRNA Feron-Zv2 were incubated with Huh7 cells prior to ZIKV infection. For the siRNA-exosome treated cells, a viability of 123% ± 46% (SEM: n = 18), relative to 8% ± 16% (SEM: n = 18) for the same concentration of control HeLa exosomes, was recorded (p = 0.0416). In each instance, 0.3 moI was used and cell viability monitored using the Pierce^TM Firefly Luciferase Glow Assay Kit by Thermo Scientific^TM. Here, we show for the first time that siRNA can significantly reduce ZIKV-induced cell killing. Future work will require quantitating ZIKV mRNA in relation to siRNA treatment, as well as testing the siRNAs and delivery systems within more complex models. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 138 KiB

Open AccessAbstract

Emergence and Selection of a Highly Pathogenic Avian Influenza H7N3 Virus

by Nancy Beerens, Rene Heutink and Ben Peeters

Proceedings 2020, 50(1), 46; https://doi.org/10.3390/proceedings2020050046 - 14 Jun 2020

Cited by 1 | Viewed by 1268

Abstract

Low pathogenic avian influenza (LPAI) viruses of subtypes H5 and H7 have the ability to spontaneously mutate into highly pathogenic (HPAI) variants, causing high mortality in poultry. The switch to high pathogenicity is poorly understood, and evidence from the field is scarce. This [...] Read more.

Low pathogenic avian influenza (LPAI) viruses of subtypes H5 and H7 have the ability to spontaneously mutate into highly pathogenic (HPAI) variants, causing high mortality in poultry. The switch to high pathogenicity is poorly understood, and evidence from the field is scarce. This study provides direct evidence for LPAI to HPAI mutation from a turkey farm during an H7N3 outbreak in the Netherlands. At the farm, only mild clinical symptoms were reported, but the intravenous pathogenicity index measured for the virus isolated from the infected turkeys was consistent with a highly pathogenic virus. Using deep-sequencing, we showed that a minority of HPAI virus (0.06%) was present in the virus preparation. Analysis of different organs of the infected turkeys showed the highest percentage of HPAI virus was present in the lung (4.4%). The HPAI virus contained a 12-nucleotide insertion in the hemagglutinin (HA) cleavage site that was introduced by a single event, as no intermediates with shorter inserts were identified. The HPAI virus was rapidly selected in chickens, after both intravenous and intranasal/intratracheal inoculation with the mixed virus preparation. Full-genome sequencing revealed that both pathotypes contained a deletion in the stalk region of the neuraminidase protein. We identified mutations in HA and polymerase basic protein 1 (PB1) in the HPAI virus, which were already present as minority variants in the LPAI virus. Our findings provide more insight into the molecular changes and mechanisms involved in the emergence of HPAI viruses. This knowledge may be used for the timely identification of LPAI viruses that pose a risk of becoming highly pathogenic in the field. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 154 KiB

Open AccessAbstract

Novel Insights for Biosurveillance of Bat-Borne Viruses

by Adrian C. Paskey, Justin H. J. Ng, Gregory K. Rice, Wan Ni Chia, Casandra W. Philipson, Randy J. H. Foo, Regina Z. Cer, Kyle A. Long, Matthew R. Lueder, Kenneth G. Frey, Theron Hamilton, Ian H. Mendenhall, Gavin J. Smith, Lin-Fa Wang and Kimberly A. Bishop-Lilly

Proceedings 2020, 50(1), 47; https://doi.org/10.3390/proceedings2020050047 - 15 Jun 2020

Viewed by 1486

Abstract

Bats are rich reservoirs of viruses, including several high consequence zoonoses. In this study, high throughput sequencing was used to characterize the virome through a longitudinal study of a captive colony of lesser dawn bats, species Eonycteris spelaea, in Singapore. This study [...] Read more.

Bats are rich reservoirs of viruses, including several high consequence zoonoses. In this study, high throughput sequencing was used to characterize the virome through a longitudinal study of a captive colony of lesser dawn bats, species Eonycteris spelaea, in Singapore. This study utilized viral RNA extracted from swabs of four body sites per bat per timepoint. Swabs of the exterior of the bat (head and body) were used to evaluate virus populations and demonstrate utility as a sample site for future surveillance to extrapolate population-level infection. Through unbiased shotgun and target-enrichment sequencing, we identified both the known and previously unknown viruses of zoonotic relevance and defined the population persistence and temporal patterns of viruses from families that have the capacity to jump the species barrier. We observed the population persistence of three zoonotic-related viral families that are known to be associated with spillover from bats to humans: Paramyxoviridae, Reoviridae, and Coronaviridae. To our knowledge, this is the first study that combines probe-based viral enrichment with high-throughput sequencing or that creates a viral profile from multiple swab sites on individual bats and their cohorts. This work demonstrates temporal patterns of the lesser dawn bat virome, including several novel viruses. Noninvasive surveillance methods that target the body of bats not only detect viruses shed within the colony but can also represent viral populations dispersed throughout the entire colony. New knowledge of persistent viral families should inform future directions for the biosurveillance of viruses that have the potential to cross the species barrier from bats to humans or other amplifying hosts. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 140 KiB

Open AccessAbstract

Characterising Interactions between Influenza A Virus and Respiratory Syncytial Virus during In Vitro Coinfection

by Joanne Haney, Kieran Dee, Colin Loney, Swetha Vijayakrishnan and Pablo R. Murcia

Proceedings 2020, 50(1), 48; https://doi.org/10.3390/proceedings2020050048 - 14 Jun 2020

Viewed by 1509

Abstract

Influenza A virus (IAV) and respiratory syncytial virus (RSV) are important respiratory pathogens that share common epidemiological features and cellular tropism within the respiratory tract. This gives rise to the potential for biological interactions between IAV and RSV during coinfection of hosts. Virus–virus [...] Read more.

Influenza A virus (IAV) and respiratory syncytial virus (RSV) are important respiratory pathogens that share common epidemiological features and cellular tropism within the respiratory tract. This gives rise to the potential for biological interactions between IAV and RSV during coinfection of hosts. Virus–virus interactions are increasingly recognised for their contribution to viral dynamics during infection, however, the molecular processes underpinning these interactions are unknown. Here, we developed an in vitro coinfection system to characterise the infection dynamics of IAV (A/Puerto Rico/8/34, H1N1) and RSV (A2) in single virus infection or coinfection in lung epithelial cells, with the aim to identify biological processes that drive virus–virus interactions during coinfection. We compared viral replication kinetics at different multiplicities of infection and observed that RSV replication was inhibited during coinfection with IAV, whilst IAV replication was facilitated by coinfection. To further characterise IAV/RSV interactions, we determined the relative proportions of single virus infected or coinfected cells during early and late timepoints post-infection and observed differences in expression of viral proteins between single and coinfected states. Additionally, cell viability was measured determine differences in viral-induced cytopathic effect. Compared with RSV infection, cell death is induced at earlier timepoints post IAV infection and coinfection, indicating that different cellular processes are initiated in response to infection. These studies highlight that both competitive and facilitative ecological interactions occur between IAV and RSV during coinfection and shed light on sources of potential interactions at the cellular and molecular level. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 133 KiB

Open AccessAbstract

Conformational Dynamics Related to Membrane Fusion Observed in Single Ebola GP Molecules

by Dibyendu Kumar Das, Uriel Bulow, Natasha D. Durham, Ramesh Govindan and James B. Munro

Proceedings 2020, 50(1), 49; https://doi.org/10.3390/proceedings2020050049 - 14 Jun 2020

Viewed by 1288

Abstract

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following the endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, exposing a binding site for the Niemann-Pick C1 (NPC1) [...] Read more.

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following the endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. The NPC1 binding to the cleaved GP1 is required for entry, but how this interaction translates to the GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a virus-liposome hemifusion assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca²⁺, and NPC1 binding act synergistically to induce conformational changes in GP2 that drive lipid mixing. Acidic pH and Ca²⁺ shift the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. GP1 cleavage and NPC1 binding enable GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the post-fusion 6-helix bundle. Thus, the GP senses the cellular environment to protect against triggering prior to the arrival of EBOV in a permissive cellular compartment. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 143 KiB

Open AccessAbstract

Revolution in Evolution of Human “Anellome”

by Joanna Kaczorowska, Mila Sparreboom, Martin Deijs, Maarten F. Jebbink and Lia van der Hoek

Proceedings 2020, 50(1), 50; https://doi.org/10.3390/proceedings2020050050 - 15 Jun 2020

Viewed by 1286

Abstract

Torque teno viruses (TTVs), the most well-known members of the family Anelloviridae, are not associated with any disease. They are one of the most abundant and divergent entities in the viral world; however, the cause of their variability is not currently known. In [...] Read more.

Torque teno viruses (TTVs), the most well-known members of the family Anelloviridae, are not associated with any disease. They are one of the most abundant and divergent entities in the viral world; however, the cause of their variability is not currently known. In this study, a set of longitudinally collected serum samples from two HIV-1 infected and two non-infected persons was analyzed for the presence of TTVs and other Anelloviridae using a genera-specific quantitative PCR. The samples positive for TTVs were selected for the quantitative heteroduplex tracking assay (QHTA), which showed repeating patterns of TTV genotypes. Sanger sequencing of the partial viral sequences revealed that the same strains of TTVs are most probably disappearing and returning in different stages of life, with scarcely any new introductions. The partial sequences were grouped into phylogenetic genogroups, and samples representing each of these genogroups were selected for full-length genome sequencing using an in-house optimized rolling circle amplification (RCA)–Illumina protocol. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 149 KiB

Open AccessAbstract

SERINC Proteins Potentiate Antiviral Type I IFN Induction and Proinflammatory Signaling Pathways

by Cong Zeng, Abdul A. Waheed, Tianliang Li, Jingyou Yu, Yi-Min Zheng, Jacob Yount, Haitao Wen, Eric O. Freed and Shan-Lu Liu

Proceedings 2020, 50(1), 51; https://doi.org/10.3390/proceedings2020050051 - 15 Jun 2020

Viewed by 1726

Abstract

T cell SERINC proteins were recently identified as human immunodeficiency virus (HIV) restriction factors that diminish viral infectivity by incorporation into virions. Here we provide evidence that SERINC3 and SERINC5 perform additional antiviral activity by enhancing the type I interferon (IFN-I) and NF-κB [...] Read more.

T cell SERINC proteins were recently identified as human immunodeficiency virus (HIV) restriction factors that diminish viral infectivity by incorporation into virions. Here we provide evidence that SERINC3 and SERINC5 perform additional antiviral activity by enhancing the type I interferon (IFN-I) and NF-κB signaling pathways. SERINC5 interacts with the mitochondrial antiviral-signaling (MAVS) and TRAF6 proteins, resulting in MAVS aggregation and TRAF6 polyubiquitination. Knockdown of SERINC5 in the target cell increases single-round HIV-1 infectivity, as well as infection by recombinant vesicular stomatitis virus (rVSV) bearing VSV-G or Ebola virus (EBOV) glycoprotein (GP). Infection by an endemic Asian strain of Zika virus (ZIKV) FSS13025 is also enhanced by SERINC5 knockdown, suggesting that SERINC5 has direct antiviral activity. Further experiments indicated that the antiviral activity of SERINC5 is IFN-I dependent. Altogether, our work uncovered a new function of SERINC proteins that promotes IFN-I and NF-B inflammatory signaling, thus contributing to SERINC-mediated antiviral activity. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 167 KiB

Open AccessAbstract

Novel Methodology for the Detection of Enveloped Viruses

by Patricia Resa-Infante, Itziar Erkizia, Jon Ander Nieto-Garai, Maier Lorizate, Nuria Izquierdo-Useros and Javier Martinez-Picado

Proceedings 2020, 50(1), 52; https://doi.org/10.3390/proceedings2020050052 - 15 Jun 2020

Viewed by 1481

Abstract

Viral infections in humans cause a huge burden in worldwide healthcare that has increased due to the emergence of new pathogenic viruses, such as in the recent Ebola virus (EBOV) outbreaks. Viral particles in body fluids are often at very low levels, making [...] Read more.

Viral infections in humans cause a huge burden in worldwide healthcare that has increased due to the emergence of new pathogenic viruses, such as in the recent Ebola virus (EBOV) outbreaks. Viral particles in body fluids are often at very low levels, making diagnosis difficult. In order to address this problem, we have developed a new detection platform to isolate and detect different enveloped viruses. We have recently identified that sialic acid-binding Ig‑like lectin 1 (Siglec-1/CD169) is one cellular receptor used by EBOV and HIV-1 to enter myeloid cells, key target cells for infection and pathogenesis. For viral uptake, the V-set domain of this myeloid cell receptor recognizes the gangliosides of viral membranes that were dragged during viral budding from the plasma membrane of infected cells. We took advantage of this specific interaction between Siglec‑1 and viral gangliosides to develop a new detection methodology. We have generated a recombinant protein that contains the V-set domain of Siglec-1 fused to the human IgG Fc domain for anchoring in latex beads. These coated beads allow the isolation of viral particles and their measurement by flow cytometry. We have tested its efficacy to detect HIV-1 and EBOV and its specificity by using anti-Siglec‑1 antibodies that prevent the interaction and serve as a negative control. To test the capacity of our method, we used synthetic liposomes to assess the effect of ganglioside concentration in membranes as well as the size of viral particles. This methodology would facilitate the diagnosis of infections by concentrating viral particles in a fast and direct method. At a time when global human mobility facilitates the dissemination of infectious agents, our approach represents a rapid and effective method to maximize the identification of both known and emerging enveloped viruses as part of public health viral surveillance strategies. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 141 KiB

Open AccessAbstract

Rotaviruses as Neonatal Vaccine Expression Vectors against Other Enteric Pathogens

by Asha A. Philip, Kaitlin K. Doucette, Tanmaya A. Rasal and John T. Patton

Proceedings 2020, 50(1), 53; https://doi.org/10.3390/proceedings2020050053 - 15 Jun 2020

Cited by 3 | Viewed by 2073

Abstract

Although the incidence of rotavirus diarrheal disease has been reduced by the introduction of neonatal rotavirus vaccines, other enteric viruses—including norovirus, hepatitis E virus (HEV), and astrovirus—remain significant causes of illness. In this study, we investigated the possibility of generating recombinant rotaviruses that [...] Read more.

Although the incidence of rotavirus diarrheal disease has been reduced by the introduction of neonatal rotavirus vaccines, other enteric viruses—including norovirus, hepatitis E virus (HEV), and astrovirus—remain significant causes of illness. In this study, we investigated the possibility of generating recombinant rotaviruses that express the capsid proteins of other enteric viruses as an approach for creating neonatal multitarget vaccines. As a first step, we examined whether the segmented dsRNA genome of rotavirus could be engineered to express a separate foreign protein through the use of a 2A translational “self-cleavage” element. These attempts were successful, allowing for the recovery of recombinant rotaviruses with modified-segment-7 RNAs that contained a single open reading frame (ORF) encoding a NSP3-2A-fluorescent protein (FP) cassette. By varying the FP introduced into the cassette, genetically-stable rotaviruses were generated which grew efficiently and directed the robust expression of FP as an independent product (e.g., UnaG (green), mRuby (red), mKate (orange), TagBFP (blue), and (YFP) yellow). Subsequently, attempts were made to recover recombinant rotaviruses with modified-segment-7 RNAs that contained a single ORF encoding NSP3-2A fused to the capsid-protein gene of norovirus (VP1, P, or P2), HEV (ORF2), or astrovirus (VP70 or VP90). These attempts resulted in the generation of recombinant viruses that efficiently expressed capsid proteins of other enteric viruses, despite the required addition of up to 2.5 kB of foreign sequence to the 18.5 kB rotavirus genome. Our findings support the idea that rotaviruses can be engineered as plug-and-play expression vectors to create next-generation neonatal vaccines that can induce immunological protection against not only rotavirus, but other enteric pathogens also. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 137 KiB

Open AccessAbstract

Nationwide Screening for Important Bee Viruses in Belgian Honey Bees

by Severine Matthijs and Nick De Regge

Proceedings 2020, 50(1), 54; https://doi.org/10.3390/proceedings2020050054 - 15 Jun 2020

Cited by 1 | Viewed by 1269

Abstract

The ecological and economic importance of bees for pollination and biodiversity is well established. The health of bees is, however, threatened by a multitude of factors, including viruses. In this study, we screened 557 colonies from 155 beekeepers distributed all over Belgium to [...] Read more.

The ecological and economic importance of bees for pollination and biodiversity is well established. The health of bees is, however, threatened by a multitude of factors, including viruses. In this study, we screened 557 colonies from 155 beekeepers distributed all over Belgium to monitor the prevalence and distribution of seven widespread viruses in Belgian honey bees (Apis mellifera). Several of these viruses have been linked with an increased risk for colony loss. Although these viruses can severely impact honey bees and can even cause the death of larvae or adults, colonies with a low viral load usually appear asymptomatic (covert infection). The presence of viruses was determined by real-time RT-PCR. The three most prevalent viruses in Belgian honey bees are Deformed wing virus B (DWV-B or VDV-1), Black queen cell virus (BQCV), and Sacbrood virus (SBV). These viruses were found in more than 90% of the honey bee colonies, but often with a high Ct value, which indicates that they are present at low viral loads (less than 3 log10 genome copies per bee). In certain colonies, however, DWV-B, BQCV, or SBV was detected with a low Ct value, representing a high viral load (in some cases, more than 7 log10 genome copies per bee) and with an increased likelihood of development of clinical symptoms. Deformed wing virus A (DWV-A), Acute bee paralysis virus (ABPV), and Chronic bee paralysis virus (CBPV) were found in less than 40% of the colonies. Kashmir bee virus (KBV) was not found in any of the analyzed Belgian honey bees. Most of the honey bee colonies are infected with multiple viruses, albeit with low virus loads. The impact of viruses can however become critical in the presence of other detrimental factors such as parasites (Nosema sp., Varroa sp.) and pesticides. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 129 KiB

Open AccessAbstract

Mycobacteriophages: Diversity, Dynamics, and Therapy

by Graham F. Hatfull

Proceedings 2020, 50(1), 55; https://doi.org/10.3390/proceedings2020050055 - 15 Jun 2020

Viewed by 1278

Abstract

Bacteriophages—viruses that infect bacteria—are the most numerous biological entities in the biosphere. The phage population is not only vast but dynamic and old, and perhaps not surprisingly, highly diverse genetically. We are exploring and defining this biological diversity by isolating and genomically characterizing [...] Read more.

Bacteriophages—viruses that infect bacteria—are the most numerous biological entities in the biosphere. The phage population is not only vast but dynamic and old, and perhaps not surprisingly, highly diverse genetically. We are exploring and defining this biological diversity by isolating and genomically characterizing individual phages, and through comparative genomic analyses. The development of large integrated research-education programs, such as the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program, have established a large collection of 15,500 individual phages—all isolated on bacterial hosts within the phylum Actinobacteria—of which over 3000 are fully sequenced. These genomes are highly diverse genetically, even if isolated on the same bacterial strain, and are richly populated in genes of unknown function. We propose that many of these genes are involved in the dynamic relationships between bacteria and their viruses, either promoting or countering viral defense systems, or denying competing phages the opportunity to superinfect infected cells. Examples of these include prophage-mediated defense systems in which integrated prophages of temperate viruses lysogenically express genes that prevent attack by unrelated (heterotypic) phages. These systems are prevalent among temperate phages and can act with exquisite specificity in their defense profiles. Phages can also encode their own counter-defense systems to negate the functions of these prophage-mediated defenses. These contributions to microbial dynamics dictate the phage susceptibility profiles of individual strains of pathogenic bacteria, adding complexity to the prospects for the broad therapeutic use of phages against bacterial infections. However, we have shown that the administration of a three-phage cocktail engineered and personalized to a specific highly antibiotic-resistant strain of Mycobacterium abscessus contributes to substantial clinical improvement. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 133 KiB

Open AccessAbstract

Conformational Dynamics Related to Membrane Fusion Observed in Single Ebola GP Molecules

by Dibyendu Kumar Das, Uriel Bulow, Natasha D. Durham, Ramesh Govindan and James B. Munro

Proceedings 2020, 50(1), 56; https://doi.org/10.3390/proceedings2020050056 - 15 Jun 2020

Viewed by 1350

Abstract

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following the endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, exposing a binding site for the Niemann-Pick C1 (NPC1) [...] Read more.

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following the endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. The NPC1 binding to the cleaved GP1 is required for entry, but how this interaction translates to the GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a virus-liposome hemifusion assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca²⁺, and NPC1 binding act synergistically to induce conformational changes in GP2 that drive lipid mixing. Acidic pH and Ca²⁺ shift the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. GP1 cleavage and NPC1 binding enable GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the post-fusion 6-helix bundle. Thus, the GP senses the cellular environment to protect against triggering prior to the arrival of EBOV in a permissive cellular compartment. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 148 KiB

Open AccessAbstract

SAMHD1 Is a Modulator of Nucleos(t)ide Analogues’ Efficacy

by Ifeanyi Ezeonwumelu, Marc Castellví, Eudald Felip, Maria Pujantell, Edurne Garcia-Vidal, Eva Riveira-Muñoz, Roger Badia, Bonaventura Clotet, Mireia Margelí and Ester Ballana

Proceedings 2020, 50(1), 58; https://doi.org/10.3390/proceedings2020050058 - 15 Jun 2020

Viewed by 1476

Abstract

Nucleos(t)ide analogues are commonly used in the treatment of infectious disease and cancer. SAMHD1 is a deoxyribonucleotide (dNTP) triphosphohydrolase which is involved in the regulation of the intracellular dNTP pool, whose function has been linked to viral restriction, cancer development, and autoimmune disorders. [...] Read more.

Nucleos(t)ide analogues are commonly used in the treatment of infectious disease and cancer. SAMHD1 is a deoxyribonucleotide (dNTP) triphosphohydrolase which is involved in the regulation of the intracellular dNTP pool, whose function has been linked to viral restriction, cancer development, and autoimmune disorders. Here, we evaluate SAMHD1 function on the antiviral and antiproliferative efficacy of a wide range of nucleos(t)ide analogues which are currently used to treat infections and cancer. The anti-HIV-1 and cytotoxic activity of compounds was assessed in primary and established cell lines in the presence or absence of SAMHD1. SAMHD1 effectively modified the anti-HIV-1 activity of all the nucleos(t)ide analogues tested, whereas sensitivity to a non-nucleoside inhibitor (nevirapine) or nucleoside phosphonates (cidofovir and tenofovir) was not affected. Interestingly, SAMHD1 could either enhance (gemcitabine, capecitabine, fluorouracil, and floxuridine) or inhibit (Ara-C, fludarabine, cladribine, clofarabine, and nelarabine) the antiviral potency of anticancer analogues, an effect that was not dependent on the specific nucleotide targeted. When cytotoxicity was evaluated, SAMHD1-dependent changes were less evident and were restricted to the increased efficacy of fluorouracil and floxuridine and reduced efficacy of nelarabine and ara-C in the presence of SAMHD1. In summary, our results demonstrate that SAMHD1 modifies the efficacy of a wide variety of nucleoside analogues which are used to treat infections, cancer, and other diseases. In addition, the anti-HIV activity of nucleos(t)ide analogues may represent a more sensitive measure of SAMHD1’s impact on drug efficacy. Thus, modulation of SAMHD1’s function may constitute a promising target for the improvement of multiple therapies. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 126 KiB

Open AccessAbstract

Lethal Encephalitis of Unknown Origin—Elucidation by Metagenomics

by Martin Beer

Proceedings 2020, 50(1), 59; https://doi.org/10.3390/proceedings2020050059 - 16 Jun 2020

Viewed by 1292

Abstract

Novel and (re)emerging viruses cause frequent threats to both human and animal health. Diagnostic metagenomics using unbiased next-generation sequencing (NGS) is the key method for the identification of new pathogens. With today’s available state-of-the-art platforms, NGS can be broadly used to also identify [...] Read more.

Novel and (re)emerging viruses cause frequent threats to both human and animal health. Diagnostic metagenomics using unbiased next-generation sequencing (NGS) is the key method for the identification of new pathogens. With today’s available state-of-the-art platforms, NGS can be broadly used to also identify novel and unknown pathogens in different sample materials (even in point-of-care diagnostics) or to characterize the complete genomes of all types of pathogens. Nevertheless, the extreme numbers of sequence fragments resulting from NGS analyses requires not only novel diagnostic pipelines including powerful software tools for big data analysis but also a new dimension of knowledge and resources. We therefore developed and validated a universal workflow for metagenome diagnostics for the analysis of disease syndromes in both animals and humans. The metagenomics pipeline will be presented, and several examples with the detection and characterization of novel viruses will be shown. The power of diagnostic metagenomics will be presented with different examples focusing on lethal encephalitis cases in both animals and humans where we were able to identify a series of novel or unexpected viral pathogens. Furthermore, the detection of zoonotic pathogens was only possible by a “one-health” approach and the close relationship between veterinary and human medicine. The major aim of the presentation is to give an idea about the capabilities of modern NGS-based metagenomics and to learn more about the newly detected viral pathogens. Since a large proportion of severe encephalitis cases still remain unexplained, a main conclusion is the recommendation that those cases should be analyzed by using a modern and powerful metagenomics workflow. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 146 KiB

Open AccessAbstract

Insights into the Activity of Second-Generation Maturation Inhibitors against HIV Clade C

by Dibya Ghimire, Yuvraj KC and Ritu Gaur

Proceedings 2020, 50(1), 60; https://doi.org/10.3390/proceedings2020050060 - 16 Jun 2020

Viewed by 1502

Abstract

Maturation inhibitors represent a new underdeveloped class of antiretroviral agents that block virus maturation by binding to the target of protease (PR)-Gag precursor (Pr55^Gag). Development of a maturation inhibitor is based on a number of small molecules that are capable of [...] Read more.

Maturation inhibitors represent a new underdeveloped class of antiretroviral agents that block virus maturation by binding to the target of protease (PR)-Gag precursor (Pr55^Gag). Development of a maturation inhibitor is based on a number of small molecules that are capable of blocking the cleavage event between p24-CA and spacer peptide 1. The bulk of the literature on HIV antiretroviral therapy and drug resistance has primarily been derived from HIV-1B, and relatively less is known about the context of HIV-1C that is responsible for more than 95% of HIV infections of India and half of these infections globally. We and others have shown that the presence of maturation inhibitor resistance mutations would make HIV-1B particles either less fit or dependent on these drugs to replicate. By contrast, it is unclear how HIV-1C would naturally acquire these mutations yet remain replication competent in the absence of the selective pressure of maturation inhibitors. Bevirimat, the first-in-class MI, was found to be inactive against HIV-C due to polymorphisms in the SP1 region. We have identified novel second generation of HIV maturation inhibitors with high potency against HIV clade C (IC₅₀ values in the low nM range). The mutations identified during selection experiments revealed the putative binding pocket of these compounds on HIV-1 subtype C Gag. While working on the role of CA-C terminal domain (CA-CTD) domain in HIV-1 Gag assembly and release, we have found that core glycine-rich residues of the β-turn motif are crucial in Gag-membrane binding, multimerization, and assembly. Furthermore, we have identified a novel mutation in CA-CTD that is dependent on both classes of MIs. In conclusion, our studies provide insights into the mechanistic action of MIs on HIV-1 Gag processing and stabilization. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 124 KiB

Open AccessAbstract

Good and Bad Neighbourhoods in Viral Sequence Space: Predicting, Altering, Targeting Virus Populations

by Marco Vignuzzi

Proceedings 2020, 50(1), 61; https://doi.org/10.3390/proceedings2020050061 - 16 Jun 2020

Viewed by 1239

Abstract

All viruses, but especially RNA viruses, generate tremendous diversity in genome composition, including point mutations, duplications, deletions, and insertions. We used in vitro and in vivo models to perform natural and directed experimental evolution. We then combined the resulting data with mathematical modelling [...] Read more.

All viruses, but especially RNA viruses, generate tremendous diversity in genome composition, including point mutations, duplications, deletions, and insertions. We used in vitro and in vivo models to perform natural and directed experimental evolution. We then combined the resulting data with mathematical modelling to determine how virus populations occupy sequence space—a multidimensional hypercube that describes all combinations of nucleotide, codon, or amino acid sequences. In this study, we demonstrate how these experimental and computational approaches can help monitor, predict, alter, and even target virus evolution and population dynamics, creating new ways to study virus–host interactions and to innovate antiviral approaches. Using arboviruses, enteroviruses, and influenza, we recreate and predict host jumps and emergence events in the lab, redirect evolution towards the ‘bad’ neighbourhoods of sequence space that represent attenuation, and poison the viral population by disturbing the balance between good and bad genomes. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 178 KiB

Open AccessAbstract

GB Virus C E2 Inhibits PD-1-Mediated T Cell Signaling Dysfunction during Chronic Viral Infection

by Nirjal Bhattarai, Jennifer L. Welch, Jinhua Xiang, Muthu Saravanan Manoharan, Jeffrey A. Martinson, Alan L. Landay, Sunil K. Ahuja, James H. McLinden and Jack T. Stapleton

Proceedings 2020, 50(1), 62; https://doi.org/10.3390/proceedings2020050062 - 16 Jun 2020

Viewed by 1445

Abstract

Background: Program death receptor 1 (PD-1) is a co-inhibitory receptor that is upregulated and contributes to T cell dysfunction (exhaustion) during chronic viral infections, including HIV and HCV. GB virus C (GBV-C) is a persistent human virus, and co-infection is associated with reduced [...] Read more.

Background: Program death receptor 1 (PD-1) is a co-inhibitory receptor that is upregulated and contributes to T cell dysfunction (exhaustion) during chronic viral infections, including HIV and HCV. GB virus C (GBV-C) is a persistent human virus, and co-infection is associated with reduced immune activation and improved clinical outcomes in HIV- and Ebola-infected individuals. Methods: PD-1 levels were measured by flow cytometry on CD38+ T cells from 45 HIV-infected individuals, 20 of whom were co-infected with GBV-C. Jurkat cell lines that stably express GBV-C E2 protein and vector control were used to purify total cellular RNA before, and 24 h following, activation using anti-CD3/CD28 treatment. Gene expression was analyzed by RNA-seq and qRT-PCR. Results: HIV-infected individuals with GBV-C viremia had reduced PD-1 expression on activated CD4+ and CD8+ T cells compared to HIV-infected GBV-C negative individuals. GBV-C particles and GBV-C E2 protein each inhibited PD-1 expression on T cells in vitro. Consistent with this, GBV-C E2 reduced gene expression of PD-1, and its ligand PD-L1, in both resting and activated T cells. GBV-C E2 regulated transcription of the PD-1 signaling pathway and T cell activation associated genes, without downregulation of the interferon-stimulated and innate immunity-related genes needed to resolve viral infections. Conclusions: Our current understanding of chronic RNA virus infections is that upregulation of PD-1 with T cell exhaustion is critical for viral persistence. However, these data demonstrate that GBV-C infection reduced PD-1 expression on activated T cells during HIV infection, and that the GBV-C E2 protein inhibits PD-1 signaling in T cells. This may preserve T cell function and contribute to the lack of immune deficiency in people with chronic GBV-C infection. Understanding the mechanisms by which GBV-C E2 alters PD-1 signaling may aid in the development of novel immunomodulatory therapeutics to prevent T cell dysfunction (exhaustion) during chronic viral infections. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 133 KiB

Open AccessAbstract

Heterologous Immunity and Hepatitis C Virus: Impact on Natural Infection, Pathogenesis and Vaccine Design

by Babita Agrawal, Shakti Singh, Nancy Gupta, Satish Vedi, Wen Li, Saurabh Garg, Jie Li and Rakesh Kumar

Proceedings 2020, 50(1), 63; https://doi.org/10.3390/proceedings2020050063 - 16 Jun 2020

Viewed by 1506

Abstract

Chronic infection with the hepatitis C virus (HCV) afflicts 1%–3% of the world’s population and can lead to serious and late-stage liver diseases. Developing a vaccine for HCV is challenging because the correlates of protection are uncertain. Host immune responses play an important [...] Read more.

Chronic infection with the hepatitis C virus (HCV) afflicts 1%–3% of the world’s population and can lead to serious and late-stage liver diseases. Developing a vaccine for HCV is challenging because the correlates of protection are uncertain. Host immune responses play an important role in the outcome of infection with HCV. They can lead to viral clearance and a positive outcome, or progression and severity of the chronic disease. Studies of natural immunity to HCV in humans have resulted in many enigmas. Extensive research in the past >25 years into understanding the immune responses against HCV have still resulted in many unanswered questions, implicating the role of unknown factors and events. Human beings are not immunologically naïve because they are continually exposed to various environmental microbes and antigens, creating large populations of memory T and B cells. This pool of memory T and B cells can cross-react against a new pathogen in an individual and thereby influence the outcome of the new infection. In our recent studies, we made the surprising discovery that peptides derived from structural and non-structural proteins of HCV have substantial amino acid sequence homologies with various proteins of adenoviruses, and that immunizing mice with a non-replicating, non-recombinant adenovirus (Ad) vector leads to induction of a robust cross-reactive cellular and humoral response against various HCV antigens. We also extended this observation to show that recombinant adenoviruses containing antigens from unrelated pathogens also possess the ability to induce cross-reactive immune responses against HCV antigens along with the induction of transgene antigen-specific immunity. This cross-reactive/heterologous immunity can a) accommodate the development of dual-pathogen vaccines, b) play an important role in the natural course of HCV infection, and c) provide a plausible answer to many unexplained questions regarding immunity to HCV. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 141 KiB

Open AccessAbstract

Serologic Analysis of Hepatitis E Virus Infection in Patients with Kidney-Related Illnesses

by Martynas Simanavičius, Arūnė Verbickaitė, Paulius Lukas Tamošiūnas, Ernesta Mačionienė and Indrė Kučinskaitė-Kodzė

Proceedings 2020, 50(1), 64; https://doi.org/10.3390/proceedings2020050064 - 16 Jun 2020

Viewed by 1364

Abstract

Hepatitis E is a globally distributed human disease caused by the hepatitis E virus (HEV). HEV is a positive-sense, single-stranded RNA virus that belongs to the family Hepeviridae. Within the genus Orthohepevirus, seven HEV genotypes infect various mammalian hosts. HEV genotypes [...] Read more.

Hepatitis E is a globally distributed human disease caused by the hepatitis E virus (HEV). HEV is a positive-sense, single-stranded RNA virus that belongs to the family Hepeviridae. Within the genus Orthohepevirus, seven HEV genotypes infect various mammalian hosts. HEV genotypes HEV-1 to HEV-4 and HEV-7 can infect humans. HEV-3 is zoonotic with the domestic pig, wild boar, deer and other mammalian species as reservoirs. HEV-3 is an underestimated emerging threat which is spread across Europe. It is transmitted through undercooked pork meat or other products, and with blood components through transfusions. HEV-3 infection in immunocompetent patients is self-limiting and clinically asymptomatic. However, immunocompromised individuals are at a high risk of developing chronic hepatitis E. Chronic infection may lead to life-threatening liver cirrhosis. Patients with kidney transplants or kidney-related illnesses are in this risk group. In this study, a serologic analysis of blood samples obtained from kidney transplant recipients, patients with chronic kidney disease, patients under dialysis and healthy controls was performed. A prevalence of anti-HEV antibodies was assessed by commercial and in-house ELISAs. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 145 KiB

Open AccessAbstract

Dynamics in HIV Gag Lattice Detected by Time-Lapse iPALM

by Ipsita Saha and Saveez Saffarian

Proceedings 2020, 50(1), 65; https://doi.org/10.3390/proceedings2020050065 - 16 Jun 2020

Viewed by 1139

Abstract

Immature HIV virions have a lattice of Gag and Gag-Pol proteins anchored to the lumen of their envelope. This lattice has significant void spaces that were previously characterized by fluorescence correlation spectroscopy, cryoelectron tomography, and iPALM imaging. In the current study, we demonstrate [...] Read more.

Immature HIV virions have a lattice of Gag and Gag-Pol proteins anchored to the lumen of their envelope. This lattice has significant void spaces that were previously characterized by fluorescence correlation spectroscopy, cryoelectron tomography, and iPALM imaging. In the current study, we demonstrate that HIV virus-like particles (VLPs,) assembled by the viral protein Gag tagged at its C terminus with the photoactivable fluorescent protein Dendra, and are of the same size as virus-like particles assembled using only HIV Gag (140 ± 15 nm). We show that the Gag-Dendra lattice observed within these VLPs has similar gaps as those observed in Gag-only VLPs. We further used time-lapse iPALM microscopy to image the Gag–Dendra lattice within the lumen of VLPs at two timepoints. The reconstruction of these time-lapse images shows significant lattice dynamics within the lumen of purified VLPs. The addition of disuccinimidyl suberate (DSS) to the purified VLPs completely abrogated these dynamics. A method to quantify the dynamics of the Gag–Dendra lattice using correlation function analysis is further presented. The HIV Gag lattice, along with the structural lattices of many other viruses, have been mostly considered static. Our study provides an important tool to investigate the dynamics within these lattices and also highlights the effects of fluorescent tags on virion structures. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 159 KiB

Open AccessAbstract

Detection of Known and Novel Viral Pathogens in Belgian Ixodes ricinus Ticks

by Bert Vanmechelen, Michelle Merino, Valentijn Vergote, Lies Laenen, Ana Rita Lopes, Edwin Claerebout and Piet Maes

Proceedings 2020, 50(1), 66; https://doi.org/10.3390/proceedings2020050066 - 16 Jun 2020

Viewed by 1410

Abstract

Recent metagenomics studies have revealed several arthropod species to be major reservoirs for RNA viruses. One of these reservoirs is Ixodes ricinus, the most prevalent tick species in Europe, which is known to be a vector for many viral and bacterial pathogens. [...] Read more.

Recent metagenomics studies have revealed several arthropod species to be major reservoirs for RNA viruses. One of these reservoirs is Ixodes ricinus, the most prevalent tick species in Europe, which is known to be a vector for many viral and bacterial pathogens. For this study, we decided to investigate the virosphere of Belgian Ixodes ricinus ticks. High-throughput sequencing of tick pools collected from six different sampling sites revealed the presence of viruses belonging to many different viral orders and families, including Mononegavirales, Bunyavirales, Partitiviridae, and Reoviridae. Of particular interest was the detection of several putative human pathogens, including members of the families Nairoviridae and Phenuiviridae as well as three new reoviruses, two of which cluster together with members of the genus Coltivirus. One of these two viruses represents a new strain of Eyach virus, a known causative agent of tick-borne encephalitis. All genome segments of this new strain are highly similar to those of previously published Eyach virus genomes, except for the fourth segment, encoding VP4, which is markedly more dissimilar, potentially indicating the occurrence of an antigenic shift. Further PCR-based screening of over 200 tick pools for 11 selected viruses showed that most viruses could be found in all six sampling sites, indicating the wide spread of these viruses throughout the Belgian tick population. Taken together, these results illustrate the role of ticks as important virus reservoirs, highlighting the need for adequate tick control measures. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 136 KiB

Open AccessAbstract

Prophage BPs Alters Mycobacterial Gene Expression and Antibiotic Resistance

by Sally Molloy, Jaycee Cushman, Emma Freeman and Keith Hutchison

Proceedings 2020, 50(1), 67; https://doi.org/10.3390/proceedings2020050067 - 16 Jun 2020

Viewed by 1341

Abstract

Diseases caused by mycobacteria such as Mycobacterium tuberculosis are the leading cause of death worldwide. With the emergence of strains that are resistant to first-line anti-tuberculosis drugs and naturally drug-resistant pathogens such as M. abscessus, there is a need to increase our [...] Read more.

Diseases caused by mycobacteria such as Mycobacterium tuberculosis are the leading cause of death worldwide. With the emergence of strains that are resistant to first-line anti-tuberculosis drugs and naturally drug-resistant pathogens such as M. abscessus, there is a need to increase our understanding of mycobacterial fitness and virulence and identify new targets for drugs. The majority of the pathogenic species of the bacterial genus Mycobacterium, including M. tuberculosis, carry integrated viral genomes (prophages) that are hypothesized to contribute to virulence. Though we know many of the ways in which phage genes directly contribute to pathogenesis, e.g., the CTX prophage encodes the toxin in Vibrio cholera, we know little about the impact of phages that encode no obvious toxin or virulence gene. Using an RNAseq approach, our lab recently showed for the first time that the presence of a prophage alters the expression of 7.4% of genes in the pathogenic mycobacterial species, M. chelonae. The presence of prophage BPs increased the expression of genes in the whiB7 regulon, including whiB7, eis2, and tap, and decreased the expression of a padR-family transcription factor. BP lysogens were more resistant to aminoglycosides (kanamycin and amikacin) and tetracycline than wild-type strains of M. chelonae. In order to determine how the BP prophage drives changes in bacterial gene expression and phenotype, we will test the effects of individual BP genes expressed during lysogeny, such as the immunity repressor, on bacterial gene expression and antibiotic resistance phenotypes. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 125 KiB

Open AccessAbstract

Regulation of RNA Virus Processes by Viral Genome Structure

by K. Andrew White

Proceedings 2020, 50(1), 68; https://doi.org/10.3390/proceedings2020050068 - 16 Jun 2020

Viewed by 1362

Abstract

The genomes of RNA viruses contain a variety of RNA sequences and structures that regulate different steps in virus reproduction. Events that are controlled by RNA elements include (i) the translation of viral proteins, (ii) the replication of viral RNA genomes, and (iii) [...] Read more.

The genomes of RNA viruses contain a variety of RNA sequences and structures that regulate different steps in virus reproduction. Events that are controlled by RNA elements include (i) the translation of viral proteins, (ii) the replication of viral RNA genomes, and (iii) the transcription of viral subgenomic mRNAs. Studies of members of the family Tombusviridae, which possess plus-strand RNA genomes, have revealed novel ways in which the RNA genome structure is utilized to control different viral processes. Recent advances in our understanding of RNA-based viral regulation in select tombusvirids will be presented. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 146 KiB

Open AccessAbstract

Influenza A Virus Disturbs the Host Cell Protein Homeostasis by Inducing the Accumulation of Insoluble Proteins

by Mariana Marques, Marisa Pereira, Maria João Amorim, Ana Raquel Soares and Daniela Ribeiro

Proceedings 2020, 50(1), 69; https://doi.org/10.3390/proceedings2020050069 - 16 Jun 2020

Viewed by 1311

Abstract

Influenza A virus (IAV) is the causative agent for most of the annual respiratory epidemics in humans and the major influenza pandemics in the last century, and is associated with high morbidity and mortality, especially in the elderly. In order to efficiently replicate, [...] Read more.

Influenza A virus (IAV) is the causative agent for most of the annual respiratory epidemics in humans and the major influenza pandemics in the last century, and is associated with high morbidity and mortality, especially in the elderly. In order to efficiently replicate, this virus hijacks the host cellular machinery and requires precise interactions with host components. However, cells have evolved specific defense mechanisms to counteract the effects induced by the virus. In fact, upon IAV infection, several processes within the cytosol and the endoplasmic reticulum, related to protein synthesis and processing, have shown to contribute either as part of an effective replication cycle or as part of an effective cellular antiviral response. Recent reports show contradictory findings regarding the control of the cellular proteostasis mechanisms by both the virus and the host cell. With this study, we aimed to further unravel the interplay between IAV and the host cell proteostasis-related mechanisms at early time-points post-infection. Our results suggest that the virus disturbs host cell protein homeostasis by inducing the accumulation of insoluble proteins in a process that seems to be related to viral RNA processing. We further analyzed the interplay between IAV infection and the endoplasmic reticulum unfolded protein response. Our results may lead to a better understanding of the interplay between IAV and the host cell and, furthermore, contribute to the development of novel antiviral strategies. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 152 KiB

Open AccessAbstract

Cell Surface-Expressed GPI-Anchored Peptides from the CHR Domain of gp41 Are Potent Inhibitors of HIV-1 Fusion

by Aleksandra Maslennikova, Dmitriy Komkov, Anastasia Zotova and Dmitriy Mazurov

Proceedings 2020, 50(1), 70; https://doi.org/10.3390/proceedings2020050070 - 16 Jun 2020

Viewed by 1397

Abstract

Current antiretroviral therapy efficiently suppresses viral replication but cannot eliminate latent HIV reservoirs. Moreover, the associated high costs, side effects, and drug resistance have stimulated a need for the development of alternative methods of HIV-1/AIDS treatment, such as peptide inhibitors or gene editing. [...] Read more.

Current antiretroviral therapy efficiently suppresses viral replication but cannot eliminate latent HIV reservoirs. Moreover, the associated high costs, side effects, and drug resistance have stimulated a need for the development of alternative methods of HIV-1/AIDS treatment, such as peptide inhibitors or gene editing. Recently, we have developed Surface Oligopeptide knock-in for Rapid Target Selection (SORTS), a method for the rapid selection of CRISPR/Cas9 gene-edited cells via knock-in of the Flag and HA epitope tags embedded into the shortest GPI-protein, CD52. By targeting the capsid region of the HIV-1 genome, we demonstrate that SORTS can be applied in provirus eradication. However, the cells with inactivated provirus will be susceptible to HIV re-infection. We hypothesized that knocking in one of the peptides from the CHR-domain of gp41, which are known potent inhibitors of HIV-1 fusion, instead of the epitope tag, will provide “post-curable” HIV-1 resistance. While these peptides were extensively studied as soluble substances, their inhibitory effects on HIV after expression on cell surfaces via GPI-anchor are largely unknown. In this study, we established HEK293T/CD4/R5 and Raji/CD4/R5 HIV-1 permissive cell lines that stably expressed one of the gp41 peptides C34, MT-C34, MT-C34-R, and MT34-15D, or alfa-helix mimetics HP23L, p52, and MT-WQ-IDL. For cell surface delivery, the indicated peptides were embedded into the CD52 molecule, and upstream GFP was used to select transformed cells. Using a single-cycle replication assay with the inLuc reporter vector and different Envs, we demonstrated that C34-based GPI-anchored peptides inhibited both cell-free and cell-to-cell HIV-1 infection by at least two orders of magnitude. With the exception of HP23L, the alfa-helix mimetics were less potent inhibitors. Thus, peptides from gp41 associated with lipid rafts and exerted a strong inhibitory activity which can far exceed that determined for soluble peptides, but this should be tested further. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 159 KiB

Open AccessAbstract

Characteristic of a Distant Relative of Teseptimavirus Genus Phages That Acquired the Ability to Lysogenize Its Host

by Agnieszka Fijałek, Radosław Giziński, Katarzyna Giermasińska-Buczek, Urszula Gągała, Jan Gawor, Andrew M. Kropinski and Małgorzata Łobocka

Proceedings 2020, 50(1), 71; https://doi.org/10.3390/proceedings2020050071 - 16 Jun 2020

Viewed by 1580

Abstract

Pseudomonas syringae is a plant pathogen, which groups over 40 pathovars. Climate change and international trade facilitate the worldwide spread of pathogenic P. syringae strains. In recent decades, infections with P. syringae have been causing large losses in vegetable growing and horticulture. With [...] Read more.

Pseudomonas syringae is a plant pathogen, which groups over 40 pathovars. Climate change and international trade facilitate the worldwide spread of pathogenic P. syringae strains. In recent decades, infections with P. syringae have been causing large losses in vegetable growing and horticulture. With the aim to look for biocontrol agents that could minimize these losses, we isolated bacteriophages infective for certain P. syringae strains. One of these phages, designated by us as vB_PsyP_3MF5 (3MF5), appeared to have atypical properties. It formed clear plaques on the layers of sensitive cells at elevated temperatures but was unable to form plaques at room temperature. It quickly adsorbed to its host cells and had a short latent period and a large burst size at permissive temperature. However, several survivors of the phage infection could be isolated in a standard killing assay. They appeared to form a lysis zone when placed on a layer of cells that were not treated with this phage, indicating that they are 3MF5 lysogens. In support of that, their DNA could serve as a template for PCR amplification with 3MF5 specific primer pairs. The analysis of the 3MF5 genomic sequence (GenBank. Acc. No. MT597419) revealed features typical of Teseptimavirus genus phages which are obligatorily lytic and are unable to lysogenize cells. Additionally, comparative analysis of the predicted 3MF5 proteins excluded the presence of any obvious homolog of a typical phage repressor that inhibits transcription of early phage genes in lysogens. Conceivably, the repression is achieved either by the interaction of a temperature-sensitive host/phage protein with a region controlling the expression of phage early genes or by temperature-induced structural changes in phage RNA, which could act by the occlusion of ribosomal binding sites of early phage genes. Surprisingly, the results of our preliminary studies indicate that despite its conditionally temperate nature, 3MF5 exhibits biocontrol properties. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 159 KiB

Open AccessAbstract

by Merve Kalkan-Yazıcı and Mehmet Ziya Doymaz

Proceedings 2020, 50(1), 72; https://doi.org/10.3390/proceedings2020050072 - 17 Jun 2020

Viewed by 1554

Abstract

Hazara virus (HAZV), a tick-borne agent of the nairoviruses, is closely related to Crimean–Congo hemorrhagic fever virus (CCHFV). Hazara virus has not been reported as a pathogen for humans and can be studied under BSL-2 conditions, whereas CCHFV causes severe hemorrhagic diseases, with [...] Read more.

Hazara virus (HAZV), a tick-borne agent of the nairoviruses, is closely related to Crimean–Congo hemorrhagic fever virus (CCHFV). Hazara virus has not been reported as a pathogen for humans and can be studied under BSL-2 conditions, whereas CCHFV causes severe hemorrhagic diseases, with up to 30% mortality rate in humans, and requires BSL-4 facilities to be handled. Serologic and phylogenetic similarities between the two viruses would therefore be an interesting area of research. In this study, we evaluated the immunological similarities between these two viruses using nucleocapsid protein as a model. Here, we evaluated cross-reactivity between CCHFV and HAZV rNP, which forms virus-like particles when expressed in Pichia pastoris. In Western blot assays using CCHFV-infected human and immunized mice and rabbit sera, cross-reactions were detected between the nucleoproteins of both viruses. Virus-like particles were visualized by transmission electron microscopy (TEM) and monitored by dynamic light scattering (DLS). These results suggest that nucleocapsid proteins of HAZV and CCHFV share similarities regarding the antiviral humoral response in both species. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 157 KiB

Open AccessAbstract

Characterization of JC Polyomavirus Entry by Serotonin Receptors

by Melissa Maginnis, Colleen L. Mayberry, Kashif Mehmood, Matthew Parent and Samuel Hess

Proceedings 2020, 50(1), 73; https://doi.org/10.3390/proceedings2020050073 - 17 Jun 2020

Viewed by 1258

Abstract

JC polyomavirus (JCPyV) causes a lifelong persistent infection in the kidney in the majority of the population. In severely immunocompromised individuals, JCPyV can become reactivated, spread in the central nervous system, and infect glial cells, astrocytes, and oligodendrocytes which are necessary for myelin [...] Read more.

JC polyomavirus (JCPyV) causes a lifelong persistent infection in the kidney in the majority of the population. In severely immunocompromised individuals, JCPyV can become reactivated, spread in the central nervous system, and infect glial cells, astrocytes, and oligodendrocytes which are necessary for myelin production. The viral infection and cytolytic destruction of glial cells leads to the development of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), for which there are currently no approved treatment options. In order to develop effective antiviral therapies, it is essential to define the virus–host cell interactions that drive infection and the virus–receptor interactions that are major regulators of tissue tropism and viral disease outcomes. Following attachment to sialic acid receptors, JCPyV requires the serotonin 5-hydroxytryptamine (5-HT₂) receptors to mediate internalization. However, the mechanism by which JCPyV utilizes 5-HT₂receptors to invade host cells is poorly understood. Using super-resolution fluorescence photoactivation localization microscopy (FPALM), we have determined that JCPyV localizes with 5-HT₂receptors at timepoints consistent with viral entry. Furthermore, we have determined that the 5-HT₂ receptor-associated scaffolding proteins beta-arrestin, adaptor protein complex 2 (AP2) and dynamin are required for viral internalization through a clathrin-mediated endocytosis pathway. Additionally, we have identified a beta-arrestin-binding motif in the intracellular loop of the 5-HT_2A receptor that is critical for JCPyV entry and infection. These findings highlight the importance of viral receptors in regulating viral infection and illuminate potential targets for antiviral treatment. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 140 KiB

Open AccessAbstract

Interferon Antagonism of Epstein–Barr Virus Tegument Proteins

by Wai-Yan Lui, Sonia Jangra, Kit-San Yuen, Michael George Botelho and Dong-Yan Jin

Proceedings 2020, 50(1), 74; https://doi.org/10.3390/proceedings2020050074 - 17 Jun 2020

Cited by 1 | Viewed by 1628

Abstract

The Epstein–Barr virus (EBV) successfully infects 95% of all adults but causes Burkitt’s lymphoma, Hodgkin’s lymphoma, gastric carcinoma, nasopharyngeal carcinoma or other malignancies in only a small subset of infected individuals. The virus must have developed effective viral countermeasures to evade host innate [...] Read more.

The Epstein–Barr virus (EBV) successfully infects 95% of all adults but causes Burkitt’s lymphoma, Hodgkin’s lymphoma, gastric carcinoma, nasopharyngeal carcinoma or other malignancies in only a small subset of infected individuals. The virus must have developed effective viral countermeasures to evade host innate immunity. In this study, we performed functional screens to identify EBV-encoded interferon (IFN) antagonists. Several tegument proteins were found to be potent suppressors of IFN production and/or signaling. The large tegument protein and deubiquitinase BPLF1 antagonized type I IFN production induced by DNA sensors cGAS and STING or RNA sensors RIG-I and MAVS. BPLF1’s ability to suppress innate immune signaling required its deubiquitinase activity. BPLF1 functioned as a catalytically active deubiquitinase for both K63- and K48-linked ubiquitin chains on STING and TBK1, with no ubiquitin linkage specificity. Induced expression of BPLF1 in EBV-infected cells through CRISPRa led to effective suppression of innate DNA and RNA sensing. Another EBV tegument protein, BGLF2, was found to suppress JAK-STAT signaling. This suppression was ascribed to more pronounced K48-linked polyubiquitination and proteasomal degradation of BGLF2-associated STAT2. In addition, BGLF2 also recruited tyrosine phosphatase SHP1 to inhibit tyrosine phosphorylation of JAK1 and STAT1. A BGLF2-deficient EBV activated type I IFN signaling more robustly. Taken together, we characterized the IFN antagonism of EBV tegument proteins BPLF1 and BGLF2, which modulate ubiquitination of key transducer proteins to counteract type I IFN production and signaling in host cells. Supported by HMRF 17160822, HMRF 18170942, and RGC C7027-16G. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 133 KiB

Open AccessAbstract

High-Resolution 3D Imaging of Virus Infections in Solvent-Cleared Organs: Novel Insights into Virus Replication and Tropism In Vivo

by Luca Zaeck, Madlin Potratz, Antonia Klein, Conrad M. Freuling, Thomas Müller and Stefan Finke

Proceedings 2020, 50(1), 75; https://doi.org/10.3390/proceedings2020050075 - 17 Jun 2020

Viewed by 1414

Abstract

The visualization of infection processes in relevant tissues and organs using microscopy methods reveals a unique link between the distribution, tropism, and abundance of pathogens and the physiological structure of the respective organ. To dissect virus replication and the host reaction in vivo [...] Read more.

The visualization of infection processes in relevant tissues and organs using microscopy methods reveals a unique link between the distribution, tropism, and abundance of pathogens and the physiological structure of the respective organ. To dissect virus replication and the host reaction in vivo at both a global and a single-cell level, conventional 2D imaging approaches can only provide limited insight. However, pathological studies of infected organ material are still mostly restricted to the immunostaining of thin sections from paraffin-embedded or frozen samples. While the 3D analysis of large tissue volumes is possible via laborious serial sectioning, a variety of problems and artifacts remain. Modern immunostaining-compatible tissue clearing techniques allow for the seamless 3D visualization of infection sites in optically cleared thick tissues sections or even entire organs. Benefiting from pure optical slicing, this approach enables the acquisition of multicolor high-volume 3D image stacks for coherent qualitative and quantitative analyses of the infection and its surrounding cellular environment. Here, we demonstrate the utility and power of this methodology by visualizing virus infections in different target tissues. For instance, we reconstructed the cellular context of rabies virus infection sites in mouse brain tissue, allowing a thorough investigation and quantitative analysis of rabies virus cell tropism. The systematic comparison of different rabies viruses with varying pathogenicity revealed a remarkable difference for highly virulent street rabies viruses and attenuated lab strains. While the virus protein expression was readily detectable at a comparable level in both neurons and non-neuronal glial cells from brains of mice infected with street rabies viruses, it was virtually absent in glial cells of lab strain-infected mice. These data provide novel and detailed insights into the pathogenesis of virus infections and substantially contribute to an improved understanding of virus–host interactions in vivo. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 136 KiB

Open AccessAbstract

Full Genomic Sequencing of Vesicular Stomatitis Virus Isolates from the 2004–2006 US Outbreaks Reveals Associations of Viral Genetics to Environmental Variables

by Rachel Palinski, Steven Pauszek, N. Dylan Burruss, Heather Savoy, Angela Pelzel-McCluskey, Jonathan Arzt, Debra P.C. Peters and Luis Rodriguez

Proceedings 2020, 50(1), 76; https://doi.org/10.3390/proceedings2020050076 - 17 Jun 2020

Viewed by 1822

Abstract

Vesicular stomatitis (VS) outbreaks in the western USA occur cyclically, approximately every 8–10 years. Phylogenetic evidence based on a 450 nt region of the P coding sequences suggests that the initial introduction was a single viral lineage closely related to those circulating in [...] Read more.

Vesicular stomatitis (VS) outbreaks in the western USA occur cyclically, approximately every 8–10 years. Phylogenetic evidence based on a 450 nt region of the P coding sequences suggests that the initial introduction was a single viral lineage closely related to those circulating in endemic areas of Mexico. In 2004, a VS outbreak was initiated in southern NM and TX, and spread as far north as northern CO. Subsequently, in 2005, VS cases appeared in nine states (AZ, CO, ID, MT, NE, NM, TX, UT and WY), and in 2006, VS reappeared only in WY. Phylogenetic analysis suggested that a single VS virus of New Jersey (VSNJV) lineage caused the 2004 outbreak, and re-emerged in 2005 and 2006. The mechanisms of VS emergence and re-emergence remain unclear. Here, we used near full-length genomic sequences of 60 viral strains isolated from 2004–2006 in the US and Mexico to determine the phylogeographic relationships and environmental variables associated with the outbreak dynamics. The results confirmed that a single VSNJV lineage caused the 2004–2006 US outbreaks, and that its closest ancestor was a virus circulating in Colima, Mexico in 2004. We also present evidence that the virus lineage overwintered in 2005 and 2006. Furthermore, rather than a simple geographic relationship, specific viral sublineages or patristic groups were associated to environmental variables, particularly precipitation and temperature. The results confirm the role of environmental factors in the evolution and spread of VSNJV in the USA. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 138 KiB

Open AccessAbstract

PSGL-1 Restricts HIV-1 Infectivity by Blocking Virus Particle Attachment to Target Cells

by Yajing Fu, Sijia He, Abdul Waheed, Deemah Dabbagh, Zheng Zhou, Benjamin Trinité, Zhao Wang, Jieshi Yu, Dan Wang, Feng Li, David N Levy, Hong Shang, Eric O Freed and Yuntao Wu

Proceedings 2020, 50(1), 77; https://doi.org/10.3390/proceedings2020050077 - 17 Jun 2020

Viewed by 1377

Abstract

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is primarily expressed on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface [...] Read more.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is primarily expressed on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits human immunodeficiency virus type 1 (HIV-1) replication, the mechanism of PSGL-1-mediated anti-HIV activity remains to be elucidated. Here, we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein, vesicular stomatitis virus G glycoprotein, or lacking a viral glycoprotein, is impaired by PSGL-1. Mapping studies show that the extracellular, N-terminal domain of PSGL-1 is necessary for its anti-HIV-1 activity, and the PSGL-1 cytoplasmic tail contributes to its inhibition. In addition, we demonstrate that the PSGL-1-related monomeric E-selectin-binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or the expression of either Vpu or Nef, downregulates PSGL-1 from the cell surface; the expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1-mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a novel mechanism of action. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 169 KiB

Open AccessAbstract

Control of Alphavirus Replication in Neurons

by Jane Yeh, Elizabeth Troisi, Rachy Abraham and Diane Griffin

Proceedings 2020, 50(1), 78; https://doi.org/10.3390/proceedings2020050078 - 17 Jun 2020

Viewed by 1318

Abstract

Sindbis virus causes age-dependent encephalomyelitis in mice. Young mice and immature neurons replicate the virus to high titers and die from infection while older mice and mature neurons restrict replication and survive infection. Studies to identify factors that affect maturation-dependent virus replication in [...] Read more.

Sindbis virus causes age-dependent encephalomyelitis in mice. Young mice and immature neurons replicate the virus to high titers and die from infection while older mice and mature neurons restrict replication and survive infection. Studies to identify factors that affect maturation-dependent virus replication in neurons have used AP-7 rat olfactory neuronal cells that can be differentiated in vitro, and have identified roles for host transcription factors interferon regulatory protein (IRF) 7 and NF-B and viral proteins E2 and nsP3. IRF7 is required for neuronal survival and deficiency leads to paralysis and death of 4–6 weeks old C57BL/6 mice. Activation of NF-κB works in conjunction with PKR to facilitate replication in mature neurons by promoting shut-off of host protein synthesis and increasing translation of the viral structural proteins from subgenomic RNA. The macro domain of nsP3 binds and hydrolyses ADP-ribose from ADP-ribosylated proteins and is important for initiation of neuronal infection and for synthesis of viral structural proteins. Thus, neurons regulate translation of the structural proteins from subgenomic RNA required for the production of the infectious virus. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 124 KiB

Open AccessAbstract

Persistent Transcriptional Alterations after Hepatitis C Virus Elimination in Cell Culture

by Victoria Castro, Gema Calvo, Juan Carlos Oliveros, Sofia Pérez-Del-Pulgar, Xavier Forns and Pablo Gastaminza

Proceedings 2020, 50(1), 79; https://doi.org/10.3390/proceedings2020050079 - 17 Jun 2020

Viewed by 1403

Abstract

Chronic hepatitis C virus (HCV) infection causes liver inflammation and fibrosis, which can lead to development of cirrhosis and hepatocellular carcinoma (HCC). The recent approval of direct-acting antiviral (DAA) drug combinations has revolutionized antiviral therapy against HCV. These drugs enable virus eradication in [...] Read more.

Chronic hepatitis C virus (HCV) infection causes liver inflammation and fibrosis, which can lead to development of cirrhosis and hepatocellular carcinoma (HCC). The recent approval of direct-acting antiviral (DAA) drug combinations has revolutionized antiviral therapy against HCV. These drugs enable virus eradication in virtually all treated patients regardless of their genotype and liver disease status. Based on clinical parameters, it has been proposed that elimination of infected cells by reactivated immune responses may be dispensable for virus eradication in contrast to previously used interferon-based therapies. It is thus formally possible that the patients, who are declared cured, do indeed carry formerly infected cells that display irreversible alterations due to prolonged chronic HCV infection. Although transcriptional profiles of biopsies from cured patients have been previously studied, it is difficult to determine the precise mechanisms by which permanent alterations are established in the context of a heterogeneous tissue, often in patients with an underlying liver disease. Thus, we used cell culture models of persistent HCV infection to determine if HCV infection causes permanent transcriptional alterations in host cells after virus eradication. In these models, HCV infection causes profound alterations of host cell transcriptome that aim at regaining cellular homeostasis in the context of intracellular membrane rearrangements, interference with homeostatic processes, and persistent stress conditions, and permit cell survival even though the virus has colonized the host cell. In this context, we asked the question of whether the original homeostasis and original transcriptomic profile are regained in formerly infected cells after DAA treatment-mediated virus eradication. Our results indicate that a minor subset of transcriptional alterations persists even after virus eradication, suggesting that DAA-mediated eradication does not ensure the normalization of formerly infected cell homeostasis. Combined analysis of the transcriptional profiles in proliferating and growth-arrested cells suggests that several mechanisms underlie the establishment of permanent alterations. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 142 KiB

Open AccessAbstract

Comparing Different Larval Food Sources and Temperature Regimes for the Rearing of Culicoides obsoletus/scoticus Complex Midges, the Predominant Bluetongue, and Schmallenberg Virus Vectors in Northern Europe

by Charlotte Sohier, Severine Matthijs and Nick De Regge

Proceedings 2020, 50(1), 80; https://doi.org/10.3390/proceedings2020050080 - 17 Jun 2020

Viewed by 1282

Abstract

During the last decade, Europe was confronted with the emergence of two Culicoides-borne viruses, bluetongue virus and Schmallenberg virus. Both diseases caused economic losses in cattle, sheep, and goats. Despite their importance, only limited knowledge is available on the developmental stages of [...] Read more.

During the last decade, Europe was confronted with the emergence of two Culicoides-borne viruses, bluetongue virus and Schmallenberg virus. Both diseases caused economic losses in cattle, sheep, and goats. Despite their importance, only limited knowledge is available on the developmental stages of Culicoides obsoletus complex midges, and a lab-reared colony has not yet been established. Therefore, this experimental study aims to compare the emergence patterns from field-collected C. obsoletus complex from egg to adult that were exposed to different combinations of temperature and larval substrates (T1: 1% liquid broth (LB) agar at 24 °C; T2: 1% LB agar with dung patches at 24 °C; T3: 1% LB agar at 28 °C). The rearing dishes were kept in an environmental chamber under an 8 h/16 h dark–light regime and 80%–85% humidity. Algae and nematodes were provided as a food source. The average period required to develop from egg to adult was 24 days and varied strongly in each of the conditions (T1: 15–47 days; T2: 14–35 days; T3: 20–37 days). A bias of sex ratio was observed towards the production of males in all three treatments with only 23.9% (T1), 23.5% (T2), and 0% (T3) females. Larval mortality was highest in T3 (25.9%), followed by T2 (8.6%), and T1 (1.8%). All pupae produced adults within 1 to 10 days, and the emergence rates for pupae varied strongly between treatments: 49.5% (T1), 71.6% (T2), and 38.5% (T3). This study shows that the C. obsoletus complex can be reared under laboratory conditions from blood-fed, wild-caught females to emerged progeny. More larval substrates and food sources should be tested with the aim of obtaining a 1:1 sex ratio to bring us one step closer to a viable lab-reared colony. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 145 KiB

Open AccessAbstract

SAMHD1 Enhances Chikungunya and Zika Virus Replication in Human Skin Fibroblasts

by Sineewanlaya Wichit

Proceedings 2020, 50(1), 81; https://doi.org/10.3390/proceedings2020050081 - 17 Jun 2020

Viewed by 1224

Abstract

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the [...] Read more.

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis. We show that the expressions of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expressions of defense response proteins DDX58, STAT1, OAS3, EIF2AK2, and SAMHD1 were significantly upregulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx resulted in a strong increase or inhibition, respectively, in both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease in viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 145 KiB

Open AccessAbstract

Cytopathic BVDV-1 Induces Type I Interferon Expression through IRF-1 and IRF-7 Transcriptional Factors in MDBK Cells

by Nicolás Maldonado, Melina Villalba, Fernanda Fredericksen, Gardenia Payne and Victor Hugo Olavarria

Proceedings 2020, 50(1), 82; https://doi.org/10.3390/proceedings2020050082 - 17 Jun 2020

Viewed by 1502

Abstract

Bovine viral diarrhea virus (BVDV-1) is responsible for the Bovine Viral Diarrhea/Mucosal Disease complex, endemic pathology of cattle, as well as for heavy losses for the livestock and dairy industry in the world. Several investigations have shown that BVDV-1 is capable of altering [...] Read more.

Bovine viral diarrhea virus (BVDV-1) is responsible for the Bovine Viral Diarrhea/Mucosal Disease complex, endemic pathology of cattle, as well as for heavy losses for the livestock and dairy industry in the world. Several investigations have shown that BVDV-1 is capable of altering the host animal's immune system, but there is little information available on the molecular and cellular mechanisms involved. The production of interferon (IFN-a/b) is considered a potent and rapid response of the innate immune system against the presence of a virus. In the case of BVDV-1, the antecedents that show whether IFN-b expression is triggered during an infection in bovine models are contradictory, and the transcription factors that regulate the expression of this key cytokine to trigger antiviral status have not been established. To investigate the effects of BVDV-1 on the activation of the immune response, Madin–Dardy bovine kidney (MDBK) cells were infected with the cytopathic biotype cpBVDV-1, and the expression of IFN-b, interferon regulatory factors (IRF) and immunity markers was analyzed. Additionally, a transient silencing of the IRF-7 factor was performed. The results obtained show that BVDV-1 is capable of inducing the production of IFN-b, IRF-1, and IRF-7 in a manner similar to polycytidylic acid, evaluated transcript, and protein level. The use of pharmacological inhibitors against IRF-1 and IRF-7 decreases the production of IFN-b, a phenomenon observed by mainly interfering with the activation pathway of IRF-7. These results propose that in an infection with cpBVDV-1, the activation of the IRF-7 factor is required and indispensable for the regulation of the transcription of the IFN-b gene in an in vitro model of infection. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 146 KiB

Open AccessAbstract

Protection of Chickens with Maternal Avian Influenza Virus (AIV) Immunity after Vaccination with a Recombinant AIV-Newcastle Disease Vector

by Magdalena Murr, Olayinka Asala, Axel Karger, Christian Grund, Thomas C. Mettenleiter and Angela Römer-Oberdörfer

Proceedings 2020, 50(1), 83; https://doi.org/10.3390/proceedings2020050083 - 17 Jun 2020

Viewed by 1605

Abstract

Highly pathogenic avian influenza virus (HPAIV) belongs to the Orthomyxoviridae family and causes a systemic and highly lethal disease in poultry. Vaccination with recombinant Newcastle disease vector viruses (NDV) expressing the hemagglutinin (HA) of HPAIV H5N1 induces high antibody titers in chickens free [...] Read more.

Highly pathogenic avian influenza virus (HPAIV) belongs to the Orthomyxoviridae family and causes a systemic and highly lethal disease in poultry. Vaccination with recombinant Newcastle disease vector viruses (NDV) expressing the hemagglutinin (HA) of HPAIV H5N1 induces high antibody titers in chickens free of specific pathogens, conveying protection against a lethal infection with HPAIV H5N1. Protection of chickens possessing maternally derived NDV immunity was achieved after the replacement of the surface proteins of NDV, the fusion protein (F), and the hemagglutinin-neuraminidase protein (HN) against those of avian paramyxovirus serotype 8. However, maternal AIV antibodies (αAIV-MDA+) still interfere with vaccine virus replication, resulting in inefficient protection. For our study, recombinant rNDVsolH5_H5 was generated. The insertion of a transgene encoding a truncated soluble HA between the NDV phosphoprotein and matrix protein genes—in addition to the gene encoding a membrane-bound HA inserted between the NDV, F and HN of the lentogenic NDV Clone 30 —was expected to increase the total amount of HA expressed by the recombinant virus. Western blot and mass spectrometry analyses confirmed the increase in HA expression compared to the parental rNDVH5 expressing only the full-length HA. The protective efficacy of the newly generated recombinant NDV was tested in an animal experiment. αAIV-MDA+ chickens were vaccinated either 7, 14, or 21 days after hatching. A homologous challenge infection was carried out three weeks later. Although the youngest chickens showed the highest titer of αAIV-MDA, there were no AIV antibodies detectable 21 days after vaccination. However, 40% of vaccinated chickens were protected, while 85% and 100% protection was observed in the middle-aged and oldest chickens, which had low and no detectable levels of αAIV-MDA, and moderate and high AIV antibody levels after vaccination, respectively. Challenge infection of non-vaccinated chickens resulted in high mortality. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 183 KiB

Open AccessAbstract

Detection and Quantification of Influenza Virus Defective Viral Genomes from NGS Datasets Obtained after RT or RT-PCR Product Sequencing

by Jeremy Boussier, Sandie Munier, Bernadette Crescenzo-Chaigne, Sylvie Behillil, Vincent Enouf, Sylvie van der Werf and Nadia Naffakh

Proceedings 2020, 50(1), 84; https://doi.org/10.3390/proceedings2020050084 - 17 Jun 2020

Viewed by 1551

Abstract

Like most RNA viruses, influenza viruses (IAV) generate defective viral genomes (DVGs) during viral replication. Although there is accumulating evidence of a biological impact of DVGs, the molecular mechanisms leading to their production remain to be unveiled. Various next-generation sequencing (NGS) technologies and [...] Read more.

Like most RNA viruses, influenza viruses (IAV) generate defective viral genomes (DVGs) during viral replication. Although there is accumulating evidence of a biological impact of DVGs, the molecular mechanisms leading to their production remain to be unveiled. Various next-generation sequencing (NGS) technologies and detection methods can be used to characterize DVGs. Here, we developed a bioinformatics pipeline called DG-seq to quickly identify and quantify DVGs in influenza viral stocks and compared two processing methods for NGS, with or without PCR amplification. To evaluate the performance of the DG-seq pipeline, we used either synthetic in-vitro-transcribed DVGs mixed with the full set of synthetic full-length genomic RNAs, or biological RNA samples extracted in duplicate from three IAV stocks: mutant viruses with a K635A or a R638A mutation in the PA subunit of the polymerase that impairs viral transcription, and their wild-type (WT) counterpart. Viral genomic RNAs were reverse-transcribed and either directly subjected to Illumina sequencing (RT-seq) or PCR-amplified prior to sequencing (RT-PCR-seq). Both methods displayed a good reproducibility between batches, with a lower detection rate but a more accurate quantification of DVGs in RT-seq samples. The PA mutants produced more DVGs than the WT virus, derived mostly from the polymerase gene segments, but also from the NA and HA segments, suggesting that an imbalance between transcription and replication can promote DVG production. Breakpoints occurred near the segment extremities, with no hotspot identified. Interestingly, we observed short direct A/T-rich repeats adjacent to the breakpoint ends at a significantly higher frequency than in the random case. This work provides the first comparison of DVG detection and quantification from NGS data obtained in the presence or absence of PCR amplification and gives novel insight into the mechanisms of influenza virus DVG production. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 131 KiB

Open AccessAbstract

Development of Zika Virus DNA Vaccine Using Envelope Modified Baculoviral Gene Delivery System

by Hanul Choi, Yuyeon Jang, Hansam Cho, Ha Youn Shin and Young Bong Kim

Proceedings 2020, 50(1), 85; https://doi.org/10.3390/proceedings2020050085 - 17 Jun 2020

Viewed by 1370

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus and the infection of pregnant women can cause a wide range of congenital abnormalities, including microcephaly in the infant. However, there is no vaccine available yet. In this study, we intended to use PrM/E, which is [...] Read more.

Zika virus (ZIKV) is a mosquito-borne flavivirus and the infection of pregnant women can cause a wide range of congenital abnormalities, including microcephaly in the infant. However, there is no vaccine available yet. In this study, we intended to use PrM/E, which is the main target gene of neutralizing antibodies, for the development of a DNA vaccine for ZIKV. To enhance the gene delivery, a recombinant baculovirus whose surface was modified to express human endogenous retrovirus (HERV) envelope was constructed. Baculovirus with HERV envelope (AcHERV) showed distinguished higher gene delivery than wild type. Using the AcHERV as a delivery vector, we constructed major antigen (prM-E)-encoding DNA under the CMV promoter, AcHERV–ZIKA. Transducing of prM/E gene in a mammalian cell was confirmed by Western blot. Immunization in mice with 10e7 of AcHERV–ZIKA elicited high IgG and neutralizing antibodies. In the challenge test, AcHERV–ZIKA immunized A129 mice showed perfect protection. These results suggest that AcHERV–ZIKA could be a potential vaccine candidate for human application. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 133 KiB

Open AccessAbstract

Complete Virus Inactivation Using a Combined Heat and Chemical Treatment

by Okechukwu Onianwa

Proceedings 2020, 50(1), 86; https://doi.org/10.3390/proceedings2020050086 - 17 Jun 2020

Viewed by 1505

Abstract

Validated inactivation protocols are required for the safe handling and disposal of virus samples. This is particularly important for hazard group 3 agents and above. Various methods are employed for virus inactivation, some of which include the use of heat or chemical chaotropic [...] Read more.

Validated inactivation protocols are required for the safe handling and disposal of virus samples. This is particularly important for hazard group 3 agents and above. Various methods are employed for virus inactivation, some of which include the use of heat or chemical chaotropic agents such as guanidine hydrochloride. It is generally accepted that these processes are sufficient to denature all viruses. While inactivation of certain viruses with such methods have been reported, validation of their activity against a wide of range of viruses is required. Here, we examined the inactivation of a panel of hazard group 2 viruses (Rhabdoviridae, Togaviridae, Peribunyaviridae, and Flaviviridae) using a combination of heat, guanidine hydrochloride-containing buffer, and ethanol. Viruses were treated with proteinase K and guanidine hydrochloride-containing buffer, and heated at 56 °C for 30 min. This was followed by additional treatment with absolute ethanol. Resulting virus–buffer–ethanol mixtures were column-purified to remove residual ethanol and other toxic reagents, before being introduced to cells. Column purification was confirmed to be insufficient for virus removal. Cultures were incubated at 37 °C for 1 h, after which media supplemented with 2% foetal bovine serum was added. Cultures were then observed daily for cytopathic effects. Samples that showed no evidence of cytopathic effects were passaged thrice to confirm absence of cytopathic effects and complete inactivation of viruses. Cultures infected with control viruses that had not been treated with buffer, heat, and ethanol but column-purified developed cytopathic effects, while cultures infected with treated viruses showed no cytopathic effects even after three passages, thus confirming complete virus inactivation. Results from this study provide evidence of the use of a combination of heat, guanidine hydrochloride-containing buffer, and ethanol for the complete inactivation of all members of the four families investigated. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 133 KiB

Open AccessAbstract

Conservation of Genetically-Embedded Virus Assembly Instructions: A Novel Route to Antiviral Therapy

by Reidun Twarock, Peter G. Stockley, Richard J. Bingham, Eric C. Dykeman and Pierre-Philippe Dechant

Proceedings 2020, 50(1), 87; https://doi.org/10.3390/proceedings2020050087 - 18 Jun 2020

Viewed by 1678

Abstract

Many single-stranded RNA viruses, including major viral pathogens, present RNA-encoded virus assembly instructions (VAIs) within their genetic message that can be isolated from the genetic code and repurposed for the design of virus-like particles. These VAIs rely on multiple dispersed RNA secondary structure [...] Read more.

Many single-stranded RNA viruses, including major viral pathogens, present RNA-encoded virus assembly instructions (VAIs) within their genetic message that can be isolated from the genetic code and repurposed for the design of virus-like particles. These VAIs rely on multiple dispersed RNA secondary structure elements with a consensus recognition motif for the capsid (core) protein, called packaging signals (PSs), which collectively promote capsid assembly. In this talk, I will provide evidence for the evolutionary conservation of the PS-encoded assembly instructions among different viruses in a viral family and discuss the implications of this discovery for viral evolution. I will then demonstrate how the VAIs can be exploited for therapy. In particular, defective interfering particles occur spontaneously in viral evolution as mutant strains lacking essential parts of the viral genome. Their ability to replicate in the presence of wild-type virus at the expense of virally produced resources can be mimicked by therapeutic interfering particles (TIPs). I will introduce a novel approach to the design of such TIPs based on synthetic nucleic acid sequences containing the VAIs but otherwise lacking genetic information. Using multiscale models of a viral infection, I will demonstrate the potential of these particles in both the prophylaxis and treatment of RNA viral infections. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 150 KiB

Open AccessAbstract

Porcine Respiratory Cell and Tissue Coinfections and Superinfections with Porcine Reproductive and Respiratory Syndrome and Swine Influenza Viruses

by Georges Saade, Caroline Hervet, Déborah Ménard, Patricia Renson, Céline Deblanc, Juliette Bougon, Gaëlle Simon, Olivier Bourry, Nicolas Bertho and François Meurens

Proceedings 2020, 50(1), 88; https://doi.org/10.3390/proceedings2020050088 - 19 Jun 2020

Viewed by 1494

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza type A virus (swIAV) are major contributors to the porcine respiratory disease complex that is still threatening porcine farming around the world. Understanding the interactions between these two viruses and the effect of [...] Read more.

Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza type A virus (swIAV) are major contributors to the porcine respiratory disease complex that is still threatening porcine farming around the world. Understanding the interactions between these two viruses and the effect of their coinfection on the porcine immune response will lead to better preventive and therapeutic measures. In vivo studies of these coinfections showed contradictory results while in vitro studies showing slight interferences between the viruses were limited due to the fact that the two viruses did not share the same host cells. SwIAV mainly infects epithelial cells, while PRRSV only infects cells expressing CD163, such as alveolar macrophages. In an attempt to evaluate the effect of PRRSV infection on the replication of swIAV, the possible induction of trained immunity, and on the host antiviral response, we carried out several coinfections and superinfections using local viral strains. Our infection protocols were performed using a tracheal epithelial cell line and precision-cut lung slices (PCLS) combining different cell types mimicking infections in real conditions. The expression of viral and cellular transcripts involved in the recognition of the virus and in the antiviral response was assessed by quantitative PCR. Immunostaining was also carried out to monitor the virus distribution in the pulmonary tissue. Interestingly, without infecting epithelial cells, PRRSV was able to interfere with swIAV infection and inhibit the antiviral response of the hosting cell. The mechanisms of this interference are still unclear and need to be investigated to ultimately apply improved preventive and therapeutic approaches. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 147 KiB

Open AccessAbstract

From Structure to Mechanisms of Zika Virus-Induced Neurodevelopmental Disease

by Diego Sebastian Ferrero, Murielle Saade, José Manuel Blanco, Elena Gonzalez-Gobartt, Victor Manuel Ruiz Arroyo, Elisa Martí and Nuria Verdaguer

Proceedings 2020, 50(1), 89; https://doi.org/10.3390/proceedings2020050089 - 19 Jun 2020

Viewed by 1437

Abstract

Zika virus (ZIKV) has explosively emerged over recent years, causing a series of epidemics across the Western world. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. As with other members in the Flaviviridae family, ZIKV [...] Read more.

Zika virus (ZIKV) has explosively emerged over recent years, causing a series of epidemics across the Western world. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. As with other members in the Flaviviridae family, ZIKV relies on its nonstructural protein 5 (NS5) for RNA genome capping (by the methyltransferase N-terminal domain) and replication (by the RNA-dependent RNA polymerase (RdRP) domain), representing an attractive crystallisable and antiviral target. The crystal structures of the ZIKV NS5 protein in two different space groups revealed conserved protein self-interactions to form dimers and higher-order fibrillar oligomers that serve as a platform for the coordination of the different enzymatic activities across neighboring molecules. The presence of dimers in solution was further verified by small angle X-ray scattering (SAXS), analytical ultracentrifugation (AU), and mass spectrometry, and ZIKV/NS5 helicoidal fibers were also observed by negative staining transmission electron microscopy (TEM) and atomic force microscopy (AFM). In addition, our preliminary data indicate that NS5 oligomerization might act as scaffold to interact with host proteins. In order to extend our findings, we have studied the in vivo effects of ZIKV NS5, both wild type and mutants in which NS5 oligomerization was disrupted, and these revealed an unexpected role of this protein in the exhaustion of neural progenitor cell (NPC) pool that may contribute to ZIKV-induced microcephaly. We have also identified a cluster of cilia/centrosome and nuclear envelope proteins of host cells as NS5 interactors. Work is currently ongoing to determine how NS5 interferes with the molecular machinery and behavior of NPCs to provide a better understanding of ZIKV–host interactions, highlighting new potential targets for therapeutic intervention. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 163 KiB

Open AccessAbstract

Siglec-1 Expressed on Dendritic Cells is a New Receptor Implicated in Arenavirus Uptake

by Xabier Muniz-Trabudua, Cristina Borio, Marcos Bilen, Itziar Erkizia, Daniel Perez-Zsolt, Susana Benet, Javier Martinez-Picado and Nuria Izquierdo-Useros

Proceedings 2020, 50(1), 90; https://doi.org/10.3390/proceedings2020050090 - 19 Jun 2020

Viewed by 1623

Abstract

Arenaviruses are enveloped viruses that cause hemorrhagic fever outbreaks in humans and still lack an effective antiviral treatment. Upon early infection, these viruses target dendritic cells (DCs), which can promote systemic viral dissemination, contributing to pathogenesis. We have previously described that Siglec-1, a [...] Read more.

Arenaviruses are enveloped viruses that cause hemorrhagic fever outbreaks in humans and still lack an effective antiviral treatment. Upon early infection, these viruses target dendritic cells (DCs), which can promote systemic viral dissemination, contributing to pathogenesis. We have previously described that Siglec-1, a sialic acid Ig-like binding lectin-1 expressed on DCs interacts with different enveloped viruses and promotes their capture within a virus-containing compartment. Such is the case of HIV-1 or Ebola virus, which display sialylated gangliosides on their viral envelope that are effectively recognized by Siglec-1. Here, we aimed to study if Siglec-1 on DCs also interacts with arenaviruses such as Junin. We produced non-infectious Junin viral-like particles (Junin-VLPs) tagged with fluorescent Egfp by transfecting a plasmid encoding the structural Junin Z protein on HEK-293T cells. Junin-VLPs were added to a Raji cell line stably transfected with Siglec-1 or to monocyte-derived DCs activated or not with either Interferon-α or lipopolysaccharide. Viral uptake was analyzed by FACS or confocal microscopy in the presence of an anti-Siglec-1 monoclonal antibody (mAb) or an isotype control. Statistical differences were assessed with the indicated tests. Raji Siglec-1 cells captured a higher number of Junin-VLPs than Raji cells, and this was blocked with an anti-Siglec-1 mAb (P = 0.0159; Mann–Whitney). On primary DCs, activation enhanced Junin-VLP capture (P = 0.0024; paired t-test) and Siglec-1 expression. Furthermore, pre-incubation with an anti-Siglec-1 mAb on activated DCs blocked Junin-VLP uptake (P ≤ 0.0002; one sample t-test), while an isotype control did not. Forty-nine percent of the activated DCs analyzed by confocal microscopy captured Junin-VLPs within a Siglec-1+ virus-containing compartment. Moreover, when HIV-1 was also added, 97% of those compartments retained both viruses. Thus, we conclude that Siglec-1 is a new receptor involved in arenavirus uptake in DCs and could represent a novel target for an anti-arenavirus treatment. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 144 KiB

Open AccessAbstract

Reactivation of Herpes Simplex Virus (HSV) from Latency in Response to Neuronal Hyperexcitability

by Sean Cuddy, Austin Schinlever, Sara Dochnal, Jon Suzich, Mina Farah, Chris Boutell and Anna Ruth Cliffe

Proceedings 2020, 50(1), 91; https://doi.org/10.3390/proceedings2020050091 - 19 Jun 2020

Viewed by 1805

Abstract

Herpes Simplex Virus (HSV) establishes a latent infection in neurons, in which viral transcription is restricted and viral promoters are associated with heterochromatin. In response to certain stimuli, the virus reactivates to permit transmission. The exact physiological triggers of reactivation, the cell signaling [...] Read more.

Herpes Simplex Virus (HSV) establishes a latent infection in neurons, in which viral transcription is restricted and viral promoters are associated with heterochromatin. In response to certain stimuli, the virus reactivates to permit transmission. The exact physiological triggers of reactivation, the cell signaling pathways involved, and how signals act on heterochromatin-associated lytic promoters, are not understood. Previously, we identified a role for a neural stress pathway involving DLK and JNK activity in HSV reactivation, triggered by nerve growth factor (NGF) deprivation. Reactivation was associated with a JNK-dependent histone phospho/methyl switch on lytic gene promoters. Because the same histone phospho/methyl switch occurs in cortical neurons following hyperexcitability (triggered by forskolin), we examined whether HSV reactivation was linked to hyperexcitability, and the contribution of JNK activity and histone phosphorylation. Using our primary neuronal model of HSV reactivation, we found that forskolin triggered DLK/JNK-dependent reactivation via a pathway that was distinct from NGF deprivation. The initial burst of HSV lytic gene expression in response to forskolin occurred independently of histone demethylase activity, and was accompanied by a histone phospho/methyl switch. To determine whether forskolin-mediated reactivation was linked to neuronal activity, we investigated the contribution of ion channel activity. Inhibition of voltage-gated potassium and sodium channels, or hyperpolarization-activated cyclic nucleotide-gated channels, prevented forskolin-mediated reactivation. In addition, hyperexcitability, resulting from the removal of a tetrodotoxin block, triggered HSV reactivation in a DLK/JNK-dependent manner. We next investigated whether physiological triggers induce HSV reactivation via hyperexcitability. IL-1 induced DNA damage associated with hyperexcitability in adult neurons. IL-1 also triggered DLK/JNK-dependent HSV reactivation, which was dependent on ion channel activity. Therefore, these data indicate that neuronal hyperexcitability in response to physiological stimuli, such as inflammation, trigger HSV reactivation, and mark out the activation of DLK/JNK and a histone phospho/methyl switch as key events in the hyperexcitability-mediated reactivation. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 149 KiB

Open AccessAbstract

RETRACTED: Isolation and Elimination of Latent and Productive Herpes Simplex Virus from the Sacral and Trigeminal Ganglions

by Bernard Middleton and Susan Michelle Cosgrove

Proceedings 2020, 50(1), 92; https://doi.org/10.3390/proceedings2020050092 - 22 Jun 2020

Cited by 1 | Viewed by 10517 | Retraction

Abstract

There is an immediate need for alternative anti-herpetic treatment options effective for both primary infections and reoccurring reactivations of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Alternatives currently approved for the purposes of clinical administration includes antivirals and a reduced set [...] Read more.

There is an immediate need for alternative anti-herpetic treatment options effective for both primary infections and reoccurring reactivations of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Alternatives currently approved for the purposes of clinical administration includes antivirals and a reduced set of nucleoside analogues. The present article tests a treatment based on a systemic understanding of how the herpes virus affects cell inhibition and breakdown, and targets different phases of the viral cycle, including the entry stage, reproductive cross mutation, and cell-to-cell infection. The treatment consisted of five immunotherapeutic core compounds (5CC), which were hypothesized to be capable of neutralizing human monoclonal antibodies. The tested 5CC were noted as being functional in the application of eliminating the DNA synthesis of herpes viral interferon (IFN) - induced cellular antiviral response. They were here found to neutralize antiviral reproduction by blocking cell-to-cell infection. The activity of the 5CC was tested on RC-37 in vitro using an assay plaque reduction and in vivo against HSV-1 and HSV-2. The 50% inhibitory concentration (IC50) of 5CC was 0.0009% for HSV-1 plaque formation and 0.0008% for HSV-2 plaque formation. Further tests were performed to evaluate the susceptibility of HSV-1 and HSV-2 to antiherpetic drugs in Vero cells after virus entry. There were high-level markers of the 5CC virucidal activity in viral suspension of HSV-1 and HSV-2. These concentrations of the 5CC are nontoxic and reduced plaque formation by 98.2% for HSV-1 and 93.0% for HSV-2. Virus HSV-1 and HSV-2 titers were reduced significantly by 5CC to the point of being negative, ranging 0.01–0.09 in 72%. The results concluded the 5CC as being an effective treatment option for the herpes simplex virus. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 132 KiB

Open AccessAbstract

Deep Mutational Scanning to Map How Zika Envelope Protein Mutations Affect Viral Growth and Antibody Escape

by Marion Sourisseau, Daniel J. P. Lawrence, Megan C. Schwarz, Carina H. Storrs, Ethan C. Veit, Jesse D. Bloom and Matthew J. Evans

Proceedings 2020, 50(1), 93; https://doi.org/10.3390/proceedings2020050093 - 23 Jun 2020

Viewed by 1642

Abstract

The Zika virus has recently been shown to be associated with severe birth defects. The virus’ envelope (E) protein mediates its ability to infect cells and is also the primary target of the antibodies that are elicited by natural infection and the vaccines [...] Read more.

The Zika virus has recently been shown to be associated with severe birth defects. The virus’ envelope (E) protein mediates its ability to infect cells and is also the primary target of the antibodies that are elicited by natural infection and the vaccines that are being developed against the virus. Therefore, determining the effects of mutations on this protein is important for understanding its function, its susceptibility to vaccine-mediated immunity, and its potential for future evolution. Functional constraints on viral proteins are often assessed by examining sequence conservation among natural strains, but this approach is relatively ineffective for the Zika virus because all known sequences are highly similar. Here, we take an alternative approach to mapping functional constraints on Zika virus’ E protein by using deep mutational scanning to measure how all amino-acid mutations in the protein affect viral growth in cell culture. The resulting sequence-function map is consistent with the existing knowledge about E protein structure and function but also provides insight into mutation-level constraints in many regions of the protein that have not been well characterized in prior functional work. In addition, we extend our approach to completely map how mutations affect viral neutralization by two monoclonal antibodies, thereby precisely defining their functional epitopes. Overall, our study provides a valuable resource for understanding the effects of mutations on this important viral protein and also offers a roadmap for future work to map functional and antigenic selection to the Zika virus at high resolution. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 137 KiB

Open AccessAbstract

Mixed Infections of Plant Viruses in Crops: Solo vs. Group Game

by Inmaculada Ferriol, Ornela Chase, María Luisa Domingo-Calap and Juan José López-Moya

Proceedings 2020, 50(1), 94; https://doi.org/10.3390/proceedings2020050094 - 23 Jun 2020

Viewed by 1589

Abstract

Plant diseases are responsible for considerable economic losses in agriculture worldwide. Recent surveys and metagenomics approaches reveal a higher than expected incidence of complex diseases, like those caused by mixed viral infections. Particularly, frequent cases of mixed infections are co-infections or superinfections of [...] Read more.

Plant diseases are responsible for considerable economic losses in agriculture worldwide. Recent surveys and metagenomics approaches reveal a higher than expected incidence of complex diseases, like those caused by mixed viral infections. Particularly, frequent cases of mixed infections are co-infections or superinfections of plant viruses belonging to different genera in the families Potyviridae (Ipomovirus or Potyvirus) and Closteroviridae (Crinivirus). The outcome of such multiple infections could modify viral traits, such as host range, titer, tissue and cell tropisms, and even vector preference and transmission rates. Therefore, we believe that understanding the virus–virus, virus–host, and virus–vector interactions would be crucial for developing effective control measures. Since there is still limited knowledge about the molecular mechanisms underlying the different interactions, and how they might contribute to specific diseases in mixed infection, we are analyzing ipomovirus–crinivirus and potyvirus–crinivirus pathosystems, to better understand single and mixed infections in selected susceptible hosts (Cucurbitaceae and Convolvulaceae plants), also incorporating in the study the interactions with insect vectors (whiteflies and aphids). Among other strategies, we are engineering new biotechnological tools, to explore the molecular biology and transmission mechanisms of several viruses implicated in complex diseases, and we are also addressing the possibility to produce virus-like particles (VLPs) through transient expression of the CP of different viruses in Nicotiana benthamiana plants, with the aim to study requirements for virion formation and determinants of transmission. Work supported by project AGL2016-75529-R and grant “Severo-Ochoa” SEV-2015-0533. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 148 KiB

Open AccessAbstract

Analysis of Humoral Immune Responses in Chikungunya Virus (CHIKV)-Infected Patients and Individuals Vaccinated with a Candidate CHIKV Vaccine

by Lisa Henss, Constanze Yue, Christine von Rhein, Roland Tschismarov, Lia-Laura Lewis-Ximenez, Albert Dölle, Sally A. Baylis and Barbara S. Schnierle

Proceedings 2020, 50(1), 95; https://doi.org/10.3390/proceedings2020050095 - 23 Jun 2020

Viewed by 1279

Abstract

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe flu-like symptoms. The acute symptoms disappear after one week, but chronic arthralgia can persist for years. Here, humoral immune responses in CHIKV-infected patients and vaccinees were analyzed. Alphavirus neutralization activity was analyzed with [...] Read more.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe flu-like symptoms. The acute symptoms disappear after one week, but chronic arthralgia can persist for years. Here, humoral immune responses in CHIKV-infected patients and vaccinees were analyzed. Alphavirus neutralization activity was analyzed with pseudotyped lentiviral vectors and antibody epitope mapping was performed with a peptide array. The greatest CHIKV neutralization activity was observed 60–92 days after onset of symptoms. The amount of CHIKV-specific antibodies, their binding avidity, and cross-reactivity with other alphaviruses increased over time. CHIKV and o’nyong-nyong virus (ONNV) were both neutralized to a similar extent. Linear antibody binding epitopes were mainly found in E2 domain B and the acid-sensitive regions (ASRs). In addition, serum samples from healthy volunteers vaccinated with a measles-vectored Chikungunya vaccine candidate, MV-CHIK, were analyzed. Neutralization activity in the samples from the vaccine cohort was lower than in samples from CHIKV-infected patients. In contrast to infection, vaccination induced cross-neutralization with ONNV and the E2 ASR1 was the major antibody target. These data could assist vaccine design and enable the identification of correlates of protection necessary for vaccine efficacy. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 169 KiB

Open AccessAbstract

Editing of the Human TRIM5 Gene Decreases the Permissiveness of Jurkat T Lymphocytic Cells to HIV-1

by Kevin Désaulniers, Levine Ortiz, Caroline Dufour, Alix Claudel, Mélodie B. Plourde, Natacha Merindol and Lionel Berthoux

Proceedings 2020, 50(1), 96; https://doi.org/10.3390/proceedings2020050096 - 24 Jun 2020

Viewed by 1421

Abstract

TRIM5α is a cytoplasmic antiviral effector induced by type I interferons (IFN-I) that has the potential to intercept incoming retroviruses by interacting with their capsid core, leading to uncoating induction and the partial degradation of core components. Most HIV-1 strains escape restriction by [...] Read more.

TRIM5α is a cytoplasmic antiviral effector induced by type I interferons (IFN-I) that has the potential to intercept incoming retroviruses by interacting with their capsid core, leading to uncoating induction and the partial degradation of core components. Most HIV-1 strains escape restriction by human TRIM5α due to a lack of interaction between TRIM5α and its viral molecular target. We previously showed, however, that two point mutations, R332G/R335G, in the capsid-binding region confer human TRIM5α with the capacity to target and strongly restrict HIV-1 upon the overexpression of the mutated protein. Here, we explored the possibility to introduce these two mutations in the endogenous human TRIM5 gene by CRISPR-Cas9-mediated gene editing. For this, we electroporated CRISPR ribonucleoproteins (RNPs) and the donor DNA into Jurkat T lymphocytic cells and isolated clones by limiting dilution. We analyzed 47 clones using specific PCR assays, and found that six clones (13%) contained at least one gene-edited allele. One clone (clone 6) had both alleles edited for R332G, but only one of the two alleles was edited for R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following IFN-β treatment, the inhibition of HIV-1 infection in clone 6 was significantly enhanced (~50-fold inhibition), whereas IFN-β treatment had no effect on TRIM5α overexpressed by retroviral transduction. Knockdown experiments confirmed that HIV-1 was inhibited by the edited TRIM5 gene products, whereas quantification of HIV-1 reverse transcription products confirmed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes, but our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 135 KiB

Open AccessAbstract

Yellow Fever Virus Vaccine Reduces T Cell Receptor Signaling and the Levels of Phosphatase PTPRE In Vivo

by Jinhua Xiang, James H. McLinden, Qing Chang, Thomas M Kaufman, Judy A. Streit and Jack T. Stapleton

Proceedings 2020, 50(1), 97; https://doi.org/10.3390/proceedings2020050097 - 24 Jun 2020

Viewed by 1366

Abstract

Background: A Src kinase-activating phosphatase (PTPRE) is targeted by a genome-derived yellow fever virus (YFV) short noncoding RNA (vsRNA) in vitro. The vsRNA reduces PTPRE translation, which leads to reduced TCR signaling. vsRNA point mutations restore PTPRE expression and T cell function. We [...] Read more.

Background: A Src kinase-activating phosphatase (PTPRE) is targeted by a genome-derived yellow fever virus (YFV) short noncoding RNA (vsRNA) in vitro. The vsRNA reduces PTPRE translation, which leads to reduced TCR signaling. vsRNA point mutations restore PTPRE expression and T cell function. We examined TCR signaling and PTPRE levels in individuals before and after YFV vaccination (YFVax). Methods: Fourteen individuals receiving YFVax (10^4.7–5.6) IM for travel prophylaxis provided written informed consent for these studies. Blood was obtained once before vaccination and four times after vaccination (days 3 to 28). Serum and PBMCs were purified and YFV was quantified by RNA and infectivity. PBMCs were assessed for activation following anti-CD3 stimulation by measuring phospho-tyrosine-394-Lck and IL-2 release. PBMC PTPRE levels were determined by immunoblot analyses (normalized to actin). A YFV-neutralizing antibody was determined by PRNT. Results: YFVax was administered alone (six out of 14 subjects) or in combination with other vaccines (eight out of 14). All subjects demonstrated reduced resting PBMC PTPRE levels and post-TCR stimulation had reduced IL-2 release between days 4 and 21 compared to pre- and day 28 samples. Phospho-Lck was reduced in all but two subjects on the same days, and both of these subjects also received an influenza vaccine. Low-level viremia was detected in 10/14 subjects, with infectious titers of 100/mL. Viremia was not detected in four out of 14 subjects. All recipients developed neutralizing antibodies by day 21. Conclusion: YFV vaccination regulates PBMC PTPRE levels 4–21 days after infection, despite the low to absent infectious YFV detected in serum, suggesting that enough YFV vsRNA is produced and released from cells to have a functional (and measurable) effect on T cell function. Studies are underway to determine if this is mediated by exosomes or defective particles containing the vsRNA that targets PTPRE. Furthermore, the association between PTPRE and TCR signaling confirms a role for PTPRE in TCR function. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 150 KiB

Open AccessAbstract

Usutu Virus NS5: Characterization of Polymerase Activity, Protein–Protein Interaction and Cellular Localization %MCEPASTEBIN%

by Laura Albentosa-González, Pilar Clemente-Casares, Rosario Sabariegos and Antonio Mas

Proceedings 2020, 50(1), 98; https://doi.org/10.3390/proceedings2020050098 - 24 Jun 2020

Viewed by 1358

Abstract

Usutu virus (USUV) is a mosquito-borne arbovirus that has rapidly propagated in birds across several European countries over the last two decades, leading to substantial avian mortalities. USUV infection in humans has been associated with a growing number of cases of neurological disease [...] Read more.

Usutu virus (USUV) is a mosquito-borne arbovirus that has rapidly propagated in birds across several European countries over the last two decades, leading to substantial avian mortalities. USUV infection in humans has been associated with a growing number of cases of neurological disease in the last years, underlining the need for increased awareness and suitable treatments. Our group is working on the characterization of the NS5 protein of USUV. This protein is responsible for the replication activity of the viral genome and can be a suitable viral target to treat the infection. NS5 contains a RNA-dependent RNA polymerase (RdRpD) and a methyltransferase domains. Recombinant NS5 and RdRpD proteins expressed in bacteria were purified and biochemically characterized to determine the best conditions for their polymerase activities. Both proteins showed de novo and primer extension activities. Optimal RNA–polymerase reaction conditions included low NaCl (less than 20 mM), 2.5 mM MgCl₂ and 5 mM MnCl₂, 30 °C, and pH 7.25. Polymerase activity was cooperative for the polymerase domain (Hill coefficient = 5.8) but not for the complete NS5 (Hill coefficient = 1.2). To study their subcellular location, suitable sequences were cloned into a pcDNA3 vector and expressed in Huh7.5 and HEK293T cells. Both proteins were preferentially located in the cytoplasmic region, although a significant amount was found in the nucleus. Preliminary results showed that the concentration of sofosbuvir (SOFTP) necessary to achieve its incorporation by NS5 in 50% of the nascent RNA is higher than 100 µM, as already observed for dengue virus DENV. In this work, we describe the main features of the full-length USUV NS5, including the polymerase activity as well as the effect of protein–protein interactions and subcellular localization. Our results will be very useful for the study of this viral enzyme as a suitable target against the infection and the effect of antiviral drugs. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 132 KiB

Open AccessAbstract

Unveiling Infection Strategies across Diverse Marine Phage–Host Systems

by Cristina Howard-Varona, Karin Holmfeldt, Melissa B Duhaime and Matthew B Sullivan

Proceedings 2020, 50(1), 99; https://doi.org/10.3390/proceedings2020050099 - 24 Jun 2020

Viewed by 1922

Abstract

Bacterial viruses (phages) are amongst the smallest, most powerful biological entities on Earth. Through infection, phages impact host metabolism, bacterial mortality, and evolution. In the oceans, 20–40% of surface microbes are infected, with 10²³ new infections each second. Yet, infections remain virtually [...] Read more.

Bacterial viruses (phages) are amongst the smallest, most powerful biological entities on Earth. Through infection, phages impact host metabolism, bacterial mortality, and evolution. In the oceans, 20–40% of surface microbes are infected, with 10²³ new infections each second. Yet, infections remain virtually uncharacterized, as the available phage isolates underrepresent the diversity of marine phage–host interactions. Additionally, while sequencing efforts reveal “who is there?”, a gap between sequence and function prevents answering “what are they doing?” and “how?”. We have developed new Bacteroidetes and Proteobacteria marine phage–host model systems with which to connect genomes, infection strategies, and functions using both traditional and genome-wide “-omics” experiments. We ask: How do infections by genomically divergent phages compare? Are there links between phage–host genomes and infection strategies? Our findings are as follows. In Bacteroidetes, a phage infecting two nearly identical strains (host38 and host18) under identical conditions is more fit and efficient on host38. By contrast, on host18, it is less fit and, except for phage transcription, it fails at efficiently mastering all stages of the infection: from adsorption through to cell lysis. In Proteobacteria, genomically unrelated podovirus and siphovirus phages infecting the same strain reprogram host metabolisms very differently. Namely, siphovirus-infected cells hardly differ from uninfected and mainly repress energy-consuming processes such as motility and translation. By contrast, podovirus-infected cells greatly differ from uninfected cells in transcription and in uniquely shifting central carbon and energy metabolism. Additionally, the siphovirus is more complementary to the host than the podovirus in %GC, amino acids, and codon usage. We found that phage–host genome complementarity may drive the resource demand and fitness of a phage: the phage most complementary to its host easily accesses intracellular resources, infects with little reprogramming, and accomplishes the largest fitness, which has not previously been shown. Together, this work helps to uncover infection efficiency strategies, and connect genomes with metabolisms in marine phage–host systems. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 137 KiB

Open AccessAbstract

Zika Virus Epidemiology in Selected West African Countries between 2007 and 2012

by Serena Marchi, Simonetta Viviani, Emanuele Montomoli, Yuxiao Tang, Adele Boccuto, Ilaria Vicenti, Maurizio Zazzi, Samba Sow, Aldiouma Diallo, Olubukola T. Idoko, Niranjan Bhat and Claudia Maria Trombetta

Proceedings 2020, 50(1), 100; https://doi.org/10.3390/proceedings2020050100 - 24 Jun 2020

Viewed by 1444

Abstract

Before its recent spread, serological investigations conducted between the 1960s and the 1990s showed the wide presence of Zika virus in Africa. According to the World Health Organization, the entire Africa continent is at risk of Zika outbreak due to the presence of [...] Read more.

Before its recent spread, serological investigations conducted between the 1960s and the 1990s showed the wide presence of Zika virus in Africa. According to the World Health Organization, the entire Africa continent is at risk of Zika outbreak due to the presence of the virus, competent vectors, and the low capacity for surveillance and containment of an epidemic. However, limited data are available on the recent prevalence in the African population. The aim of this study was to evaluate the immunity against Zika virus in samples of a selected cohort from West Africa, in order to investigate the circulation of the virus in the region during the first years of its emergence in the Pacific. Human serum samples were collected in 2007 and between 2011 and 2012 from a cohort of subjects from Mali, Senegal, and The Gambia. The samples were tested using an enzyme-linked immunosorbent assay (ELISA) detection kit and positives were further confirmed by microneutralization test. The results indicate that Zika virus is present and actively circulating in Senegal and The Gambia, with prevalence values of 13.7% and 6.9% in 2012, respectively. Although no significant differences in prevalence were found for the considered time period, seroconversion of some subjects showed the active circulation of Zika virus in the West African area. Analysis by age showed an increase in immunity in relation to increasing age, demonstrating that the population is consistently exposed to the virus throughout life and with a high possibility of being infected during reproductive age. In conclusion, the obtained results allow for better knowledge of the circulation of Zika virus within three different ecological and demographic contexts, and represent an update to the limited data currently available. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 128 KiB

Open AccessAbstract

Structural Basis for Capsid Recruitment and Coat Formation during HSV-1 Nuclear Egress

by Elizabeth Draganova, Jiayan Zhang, Hong Zhou and Ekaterina Heldwein

Proceedings 2020, 50(1), 101; https://doi.org/10.3390/proceedings2020050101 - 24 Jun 2020

Viewed by 1339

Abstract

During herpesvirus infection, nascent viral capsids egress the nucleus into the cytoplasm by an unusual mechanism whereby capsids bud at the inner nuclear membrane. This process is mediated by the conserved heterodimeric nuclear egress complex (NEC), anchored to the inner nuclear membrane, that [...] Read more.

During herpesvirus infection, nascent viral capsids egress the nucleus into the cytoplasm by an unusual mechanism whereby capsids bud at the inner nuclear membrane. This process is mediated by the conserved heterodimeric nuclear egress complex (NEC), anchored to the inner nuclear membrane, that deforms the membrane around the capsid by forming a hexagonal array. However, how the NEC coat interacts with the capsid and how proper curvature of the coat is achieved to enable budding are yet unclear. Here, we show that the binding of a capsid protein, UL25, promotes the formation of a pentagonal rather than hexagonal NEC arrangement. Our results suggest that during nuclear budding interactions between the UL25 bound to the pentagonal capsid vertices and the NEC introduce pentagonal insertions into the hexagonal NEC array to yield an NEC coat of the appropriate size and curvature, leading to the productive budding and egress of UL25-decorated capsids. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 171 KiB

Open AccessAbstract

Specific Lipid Recruitment by the Retroviral Gag Protein upon HIV-1 Assembly: From Model Membranes to Infected Cells

by Cyril Favard, Jakub Chojnacki, Naresh Yandrapalli, Johnson Mak, Christian Eggeling and Delphine Muriaux

Proceedings 2020, 50(1), 102; https://doi.org/10.3390/proceedings2020050102 - 24 Jun 2020

Viewed by 1559

Abstract

The retroviral Gag protein targets the plasma membrane of infected cells for viral particle formation and release. The matrix domain (MA) of Gag is myristoylated for membrane anchoring but also contains a highly basic region that recognizes acidic phospholipids. Gag targets lipid molecules [...] Read more.

The retroviral Gag protein targets the plasma membrane of infected cells for viral particle formation and release. The matrix domain (MA) of Gag is myristoylated for membrane anchoring but also contains a highly basic region that recognizes acidic phospholipids. Gag targets lipid molecules at the inner leaflet of the plasma membrane including phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and cholesterol. Here, we addressed the question whether HIV-1 Gag was able to trap PI(4,5)P2 and/or other lipids during HIV-1 assembly in silico, in vitro on reconstituted membranes and in cellulo at the plasma membrane of the host CD4+ T cells. In silico, we could observe the first PI(4,5)P2 preferential recruitment by HIV-1 MA or Gag while protein docked on artificial membranes. In vitro, using biophysical techniques, we observed the specific trapping of PI(4,5)P2, and, to a lesser extent, cholesterol and the exclusion of sphingomyelin, during HIV-1 myr(-)Gag self-assembly on LUVs and SLBs. Finally, in infected living CD4+ T cells, we measured lipid dynamics within and away from HIV-1 assembly sites using super-resolution stimulated emission depletion (STED) microscopy coupled with scanning Fluorescence Correlation Spectroscopy (sSTED-FCS). The analysis of HIV-1 infected CD4+ T lymphocytes revealed that, upon virus assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol but not phosphatidylethanolamine (PE) or sphingomyelin (SM) at the cellular membrane. Furthermore, analyzing CD4+ T cells expressing only HIV-1 Gag protein showed that Gag is the main driving force restricting the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. Our data provide the first direct evidence showing that HIV-1 Gag creates its own specific lipid environment for virus assembly by selectively recruiting lipids to generate PI(4,5)P2/cholesterol-enriched nanodomains favoring virus assembly, and that HIV-1 does not assemble on pre-existing lipid domains. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 164 KiB

Open AccessAbstract

Small RNAs Virome Characterization Reveals Arthropod-Associated Viruses in Anopheles atroparvus from the Ebro Delta, Spain

by Lotty Birnberg, Francisco M. Codoñer, Raúl Escosa, Carles Aranda, Ana Isabel Núñez, Sandra Talavera and Núria Busquets

Proceedings 2020, 50(1), 103; https://doi.org/10.3390/proceedings2020050103 - 24 Jun 2020

Viewed by 1429

Abstract

Even though malaria was eradicated from Europe after the mid-20th century, in 2017, more than 8000 imported cases were reported in the continent. Due to travel routes to endemic areas, climate change, and the presence of native vector mosquitoes (genus Anopheles), the [...] Read more.

Even though malaria was eradicated from Europe after the mid-20th century, in 2017, more than 8000 imported cases were reported in the continent. Due to travel routes to endemic areas, climate change, and the presence of native vector mosquitoes (genus Anopheles), the re-establishment of autochthonous malaria transmission is a current concern. Anopheles atroparvus (Van Thiel, 1972) is one of the 11 sibling species within the Palearctic Anopheles maculipennis complex, which formerly were considered the main vectors of the disease in the European continent. The microbiota (bacteria and viruses) of vector species has been demonstrated to play a significant role in the biology of these organisms, including their infection susceptibility and their capacity to transmit disease-causing agents. Recently, with the improvement of metagenomics techniques, several viruses that naturally infect vector mosquitoes have been identified. The purpose of the present study was to characterize, for the first time, the virome present in An. atroparvus from the Ebro Delta and assess its evolution after ten generations in the laboratory. Small RNA sequencing was used to characterize the virome from wild-caught An. atroparvus females and from the tenth generation produced under controlled laboratory conditions. Through this approach, we were able to identify viral linages previously reported in other invertebrates, such as Chaq virus and several Partiti-like viruses. A reduction in the viral composition was observed during the colonization process. The present study contributes to the understanding of the viral diversity of a medically relevant vector species in its natural setting and under confinement, and sets a baseline for further studies to assess the potential implications of these viruses in the transmission of pathogens. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 129 KiB

Open AccessAbstract

Vaccination with Single Plasmid DNA Encoding IL-12 and Antigens of Severe Fever with Thrombocytopenia Syndrome Virus Elicits Complete Protection in IFNAR Knockout Mice

by Nam-Hyuk Cho

Proceedings 2020, 50(1), 104; https://doi.org/10.3390/proceedings2020050104 - 24 Jun 2020

Viewed by 1504

Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by SFTS virus (SFTSV) infection. Despite the gradual increase in SFTS cases and high mortality in the endemic region, no specific viral therapy nor vaccine is available. Here, we developed single [...] Read more.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by SFTS virus (SFTSV) infection. Despite the gradual increase in SFTS cases and high mortality in the endemic region, no specific viral therapy nor vaccine is available. Here, we developed single recombinant plasmid DNA encoding SFTSV genes Gn and Gc together with the NP–NS fusion antigen as a vaccine candidate. The viral antigens were fused with Fms-like tyrosine kinase-3 ligand (Flt3L) and the IL-12 gene was incorporated into the plasmid to enhance cell-mediated immunity. Vaccination with the DNA can provide complete protection of IFNAR KO mice upon lethal SFTSV challenge, whereas immunization with a plasmid lacking the IL-12 gene resulted in partial protection. Since we failed to detect antibodies against the surface glycoproteins Gn and Gc in the immunized mice, antigen-specific cellular immunity, as confirmed by enhanced antigen-specific T cell responses, might play a major role in protection. Finally, we evaluated the degree of protective immunity after protein immunization that was provided by individual glycoproteins Gn or Gc. Although both protein antigens induced significant levels of neutralizing activity against SFTSV, Gn vaccination resulted in relatively higher neutralizing activity and better protection than Gc vaccination. However, both antigens failed to provide complete protection. Given that the DNA vaccines have failed to induce sufficient immunogenicity in human trials when compared with protein vaccines, optimal combinations of DNA and protein vaccine, proper selection of target antigens, and incorporation of an efficient adjuvant need to be further investigated for SFTS vaccine development. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 147 KiB

Open AccessAbstract

Ubiquity, Diversity, and Genomic Complexity of Cyanophages in Freshwater Environments

by Qi-Ya Zhang

Proceedings 2020, 50(1), 105; https://doi.org/10.3390/proceedings2020050105 - 24 Jun 2020

Viewed by 1230

Abstract

Cyanophages are viruses that infect cyanobacteria (also known as blue-green algae) and are ubiquitious in marine and freshwater environments. In recent years, freshwater cyanophages have attracted more attention because they can affect global freshwater ecosystems. The spatial distribution and morphological diversity of cyanophage [...] Read more.

Cyanophages are viruses that infect cyanobacteria (also known as blue-green algae) and are ubiquitious in marine and freshwater environments. In recent years, freshwater cyanophages have attracted more attention because they can affect global freshwater ecosystems. The spatial distribution and morphological diversity of cyanophage populations were examined in Lake Donghu with three trophic regions: hypertrophic, eutrophic, and mesotrophic regions. The surprisingly high viral abundance (ranging from 10⁸ to 10⁹ phage mL⁻¹) and morphological diversity were detected. Most of them have tails and belong to the families Siphoviridae, Myoviridae, and Podoviridae. Various morphotypes were observed, such as prolate-headed virus-like particles and lemon-shaped virus-like particles. In addition, some cyanophages were studied by virological experiments and whole-genome analyses, combined with morphological observation. For example, three cyanophages were isolated and their whole genomes were sequenced. Contractile tail myonophage MaMV-DC infects bloom-forming cyanobacterium Microcystis aeruginosa. Tailless cyanophage Planktothrix agardhii virus isolated from Lake Donghu (PaV-LD) infects filamentous cyanobacterium. Short-tail podovirus A-4L can infect the model cyanobacterium Anabaena sp. strain PCC 7120. The MaMV-DC genome contains 169,223 bp encoding 170 putative open reading frames (ORFs). The PaV-LD genome posseses 95,299 bp encoding 142 putative ORFs. The genome of short-tail podovirus A-4L has 41,750 bp encoding 38 putative ORFs. There are significant differences in their genomic size and encoded tail proteins, but all three cyanophages contain genes that are not commonly found in phages. By studying the vast biodiversity of viruses in freshwater environments, these novel findings of cyanophages broaden our insights, and allow us to gain more useful knowledge about the global impact of these viruses in freshwater ecosystems. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 179 KiB

Open AccessAbstract

Ts2631 Endolysin from the Extremophilic Thermus scotoductus Bacteriophage vB_Tsc2631 as an Antimicrobial Agent against Gram-Negative Multidrug-Resistant Bacteria

by Magdalena Plotka, Malgorzata Kapusta, Sebastian Dorawa, Anna-Karina Kaczorowska and Tadeusz Kaczorowski

Proceedings 2020, 50(1), 106; https://doi.org/10.3390/proceedings2020050106 - 26 Jun 2020

Viewed by 1357

Abstract

Plotka, M.; Kapusta, M.; Dorawa, S.; Kaczorowska, A.-K.; Kaczorowski, T. Ts2631 Endolysin from the Extremophilic Thermus scotoductus Bacteriophage vB_Tsc2631 as an Antimicrobial Agent against Gram-Negative Multidrug-Resistant Bacteria. Viruses 2019, 11, 657. doi: 10.3390/v11070657 Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 158 KiB

Open AccessAbstract

Ultrastructural Characterization of the Frontal Lobe in the Case of Human Herpes Virus-6 Infection

by Sandra Skuja, Valerija Groma and Modra Murovska

Proceedings 2020, 50(1), 107; https://doi.org/10.3390/proceedings2020050107 - 24 Jun 2020

Viewed by 1191

Abstract

The vast majority of the world’s population is exposed to beta-herpesviruses during early childhood. After the primary infection, human herpesvirus-6 (HHV-6) can establish a lifelong persistence. The role of HHV-6 in the development of neurodegenerative disorders is not completely clarified. Postmortem samples of [...] Read more.

The vast majority of the world’s population is exposed to beta-herpesviruses during early childhood. After the primary infection, human herpesvirus-6 (HHV-6) can establish a lifelong persistence. The role of HHV-6 in the development of neurodegenerative disorders is not completely clarified. Postmortem samples of brain tissue obtained from 24 elderly subjects with unspecified encephalopathy were used in the study. Nested (nPCR) and real-time polymerase chain reaction (RT PCR) were used for the qualitative and quantitative detection of viral genomic sequences in isolated DNA from frontal lobe samples. For ultrastructural examination, transmission electron microscopy (TEM), nPCR, and immunohistochemically confirmed HHV-6-positive tissue samples were used. Immunogold (IG) labeling using anti-HHV-6 (20) mouse monoclonal antibodies, raised against viral lysate (Santa Cruz Biotechnology, dilution 1:30), was performed. HHV-6 DNA was detected in 38% (9/24) of the frontal lobe tissue samples. The HHV-6 load in the nPCR-positive samples ranged from 10 to 3878.5 (copies/10⁶ cells). A TEM examination of the frontal cortex revealed lipofuscin containing neurons, glial cells, unmyelinated and small myelinated axons, and symmetric synapses. Subcortical brain regions revealed glial cells interspersed by myelinated axons. The expression of viral proteins was found in the nuclei of neurons, demonstrating disarranged chromatin. HHV-6 positivity was detected between the adjacent cisternae of the rough endoplasmic reticulum of neurons displaying IG labeling. Furthermore, products of IG labeling were found in nuclei and cytoplasm of oligodendrocytes. The cytoplasm of astrocytes was IG labeled as well. IG labeling was used to determine the presence and intracellular localization of HHV-6 proteins in the human brain. HHV-6 possibly contributes to the demyelination process via entry into and affection of oligodendrocytes. Finally, neural susceptibility to HHV-6 may be linked to an invalid cellular immune response, followed by the development of a persistent viral infection. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 127 KiB

Open AccessAbstract

Valosin-Containing Protein (VCP/p97) Is a Potential Antiviral Target against Mononegavirales

by Victor Latorre and Ron Geller

Proceedings 2020, 50(1), 108; https://doi.org/10.3390/proceedings2020050108 - 30 Jun 2020

Viewed by 1313

Abstract

The viral order Mononegavirales consist of eight virus families. Members of these families include some of the most infectious (Measles, lethal (Ebola and Rabies), and most common viruses (Respiratory syncytial virus, RSV). Despite their medical importance, few vaccines and no antiviral treatments are [...] Read more.

The viral order Mononegavirales consist of eight virus families. Members of these families include some of the most infectious (Measles, lethal (Ebola and Rabies), and most common viruses (Respiratory syncytial virus, RSV). Despite their medical importance, few vaccines and no antiviral treatments are available for treating infections with these viruses. Being obligate cellular parasites, viruses must rely on the cellular machinery for their replication. One example of this is the widespread use of molecular chaperones, which assist the correct folding of newly synthesized proteins, refold misfolded or aggregated proteins, and play key roles in maintaining proteostasis in cells. Targeting chaperones required for viral replication may, therefore, provide an antiviral approach. In this work, we set out to identify all the members of the cytoplasmic chaperone network that are involved in the replication of RSV using an RNA interference screen. Among our hits is valosin-containing protein (VCP; also known as p97), a chaperone involved in ubiquitin-mediated protein degradation, which has been shown to play a role in the life cycle of several viruses. We investigated the role of VCP during RSV and vesicular stomatitis virus (VSV) infections using specific VCP inhibitors. Our results suggest that VCP activity is necessary for RSV and VSV replication and may constitute a promising antiviral approach for the Mononegavirales. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 149 KiB

Open AccessAbstract

Viral Genome Conformations and Contacts across Different Lifecycle Stages

by Peter G. Stockley, Nikesh Patel, Emma L. Wroblewski, Andrew J. P. Scott, Carlos P. Mata, Rebecca Chandler-Bostock, Amy M. Barker, Reidun Twarock, Sam Clark and Richard J. Bingham

Proceedings 2020, 50(1), 109; https://doi.org/10.3390/proceedings2020050109 - 30 Jun 2020

Viewed by 1551

Abstract

Single-stranded RNA viral genomes (gRNA) are dynamic molecules that permit packaging into virions and their subsequent extrusion during infection. For viruses with such genomes, we discovered a previously unsuspected mechanism that regulates their assembly. This regulation is the result of multiple cognate coat [...] Read more.

Single-stranded RNA viral genomes (gRNA) are dynamic molecules that permit packaging into virions and their subsequent extrusion during infection. For viruses with such genomes, we discovered a previously unsuspected mechanism that regulates their assembly. This regulation is the result of multiple cognate coat protein (CP)–gRNA contacts distributed across the RNA. Collectively, these interactions make the assembly highly efficient and specific. The regions of the gRNA packaging signals (PSs) driving this assembly are potential drug targets, whilst the manipulation of PS–CP contacts with nonviral RNA cargos is a route towards bespoke virus-like particles. Infectivity depends on the virions being able to transfer their gRNAs into host cells. The starting point for this transfer appears to be an encapsidated RNA with a defined three-dimensional structure, especially around the PSs. A combination of asymmetric cryo-electron microscopy structure determination and X-ray synchrotron footprinting were used to define these contacts and structures in a number of viral examples, including hepatitis B virus and enteroviruses. These tools allow us to look beyond the outer CP layer of the virion shell and to see the functional, asymmetric components that regulate viral infectivity. This revealed yet more unexpected aspects of critical infection mechanisms, such as the RNA conformational changes required for encapsidation, the details of PS–CP contacts regulating the assembly, and the conformational “memory” imposed by encapsidation. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 122 KiB

Open AccessAbstract

Study of the Retrotransposon-Derived Human PEG10 Protease

by Mária Golda, János András Mótyán, Mohamed Mahdi and József Tőzsér

Proceedings 2020, 50(1), 110; https://doi.org/10.3390/proceedings2020050110 - 1 Jul 2020

Viewed by 1793

Abstract

Paternally expressed gene 10 (PEG10) is a human retrotransposon-derived imprinted gene. Previous works have demonstrated that a mutation in the coding sequence of this gene is lethal with regard to embryological age due to defects of placental development. In addition, PEG10 [...] Read more.

Paternally expressed gene 10 (PEG10) is a human retrotransposon-derived imprinted gene. Previous works have demonstrated that a mutation in the coding sequence of this gene is lethal with regard to embryological age due to defects of placental development. In addition, PEG10 is implicated in several malignancies, such as pancreatic cancer and hepatocellular carcinoma. The PEG10 gene encodes two protein isoforms, which are translated by a typical retroviral frameshift mechanism. The Gag-like protein (RF1_PEG10) is encoded by reading frame 1, whilst reading frames 1 and 2 accounts for the Gag-Pol-like polyprotein (RF1/RF2_PEG10). The protease (PR) domain of RF2_PEG10 contains an -Asp-Ser-Gly- sequence, which refers to the conservative -Asp-Ser/Thr-Gly- active-site motif of retroviral aspartic proteases. The function of the aspartic protease domain of RF2_PEG10 remains unclear. In order to further investigate the function of the PEG10 protease (PR_PEG10), a frameshift mutant was generated (_fsRF1/RF2_PEG10) for comparison with the RF1/RF2_PEG10 form. To study the effects of PR_PEG10on cellular proliferation and viability, mammalian HEK293T and HaCaT cells were transfected with plasmids encoding for either the frameshift mutant (_fsRF1/RF2_PEG10) or a PR active-site (D370A) mutant _fsRF1/RF2_PEG10. Based on our findings, an _fsRF1/RF2_PEG10overexpression resulted in an increased cellular proliferation, compared to the mutant form. Interestingly, transfection with _fsRF1/RF2_PEG10had a detrimental effect on cell viability. We hypothesize that PR_PEG10 may play a cardinal role in the function of this retroviral remnant, possibly implicated in cellular proliferation and the inhibition of apoptosis. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 125 KiB

Open AccessAbstract

Globally Defining the Effects of Amino Acid Mutations across a Picornavirus Capsid

by Florian Mattenberger and Ron Geller

Proceedings 2020, 50(1), 111; https://doi.org/10.3390/proceedings2020050111 - 1 Jul 2020

Viewed by 1208

Abstract

RNA viruses are characterized by their extreme mutation rates, which play key roles in their biology and give them the ability to rapidly adapt to new environments. However, non-synonymous mutations tend to be largely deleterious to protein function, raising the question of how [...] Read more.

RNA viruses are characterized by their extreme mutation rates, which play key roles in their biology and give them the ability to rapidly adapt to new environments. However, non-synonymous mutations tend to be largely deleterious to protein function, raising the question of how the proteins of RNA viruses maintain functionality in the face of high mutation rates. This is of particular relevance to the capsids of non-enveloped RNA viruses, which form highly complex protein structures that assemble from numerous subunits, interact with cellular host factors to mediate entry and uncoating, and are under strong immune selection. To better understand how viral capsids accommodate mutations, we generated viral populations harboring a large fraction of all possible single amino acid mutations in a picornavirus capsid. We then used high-fidelity next-generation sequencing to derive the relative fitness of these mutations compared to the wildtype sequence. Combining our results with available structural, genetic, and phenotypic data, we are able to provide a comprehensive understanding of the ability of a viral capsid to accommodate mutations. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 137 KiB

Open AccessAbstract

Zika Virus sfRNA Plays an Essential Role in the Infection of Insects and Mammals

by Andrii Slonchak, Leon E. Hugo, Morgan Freney, Alberto A Amarilla, Sonja Hall-Mendelin, Kexin Yan, Francisco Torres, Yin Xiang Setoh, Nias Peng, Harman K Chaggar, Andrew Van Den Hurk, Ernst Wolvetang, Andreas Suhrbier, Gregor J. Devine and Alexander A Khromykh

Proceedings 2020, 50(1), 112; https://doi.org/10.3390/proceedings2020050112 - 1 Jul 2020

Viewed by 2049

Abstract

Similar to other flaviviruses, Zika virus (ZIKV) produces abundant subgenomic flavivirus RNA (sfRNA) derived from the 3’ untranslated region. The molecular mechanisms that determine the functions of sfRNA are currently not completely understood. Here, we created ZIKV mutants deficient in sfRNA production and [...] Read more.

Similar to other flaviviruses, Zika virus (ZIKV) produces abundant subgenomic flavivirus RNA (sfRNA) derived from the 3’ untranslated region. The molecular mechanisms that determine the functions of sfRNA are currently not completely understood. Here, we created ZIKV mutants deficient in sfRNA production and employed them to investigate the role of this RNA in virus interactions with mammalian and insect hosts. We found that in mosquitoes, sfRNA facilitates virus replication and is required for ZIKV dissemination into saliva and virus transmission. The production of sfRNA was found to have no effect on the RNAi pathway, but instead downregulated the expression of genes involved in the regulation of apoptosis. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of histological sections from infected mosquitoes confirmed that sfRNA prevents the apoptotic death of infected cells, thus identifying inhibition of apoptosis as a novel mechanism of sfRNA action in mosquitoes. We also found that sfRNA facilitates ZIKV replication in mammalian cells, mice, and human brain organoids. Moreover, ZIKV mutants deficient in sfRNA production were unable to form plaques, cause the death of human brain organoids, or establish infection in the mouse foetal brain. We then found that the proviral activity of sfRNA in mammalian cells relies on its ability to suppress type I interferon signalling. We showed that this is achieved via the inhibition of phosphorylation and the nuclear translocation of STAT1. In addition, we found that the production of sfRNA in the ZIKV infection of human brain organoids is associated with the suppression of multiple genes involved in brain development, indicating that sfRNA can be involved in the disruption of brain development associated with ZIKV infection. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 147 KiB

Open AccessAbstract

Structural and Functional Studies of Chikungunya Virus nsP2

by Yee Song Law, Yaw Bia Tan, Orion Shih, Age Utt, Jie Zheng, Sainan Wang, Ming Wei Chen, Patrick R. Griffin, U-Ser Jeng, Andres Merits and Dahai Luo

Proceedings 2020, 50(1), 113; https://doi.org/10.3390/proceedings2020050113 - 1 Jul 2020

Viewed by 2548

Abstract

Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural protein 2 (nsP2) contains an N-terminal RNA helicase with both nucleotide triphosphatase and RNA triphosphatase activities, and a C-terminal cysteine protease that is responsible for polyprotein processing. Both N-terminal [...] Read more.

Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural protein 2 (nsP2) contains an N-terminal RNA helicase with both nucleotide triphosphatase and RNA triphosphatase activities, and a C-terminal cysteine protease that is responsible for polyprotein processing. Both N-terminal RNA helicase and C-terminal cysteine protease are connected through a flexible linker. Although the structure of the C-terminal cysteine protease has been solved, the structure and the conformational arrangement of full-length nsP2 remains elusive. Here, we determined the crystal structure of the helicase part of the CHIKV nsP2 (nsP2h) bound to the conserved 3′-end of the genomic RNA and the nucleotide analogue ADP-AlF₄. The structure of this ternary complex revealed the molecular basis for viral RNA recognition and ATP hydrolysis by the nsP2h. Unique hydrophobic protein–RNA interactions play essential roles in viral RNA replication. We also determined the solution structure of full-length nsP2 using small-angle X-ray scattering (SAXS). The solution architecture of the nsP2 was modeled using the available high-resolution structures and program CORAL (complexes with random loops). The CORAL model revealed that nsP2 is partially unfolded and the N-terminal protease domain is arranged near the N-terminal domain of the helicase domain. These findings expand our knowledge of CHIKV and related alphaviruses and might also have broad implications for antiviral and vaccine developments against pathogenic alphaviruses. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 112 KiB

Open AccessAbstract

Structural Basis for Env Incorporation into HIV-1 Particles

by R. Elliot Murphy, Alexandra B. Samal, Gunnar Eastep, Ruba H. Ghanam, Peter E. Prevelige and Jamil S. Saad

Proceedings 2020, 50(1), 114; https://doi.org/10.3390/proceedings2020050114 - 2 Jul 2020

Viewed by 1353

Abstract

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂). Subsequent [...] Read more.

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂). Subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Evidence suggests that the incorporation of the Env protein is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of the gp41 subunit of Env (gp41CT), a mechanism that remains to be elucidated. Trimerization of the MA domain of Gag appears to be an obligatory step for this interaction. The interplay between gp41CT, the MA trimer, and the membrane has yet to be determined. Our lab has pioneered methods and approaches to investigate, at the molecular level, how the retroviral MA domains of Gag interact with membranes, a key requirement for understanding the Gag assembly and Env incorporation. Herein, we devised innovative approaches that will enable the structural characterization of the gp41CT–MA–membrane interactions. We employed structural biology (NMR and cryo-electron microscopy, biophysical methods, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to the membrane and how it interacts with gp41CT. To this end, we: (i) determined the three-dimensional structure of HIV-1 gp41CT and characterized its interaction with the membrane, (ii) engineered trimeric constructs of gp41CT and the MA to recapitulate the native and functional states of the proteins, and (iii) utilized membrane nanodisc technology to anchor the MA and gp41CT proteins. Our studies will allow for a detailed structural characterization of the gp41CT–MA–membrane interactions, which will advance our knowledge of HIV-1 Gag assembly and Env incorporation. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 106 KiB

Open AccessAbstract

Structure of the RSV Polymerase Complex Reveals a Tentacular Arrangement of the Viral Phosphoprotein

by Jason S. McLellan

Proceedings 2020, 50(1), 115; https://doi.org/10.3390/proceedings2020050115 - 3 Jul 2020

Viewed by 1514

Abstract

Numerous interventions are currently in the process of clinical development for respiratory syncytial virus (RSV) infection, including the use of small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the [...] Read more.

Numerous interventions are currently in the process of clinical development for respiratory syncytial virus (RSV) infection, including the use of small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). The RSV P performs many functions, including the recruitment of viral proteins to the polymerase complex. Despite their critical roles in RSV transcription and replication, the structures of L and P have remained elusive, though RSV P is thought to be intrinsically disordered in solution, with the exception of its oligomerization domain. Here, we describe the 3.2 Å cryo-EM structure of RSV L bound to the tetrameric P. The structure reveals a striking tentacular arrangement of P in which each of the four monomers adopts a distinct conformation. The structure also provides a rationale for the inhibitor-escape mutants and mutations observed in live attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 135 KiB

Open AccessAbstract

KSHV Activates Unfolded Protein Response Sensors but Suppresses Downstream Transcriptional Responses to Support Lytic Replication

by Benjamin P. Johnston, Eric S. Pringle and Craig McCormick

Proceedings 2020, 50(1), 116; https://doi.org/10.3390/proceedings2020050116 - 3 Jul 2020

Viewed by 1251

Abstract

Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure the proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading the ER folding capacity activates the unfolded protein response [...] Read more.

Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure the proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading the ER folding capacity activates the unfolded protein response (UPR), whereby sensor proteins, ATF6, PERK and IRE1, initiate a stress-mitigating transcription program that accelerates the catabolism of misfolded proteins, while increasing the ER folding capacity. Kaposi’s sarcoma-associated herpesvirus (KSHV) can be reactivated from latency through the chemical induction of ER stress, which causes an accumulation of the XBP1s transcription factor that transactivates the viral RTA lytic switch gene. The presence of XBP1s-responsive elements in the RTA promoter suggests that KSHV evolved a mechanism to respond to ER stress. Here, we report that ATF6, PERK and IRE1 were activated upon reactivation from latency and were required for efficient KSHV lytic replication. The genetic or pharmacologic inhibitions of each UPR sensor reduced virion production. Despite UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted: (1) ATF6 was cleaved to activate the ATF6(N) transcription factor but ATF6(N)-responsive genes were not transcribed; (2) PERK phosphorylated eIF2, but ATF4 did not accumulate; (3) IRE1 caused XBP1–mRNA splicing, but the XBP1s protein did not accumulate and the XBP1s-responsive genes were not transcribed. The complementation of XBP1s deficiency during KSHV lytic replication inhibited virion production in a dose-dependent manner in epithelial cells. Taken together, these findings indicate that, while XBP1s plays an important role in reactivation from latency, it can inhibit virus replication at a later step, which the virus overcomes by preventing its synthesis. These findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 141 KiB

Open AccessAbstract

VANIR—NextFlow Pipeline for Viral Variant Calling and de Novo Assembly of Nanopore and Illumina Reads for High-Quality dsDNA Viral Genomes

by Joan Martí-Carreras and Piet Maes

Proceedings 2020, 50(1), 117; https://doi.org/10.3390/proceedings2020050117 - 3 Jul 2020

Cited by 2 | Viewed by 1903

Abstract

Human cytomegalovirus (HCMV), like other herpes and dsDNA viruses, possesses unique properties derived from their genome architecture. The HCMV genome is composed of two unique domains: long (L) and short (S). Each domain contains a central unique region (U; thus, UL and US, [...] Read more.

Human cytomegalovirus (HCMV), like other herpes and dsDNA viruses, possesses unique properties derived from their genome architecture. The HCMV genome is composed of two unique domains: long (L) and short (S). Each domain contains a central unique region (U; thus, UL and US, respectively) and two repeated regions (thus, TRL/IRL and TRS/IRS). Recombination between repetitive regions is possible, yielding four possible genomic isomers, found in equimolar proportion in any viral infective population. Frequent recombination and an altered selective landscape can give rise to the persistence, if not fixation, of diverse variants in culturized HCMV isolates. This phenomenon has already been discovered in AD169 and Towne strains, characterizing a 10 kbp deletion (ΔUL/b’) in commonly used viral strains. Other dsDNA viruses are known for their structural rearrangements and frequent recombination. VANIR (viral variant calling and de novo assembly using nanopore and illumina reads) is a novel analysis pipeline that benefits from both short-read (Illumina) and long-read sequencing technologies (Oxford Nanopore Technologies Ltd.) to assemble high-quality dsDNA viral genomes and detection of variants. Illumina and nanopore sequencing provide complementary information to the assembly and variant discovery. Assembly contiguity, structural variant, and repeat calling are greatly improved by nanopore read-length and base-calling and base confidence by Illumina reduced error rate and increased yield. This specialized bioinformatic analysis pipeline is encoded in the NextFlow pipeline manager and containerized in a Singularity image. This set-up allows for improved traceability, reproducibility, transportability, and speed. Through VANIR, novel point mutations and structural genome rearrangements are called from sequencing data, benefiting diversity research with attenuated lab-strains and wild-type viruses. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 127 KiB

Open AccessAbstract

The European Virus Archive Goes Global: A Growing Resource for Research

by Christine MA Prat and Jean-Louis Romette

Proceedings 2020, 50(1), 118; https://doi.org/10.3390/proceedings2020050118 - 3 Jul 2020

Viewed by 1348

Abstract

The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. Within [...] Read more.

The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. Within three years, it developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. In 2014, the H2020 Research and Innovation Framework Programme (INFRAS projects) provided support for the transformation of the EVA from a European to a global organisation (EVA-GLOBAL). The EVA now operates as a non-profit consortium, with 37 partners and associated partners from 29countries. In this communication, we outline the structure and goals of the EVA, and how the EVA has helped to respond to Viral Public Health Emergencies. Our aim is to bring to the attention of researchers the wealth of products it can provide and to illustrate how they can gain access to these resources, free of charge. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 138 KiB

Open AccessAbstract

Composition of Herpesvirus Ribonucleoprotein Complexes

by Eric S. Pringle and Craig McCormick

Proceedings 2020, 50(1), 119; https://doi.org/10.3390/proceedings2020050119 - 4 Jul 2020

Viewed by 1699

Abstract

Herpesvirus genomes are decoded by host RNA polymerase enzymes, generating messenger ribonucleotides (mRNA) that are post-transcriptionally modified and exported to the cytoplasm through the combined work of host and viral factors. These viral mRNA bear 5′-m⁷GTP caps and poly(A) tails that [...] Read more.

Herpesvirus genomes are decoded by host RNA polymerase enzymes, generating messenger ribonucleotides (mRNA) that are post-transcriptionally modified and exported to the cytoplasm through the combined work of host and viral factors. These viral mRNA bear 5′-m⁷GTP caps and poly(A) tails that should permit the assembly of canonical host eIF4F cap-binding complexes to initiate protein synthesis. However, the precise mechanisms of translation initiation remain to be investigated for Kaposi’s sarcoma-associated herpesvirus (KSHV) and other herpesviruses. During KSHV lytic replication in lymphoid cells, the activation of caspases leads to the cleavage of eIF4G and depletion of eIF4F. Translating mRNPs depleted of eIF4F retain viral mRNA, suggesting that non-eIF4F translation initiation is sufficient to support viral protein synthesis. To identify proteins required to support viral protein synthesis, we isolated and characterized actively translating messenger ribonucleoprotein (mRNP) complexes by ultracentrifugation and sucrose-gradient fractionation followed by quantitative mass spectrometry. The abundance of host translation initiation factors available to initiate viral protein synthesis were comparable between cells undergoing KSHV lytic or latent replication. The translation initiation factors eIF4E2, NCBP1, eIF4G2, and eIF3d were detected in association with actively translating mRNP complexes during KSHV lytic replication, but their depletion by RNA silencing did not affect virion production. By contrast, the N6-methyladenosine methyltransferase METTL3 was required for optimal late gene expression and virion production, but was dispensable for genome replication. Furthermore, we detected several KSHV proteins in actively translating mRNP complexes that had not previously been shown to play roles in viral protein synthesis. We conclude that KSHV usurps distinct host translation initiation systems during latent and lytic phases of infection. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 145 KiB

Open AccessAbstract

Coxsackieviruses Undergo Intercellular Transmission as Pools of Sibling Viral Genomes Associated to Membranes

by Juan-Vicente Bou, Ron Geller and Rafael Sanjuán

Proceedings 2020, 50(1), 120; https://doi.org/10.3390/proceedings2020050120 - 4 Jul 2020

Viewed by 1257

Abstract

Some viruses are released from cells as pools of membrane-associated virions. By increasing the multiplicity of infection, this type of collective dispersal could favor viral cooperation, but also the emergence of cheater-like viruses, such as defective interfering particles. To better understand this process, [...] Read more.

Some viruses are released from cells as pools of membrane-associated virions. By increasing the multiplicity of infection, this type of collective dispersal could favor viral cooperation, but also the emergence of cheater-like viruses, such as defective interfering particles. To better understand this process, we examined the genetic diversity of membrane-associated coxsackievirus infectious units. We found that infected cells released large membranous structures containing 8–21 infectious particles on average, including vesicles. However, in most cases (62–93%), these structures did not promote the co-transmission of different viral genetic variants present in a cell. Furthermore, collective dispersal had no effect on viral population sequence diversity. Our results indicate that membrane-associated collective infectious units typically contain viral particles derived from the same parental genome. Hence, if cooperation occurred, it should probably involve sibling viral particles rather than different variants. As shown by social evolution theory, cooperation among siblings should be robust against cheater invasion. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 150 KiB

Open AccessAbstract

The First Complete Genome Sequences of Hepatitis C Virus Subtype 2b from Latin America: Molecular Characterization and Phylogeographic Analysis

by Natália Spitz, José J. Barros, Kycia M. do Ó, Carlos E. Brandão-Mello and Natalia M. Araujo

Proceedings 2020, 50(1), 121; https://doi.org/10.3390/proceedings2020050121 - 6 Jul 2020

Viewed by 1233

Abstract

The hepatitis C virus (HCV) has remarkable genetic diversity and exists as eight genotypes (1 to 8) with distinct geographic distributions. No complete genome sequence of HCV subtype 2b (HCV-2b) is available from Latin American countries, and the factors underlying its emergence and [...] Read more.

The hepatitis C virus (HCV) has remarkable genetic diversity and exists as eight genotypes (1 to 8) with distinct geographic distributions. No complete genome sequence of HCV subtype 2b (HCV-2b) is available from Latin American countries, and the factors underlying its emergence and spread within the continent remain unknown. The present study was conducted to determine the first full-length genomic sequences of HCV-2b isolates from Latin America and reconstruct the spatial and temporal diversification of this subtype in Brazil. Nearly complete HCV-2b genomes isolated from two Brazilian patients were obtained by direct sequencing of long PCR fragments and analyzed together with reference sequences using the Bayesian coalescent and phylogeographic framework approaches. The two HCV-2b genomes were 9318 nucleotides (nt) in length (nt 37–9354). Interestingly, the long RT-PCR technique was able to detect the co-circulation of viral variants that contained an in-frame deletion of 2022 nt, encompassing E1, E2, and p7 proteins. Spatiotemporal reconstruction analyses suggest that HCV-2b had a single introduction in Brazil during the early 1980s, displaying an epidemic history characterized by a low and virtually constant population size to date. These results coincide with epidemiological data in Brazil and may explain the low national prevalence of this subtype. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 151 KiB

Open AccessAbstract

The Persistent Infection of Tick Cells by Hazara orthonairovirus Is Mediated by Virus-Derived DNA Forms

by Maria Vittoria Salvati, Claudia Del Vecchio, Lesley Bell-Sakyi, Cristina Parolin, Arianna Calistri, Giorgio Palù, Ali Mirazimi and Cristiano Salata

Proceedings 2020, 50(1), 122; https://doi.org/10.3390/proceedings2020050122 - 6 Jul 2020

Viewed by 1645

Abstract

Introduction: Crimean–Congo hemorrhagic fever (CCHF) is a severe disease of humans caused by CCHF orthonairovirus (CCHFV), a class 4 pathogen. Hyalomma ticks are the viral reservoir, and they represent the main vector. CCHFV can be transmitted to its hosts during tick blood feeding. [...] Read more.

Introduction: Crimean–Congo hemorrhagic fever (CCHF) is a severe disease of humans caused by CCHF orthonairovirus (CCHFV), a class 4 pathogen. Hyalomma ticks are the viral reservoir, and they represent the main vector. CCHFV can be transmitted to its hosts during tick blood feeding. We have previously shown that CCHFV can persistently infect Hyalomma-derived tick cell lines without any cytopathic effect. However, the mechanism allowing for the establishment of a persistent viral infection in ticks is still unknown. It has been recently reported that Hazara orthonairovirus (HAZV) can be used as a BSL-2 model virus instead of CCHFV to study viral/vector interaction. The aim of our study is to elucidate the mechanism that allows establishment of persistent CCHFV infection in ticks using HAZV as a model. Methods: We used classical and molecular methods applied to virology to characterize the establishment of persistent HAZV infection in two Hyalomma anatolicum-derived cell lines—HAE/CTVM8 and HAE/CTVM9. Results: As for CCHFV, we showed that HAZV persistently infects tick cells without any sign of cytopathic effect and that infected cells can be cultured for more than one year. The persistent infection is characterized by a low viral titer compared to the initial time points. Interestingly, short viral-derived DNA forms (vDNAs) start to be detected in parallel with the beginning of viral replication and are maintained in persistently-infected cells. Experiments with the antiretroviral drug AZT suggest that vDNAs are produced by retrotranscriptase activity. Furthermore, we collected evidence that vDNAs are not integrated and seem to be involved in the downregulation of viral replication by promoting cell survival. Conclusion: vDNA synthesis might represent a strategy to control the replication of RNA viruses in ticks, as recently demonstrated in insects, allowing for persistent infection of virus vectors. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 167 KiB

Open AccessAbstract

MARCH8 Restricts Ebola Virus Replication by Blocking the Viral Glycoprotein Processing and Glycosylation

by Changqing Yu, Sunan Li, Omid Madadgar, Iqbal Ahmad, Xianfeng Zhang, Jing Shi, Yu Wang, Yulong Zhou and Yong-Hui Zheng

Proceedings 2020, 50(1), 123; https://doi.org/10.3390/proceedings2020050123 - 7 Jul 2020

Viewed by 1976

Abstract

Ebola virus (EBOV) glycoprotein (GP) is a class I fusion protein whose maturation is dependent on furin-mediated processing. EBOV-GP is heavily glycosylated, with glycans constituting ~50% of its molecular mass. Compared with 15 N-linked glycosylation sites, EBOV-GP₁ has ~80 potential O [...] Read more.

Ebola virus (EBOV) glycoprotein (GP) is a class I fusion protein whose maturation is dependent on furin-mediated processing. EBOV-GP is heavily glycosylated, with glycans constituting ~50% of its molecular mass. Compared with 15 N-linked glycosylation sites, EBOV-GP₁ has ~80 potential O-linked glycosylation sites in the mucin-like domain (MLD), suggesting that O-linked glycans are dominated. The membrane-associated RING-CH (MARCH) family consists of 11 members that are RING-finger ubiquitin E3 ligases. Recently, human MARCH1, MARCH2, and MARCH8 were reported to inhibit HIV-1 replication by targeting its Env. Here, we show that human MARCH8 also inhibits EBOV replication by blocking GP incorporation into virions via downregulating its cell surface expression. To understand how the downregulation occurs, we investigated EBOV-GP subcellular localization, processing, glycosylation, and intracellular trafficking in the presence of human MARCH8. We find that MARCH8 interacts with GP and retains GP in the Golgi. MARCH8 also interacts with the homoB domain of furin that blocks its convertase activity. In consequence, MARCH8 blocks GP processing in an MLD-independent manner. Consistently, MARCH8 also blocks the O-linked, but not the N-linked glycosylation of GP. Importantly, in the presence of MARCH8, the shedding of GP₁ but not the secreted GP (sGP) is blocked, suggesting that MARCH8 targets the GP₁ C-terminal region. The MARCH8 activity is extended to its orthologs from Bos taurus and mice, and its paralogs MARCH1 and MARCH2. In addition, MARCH8 inhibits the processing of two other class I fusion proteins, including HIV-1 Env and IAV HA, and it triggers the degradation of the class III fusion protein VSV-G. We conclude that MARCH8 exerts a very broad and conserved antiviral activity by inhibiting the maturation of class I fusion proteins, which blocks their secretion to the cell surface and incorporation into virions. It should also target class III fusion proteins by triggering their degradation. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 129 KiB

Open AccessAbstract

Single-Particle Tracking Porcine Epidemic Diarrhea Virus Moving along Microtubules in Living Cells

by Yangyang Li, Wei Hou, Jian Wang and Fei Liu

Proceedings 2020, 50(1), 124; https://doi.org/10.3390/proceedings2020050124 - 7 Jul 2020

Viewed by 1412

Abstract

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, has caused severe damage to the swine industry. Although viruses are believed to hijack the microtubule-based transport system, the exact manner of PEDV moving along microtubules has not been fully characterized. [...] Read more.

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, has caused severe damage to the swine industry. Although viruses are believed to hijack the microtubule-based transport system, the exact manner of PEDV moving along microtubules has not been fully characterized. In this study, PEDV was labeled with quantum dots which have great brightness and photostability. By using quantum dot-labeled PEDV and single-particle tracking, we were able to systematically dissect the dynamic behaviors of PEDV moving along the microtubules in living cells. We found that PEDVs maintained a restricted motion mode with a relatively stable speed in the cell membrane region while displaying a slow–fast–slow velocity pattern with different motion modes in the cell cytoplasm region and near the microtubule-organizing center. The return movements of small amounts of PEDVs were also observed in living cells. Collectively, our work is crucial for understanding the movement of PEDV in living cells; the proposed work also provides important references for further analysis and studies of the infection mechanism of PEDV. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 125 KiB

Open AccessAbstract

The Long Road to a Universal Influenza Virus Vaccine

by Peter Palese

Proceedings 2020, 50(1), 125; https://doi.org/10.3390/proceedings2020050125 - 8 Jul 2020

Viewed by 1268

Abstract

Seasonal and pandemic influenza virus infections can cause significant disease worldwide. Current vaccines only provide limited, short-lived protection, and antigenic drift/shift in the hemagglutinin (HA) surface glycoprotein necessitates their annual reformulation and re-administration. To overcome these limitations, universal influenza virus vaccine strategies aim [...] Read more.

Seasonal and pandemic influenza virus infections can cause significant disease worldwide. Current vaccines only provide limited, short-lived protection, and antigenic drift/shift in the hemagglutinin (HA) surface glycoprotein necessitates their annual reformulation and re-administration. To overcome these limitations, universal influenza virus vaccine strategies aim at eliciting broadly protective antibodies to conserved epitopes of the HA. We have developed two approaches. (1) The first is based on “chimeric” HA constructs that retain the conserved stalk domain of the HA and have exotic HA heads. Vaccination and boosting with such constructs successfully redirects the immune system in animals and in humans towards the conserved immune sub-dominant domains of the HA stalks; this results in an antigenic silencing of the HA heads and a protective immune response facilitated by the conserved HA stalks. In mice and ferrets, such a strategy protects the animals against homo-subtypic and hetero-subtypic challenge with influenza A strains as well as against influenza B variants. It is hoped that vaccine constructs expressing three components (i.e., conserved group 1 HA stalks, conserved group 2 HA stalks, and conserved influenza B HA stalks) will be protective against all future seasonal and pandemic strains. (2) The “mosaic” HA approach is based on antigenic silencing of the major immunodominant antigenic sites of the HA heads by only replacing those epitopes with corresponding sequences of exotic avian HAs, yielding “mosaic” HAs. In mice, a prime-boost vaccination regime with inactivated viruses expressing “mosaic” HAs elicited highly cross-reactive antibodies against the stalk domain of the HAs that were capable of eliciting Fc-mediated effector functions in vitro. Extensive trials will be necessary in the future in order to identify the optimal vaccination regime (“chimeric” HA-based versus “mosaic” HA-based) in humans. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 143 KiB

Open AccessAbstract

Teicoplanin Derivatives Impact on West Nile Virus Pathogenesis

by Henrietta Papp, Ilona Bereczki, Pál Herczegh, Mónika Madai, Gábor Kemenesi, Eszter Boglárka Lőrincz, Anikó Borbás and Ferenc Jakab

Proceedings 2020, 50(1), 126; https://doi.org/10.3390/proceedings2020050126 - 8 Jul 2020

Viewed by 1874

Abstract

West Nile virus (WNV) is an emerging arbovirus that causes infections worldwide. Clinical manifestations of the infection vary from asymptomatic to fatal illness when it reaches the central nervous system. To date, vaccine and specific antiviral treatments are not available. Teicoplanin is already [...] Read more.

West Nile virus (WNV) is an emerging arbovirus that causes infections worldwide. Clinical manifestations of the infection vary from asymptomatic to fatal illness when it reaches the central nervous system. To date, vaccine and specific antiviral treatments are not available. Teicoplanin is already used to treat Gram-positive bacterial infections. Furthermore, it has been reported to block the entry of pseudotyped Ebola, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus. Moreover, teicoplanin derivatives showed anti-influenza virus, anti-human immunodeficiency virus, anti-hepatitis C virus, and anti-dengue virus activity. In total, 12 teicoplanin derivatives have been tested against our West Nile virus isolate. Vero E6 cells were simultaneously treated with 50 µM of teicoplanin derivatives and infected with WNV at the same time. Virus-induced cytopathic effect and cytotoxicity were examined 4 days post-infection. One compound completely blocked virus pathogenesis, while five compounds reduced the viral titer. Further studies will be conducted to unravel the mode of action of these promising derivatives. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 144 KiB

Open AccessAbstract

The AP-1 Transcription Factor Is a Key Determinant of Human Cytomegalovirus Latency and Reactivation

by Benjamin A. Krishna, Amanda B. Wass and Christine M. O’Connor

Proceedings 2020, 50(1), 127; https://doi.org/10.3390/proceedings2020050127 - 13 Jul 2020

Viewed by 1393

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic progenitor cells (HPCs). Individuals with a competent immune system are, for the most part, asymptomatic for the disease. However when a latently infected individual becomes immunosuppressed, HCMV can reactivate, causing severe morbidity [...] Read more.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic progenitor cells (HPCs). Individuals with a competent immune system are, for the most part, asymptomatic for the disease. However when a latently infected individual becomes immunosuppressed, HCMV can reactivate, causing severe morbidity and mortality. While much of the viral genome is transcriptionally silenced during latency, some genes are expressed, including the HCMV-encoded G-protein coupled receptor US28. We showed that US28 expression is required for latency, as it suppressed the activator protein-1 (AP-1) transcription factor by attenuating the AP-1 subunit, fos. In turn, this prevents AP-1 from binding and activating the major immediate early promoter (MIEP), the key promoter regulating the latent-to-lytic transcriptional “switch”. Our new data suggest that US28-mediated signaling during latency attenuates the Src-MAPK signaling axis to regulate AP-1. We find that US28 expression suppresses Src, MEK, and ERK, as well as fos phosphorylation and AP-1 binding to the MIEP. Conversely, the pharmacological inhibition of Src, MEK, or ERK in US28Δ-latently infected HPCs suppresses infectious virus production, demonstrating the important role for this signaling axis during latency. Our recent data also reveal that regulating AP-1 is a key determinant in balancing HCMV latency and reactivation. Infection with a virus in which we disrupted the proximal AP-1 binding site in the MIEP (AP-1Δp) leads to reduced AP-1 binding and inefficient viral reactivation compared to wild type. Furthermore, AP-1 is critical for the de-repression of MIEP-driven transcripts and downstream early and late genes, while other immediate early genes remain unaffected. Collectively, these data suggest that AP-1 binding to the MIEP is suppressed during latency, but is required for the efficient transactivation of the MIEP during reactivation. We are currently elucidating US28’s involvement in recruiting AP-1 to the MIEP during reactivation. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 141 KiB

Open AccessAbstract

Neutralizing Antibodies against Crimean–Congo Hemorrhagic Fever Virus Derived from a Human Survivor

by J. Maximilian Fels, Daniel Maurer, Ana I. Kuehne, Dafna M. Abelson, Noel T. Pauli, Andrew S. Herbert, John M. Dye, Robert W. Cross, Thomas W. Geisbert, Leslie Lobel, Zachary A. Bornholdt, Laura M. Walker and Kartik Chandran

Proceedings 2020, 50(1), 128; https://doi.org/10.3390/proceedings2020050128 - 13 Jul 2020

Viewed by 1685

Abstract

Crimean–Congo hemorrhagic fever virus (CCHFV) is an arbovirus belonging to the Nairoviridae family. The virus, as well as ticks of the Hyalomma genus, which serve as its reservoir host, are found in parts of Africa, western Asia, and southern Europe. Following sporadic [...] Read more.

Crimean–Congo hemorrhagic fever virus (CCHFV) is an arbovirus belonging to the Nairoviridae family. The virus, as well as ticks of the Hyalomma genus, which serve as its reservoir host, are found in parts of Africa, western Asia, and southern Europe. Following sporadic zoonotic or human-to-human transmission, infection is characterized by fever, fatigue, vomiting, diarrhea, and in fatal cases, often hemorrhagic symptoms. There are currently no vaccines or targeted treatments available against CCHFV, leading the WHO to declare it a Blueprint priority pathogen in 2017. Here, we report the isolation and characterization of a panel of human monoclonal antibodies (mAbs) against CCHFV. Using a novel soluble Gn/Gc sorting antigen, we were able to isolate memory B cells specific for CCHFV from four convalescent donors. From each patient sample, we were able to derive several potently neutralizing antibodies with IC₅₀ in the nanomolar range as determined by neutralization of CCHFV virus-like particles. Neutralization by candidate hits was also confirmed using authentic CCHFV. We further show that several of the most potently neutralizing mAbs possess a breadth of neutralization spanning three clades of CCHFV strains. These broadly neutralizing mAbs are currently being tested in a mouse model of CCHFV infection, with preliminary results indicating that they have protective potential. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 132 KiB

Open AccessAbstract

Visualizing HCV Core Protein via Fluorescent Unnatural Amino Acid Incorporation

by Leah Jane Fitzgerald Curnew, Kate McNicholas, Bridgette Green, Jackie Barry, Hannah L Wallace, Lingyan Wang, Cassandra Davidson, John P Pezacki and Rodney S Russell

Proceedings 2020, 50(1), 129; https://doi.org/10.3390/proceedings2020050129 - 14 Jul 2020

Viewed by 1504

Abstract

Introduction: Unnatural amino acids (UAAs) share the same basic structure as proteinogenic amino acids. However, UAAs permit additional functions and applications to proteins due to their different side chains. Recent UAA applications include using fluorescent UAAs to label proteins. The UAA [...] Read more.

Introduction: Unnatural amino acids (UAAs) share the same basic structure as proteinogenic amino acids. However, UAAs permit additional functions and applications to proteins due to their different side chains. Recent UAA applications include using fluorescent UAAs to label proteins. The UAA system provides an alternative method to traditional protein labeling mechanisms (antibodies, GFP, and tags, such as HA and HIS), which can affect protein functionality and topology. The purpose of this study was to visualize the hepatitis C virus (HCV) core protein using the fluorescent UAA Anap (3-[(6-acetyl-2-naphthalenyl)amino]-L-alanine). Methods: Huh-7.5 cells were co-transfected with HCV core plasmids containing amber stop codons at various positions throughout the coding sequence and a second plasmid encoding the orthogonal tRNA/synthetase pair that facilitates Anap incorporation. Three days post transfection, cells were stained for core protein and lipid droplets (LDs) and visualized using immunofluorescence or confocal microscopy. Results: We have optimized transfection protocols for the efficient expression of the tRNA/synthetase pair required for Anap incorporation and are able to visualize our core mutant proteins containing Anap. We have successfully substituted Anap into 11 different positions within the core, including substitutions for tryptophan, tyrosine, and phenylalanine residues. In addition, we have shown that our core mutants associate with cellular LDs, suggesting that the incorporation of the UAA did not disrupt core protein expression, stability, or cellular localization. Conclusions: We have demonstrated the establishment of a UAA incorporation system in an HCV protein without any obvious impact on core protein function. The ability to label viral proteins using fluorescent UAAs eliminates the requirement of antibodies or tags for protein visualization. In conclusion, the UAA system is a useful method to study HCV proteins and can potentially be used to label viruses for live cell and animal studies. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 168 KiB

Open AccessAbstract

A Center for the Rapid Analysis of Clinical-Grade Biologics: The Biophysical Characterization of HIV-1 Env Protein

by José A. Lasalde-Dominicci, Abel Baerga-Ortiz, Pearl Akamine, José A. González-Feliciano, Coral Capó-Vélez and Manuel Delgado-Vélez

Proceedings 2020, 50(1), 130; https://doi.org/10.3390/proceedings2020050130 - 15 Jul 2020

Cited by 1 | Viewed by 1424

Abstract

The development of a prophylactic vaccine against the human immunodeficiency virus (HIV) is of paramount importance in the global drive to halt the spread of the virus. Even after the successful discovery and initial testing of a vaccine candidate, there are hurdles associated [...] Read more.

The development of a prophylactic vaccine against the human immunodeficiency virus (HIV) is of paramount importance in the global drive to halt the spread of the virus. Even after the successful discovery and initial testing of a vaccine candidate, there are hurdles associated with production yield, purification strategy, and in vitro stability that may hinder its development as a biological product. The goal of the Clinical Bioreagent Center (CBC) is to streamline the vaccine development pipeline from a promising lead to the clinic, in part by developing state-of-the-art analytical tools to characterize and to quickly monitor the quality of the HIV-1 Env protein, a new vaccine candidate. A method was developed to determine the purity of the HIV-1 Env glycoprotein by capillary electrophoresis that provides higher sensitivity of detection of impurities and better resolution as compared to regular gel electrophoresis. Using an Octet QKe system, host cell protein content was confirmed using a kit that has greater precision and linear range than available kits based on enzyme-linked immunosorbent assay (ELISA). Imaged capillary isoelectric focusing results highlight the charge heterogeneity of the recombinant HIV-1 Env protein. The binding affinity of the broadly neutralizing antibody, 4E10, to the HIV-1 Env protein was determined by biolayer interferometry. The glycan profile obtained by matrix-assisted laser desorption/ionization (MALDI) spectrometry showed that the recombinant HIV-1 Env protein glycans are distinct from SF162 gp140. These analytical tools can be implemented to ensure that the protein expression and purification conditions do not change the integrity, bioactivity, and therapeutic properties of the vaccine. The methods developed here can be qualified with current good manufacturing practices to facilitate their transfer to a biomanufacturing facility. Our experimental tools were developed to monitor the quality of the HIV-1 Env protein, with the goal of boosting production yields to expedite its success onto clinical trials. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 163 KiB

Open AccessAbstract

A Moonlighting microRNA: Mechanism(s) of miR-122-Mediated Viral RNA Accumulation

by Jasmin Chahal, Luca FR Gebert, Hin Hark Gan, Kristin C Gunsalus, Ian J MacRae and Selena M Sagan

Proceedings 2020, 50(1), 131; https://doi.org/10.3390/proceedings2020050131 - 15 Jul 2020

Viewed by 1569

Abstract

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a human-liver-specific microRNA, termed miR-122. miR-122 binds to two sites in the 5' untranslated region (UTR) of the viral genome, and this interaction promotes HCV RNA accumulation. This interaction is important [...] Read more.

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a human-liver-specific microRNA, termed miR-122. miR-122 binds to two sites in the 5' untranslated region (UTR) of the viral genome, and this interaction promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been demonstrated to be efficacious in reducing HCV titers in chronic HCV-infected patients. However, the precise mechanism(s) of miR-122-mediated viral RNA accumulation have remained elusive. We have used biophysical analysis and assays for viral replication in cell culture to understand the interactions between the human Argonaute 2 (hAgo2):miR-122 complex and the HCV genome. In addition, we have analyzed several resistance-associated variants which were isolated from patients who underwent miR-122 inhibitor-based therapy in order to shed light on novel mechanisms of antiviral resistance. Our results provide a new model for miR-122:HCV RNA interactions and demonstrate that miR-122 plays at least three roles in the HCV life cycle: (1) miR-122 acts as an RNA chaperone to suppress an energetically favorable secondary structure and allows the viral internal ribosomal entry site (IRES) to form; (2) miR-122 binding to the 5' terminus protects the genome from the activity of cellular pyrophosphatases (DOM3Z and DUSP11) and subsequent exonuclease-mediated decay; and (3) the Argonaute (Ago) protein at Site 2 makes direct contact with the HCV IRES, enhancing viral translation. In addition, analyses of several resistance-associated variants that were isolated from patients that underwent miR-122 inhibitor-based therapy suggests that mutations in the 5' terminus alter the structure of the 5' UTR in a manner that promotes RNA chaperone activity or viral genome stability, even in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122-mediated viral RNA accumulation and suggest new mechanisms of antiviral resistance which are mediated by changes in RNA structure. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 118 KiB

Open AccessAbstract

A Cross-Reactive Mouse Monoclonal Antibody against Rhinovirus Mediates Phagocytosis In Vitro

by Mohammad Amin Behzadi, James Duehr, Angela Choi, Michael Schotsaert, Adolfo García-Sastre, Peter Palese and Raffael Nachbagauer

Proceedings 2020, 50(1), 132; https://doi.org/10.3390/proceedings2020050132 - 15 Jul 2020

Viewed by 1453

Abstract

Human rhinoviruses (RVs) are the main cause of the common cold worldwide. To date, more than 160 serotypes of the virus have been recognized. These viruses are categorized into three major groups: A, B, and C. There are currently no approved vaccines available [...] Read more.

Human rhinoviruses (RVs) are the main cause of the common cold worldwide. To date, more than 160 serotypes of the virus have been recognized. These viruses are categorized into three major groups: A, B, and C. There are currently no approved vaccines available to prevent infection with RVs. We designed a mouse immunization strategy that aimed to elicit a humoral response against conserved regions of capsid proteins of RV-A viruses. To this end, recombinant DNA plasmids expressing the capsid proteins (VP1-4) and two proteases (2A and 3C) of RV1A, 16, 49, 68, and 71 were engineered. Mice were sequentially vaccinated with these DNA plasmids at three-week intervals. After a final boost with purified whole virus using the RV15 strain, mice spleens were extracted and cells expressing monoclonal antibodies (mAbs) were generated by hybridoma fusion. A total of 98 mAbs with reactivity to different strains of RV-A were isolated. After isotyping, 22 mAbs expressing an IgG Fc-domain were selected for further expansion and purification. Three mAbs showed cross-reactivity against multiple strains of RV-A viruses by ELISA, including 1A, 1B, 15, 16, and 49. Additional mAbs had strain-specific binding patterns, with a surprising number of mAbs showing reactivity to RV15, the strain used for the final vaccination. Using a microneutralization assay, we found that the RV15-specific mAbs, but not the cross-reactive mAbs, were highly neutralizing. Additional testing in a flow cytometry-based antibody-dependent cellular phagocytosis (ADCP) assay revealed a high degree of ADCP activity for one of the cross-reactive mAbs. Epitope mapping of the neutralizing mAbs via escape mutant viruses revealed binding sites with a shared epitope on VP1 of RV15. The epitope of the ADCP-active, non-neutralizing mAb was determined by the microarray analysis of cyclic constrained peptides generated from the VP1 capsid protein. This study identified a cross-reactive mAb that mediates phagocytosis. These findings could be used toward the development of vaccines against RV. The full study results have since been published (https://doi.org/10.1038/s41598-020-66600-x). Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 142 KiB

Open AccessAbstract

The Role of DNA Repair Complex DNA-PK in HIV-1 Transcription

by Olga Shadrina, Andrey Anisenko and Marina Gottikh

Proceedings 2020, 50(1), 133; https://doi.org/10.3390/proceedings2020050133 - 16 Jul 2020

Viewed by 1428

Abstract

The human DNA-dependent protein kinase (DNA-PK), composed of the heterodimeric protein Ku and catalytic subunit DNA-PKcs, is a sensor of double-strand DNA breaks in the non-homologous end-joining DNA repair pathway. The key role of DNA-PK in the post-integrational repair of HIV-1 has been [...] Read more.

The human DNA-dependent protein kinase (DNA-PK), composed of the heterodimeric protein Ku and catalytic subunit DNA-PKcs, is a sensor of double-strand DNA breaks in the non-homologous end-joining DNA repair pathway. The key role of DNA-PK in the post-integrational repair of HIV-1 has been shown. It has also been suggested that DNA-PK can participate in the regulation of HIV transcription, although the mechanism is unclear. To clarify the impact of each DNA-PK subunit on the transcription of HIV-1, HEK 293T cells, in which each of the DNA-PK components was depleted, were transfected with reporter vectors containing firefly luciferase under the control of HIV LTR promoter. We detected a positive influence of both Ku subunits, but not of DNA-PKcs, on the transcription from the HIV promoter. Ku is known to interact with HIV-1 TAR RNA, playing an essential role in viral transcription; nonetheless, the deletion of the TAR-coding region from LTR did not alter the Ku effect. Human small noncoding 7SK RNA participates in HIV-1 transcription. The direct binding of recombinant Ku and in vitro transcribed 7SK RNA was demonstrated using EMSA. In addition, we identified the interactions of endogenous Ku with proteins HEXIM1 and Cdk9 from the 7SK RNP complex. These results suggest that Ku exerts its effects on HIV-1 transcription via interaction with the 7SK RNP complex. However, we cannot rule out an indirect effect of Ku on transcription via the regulation of the levels of some transcription factors participating in HIV-1 transcription. We performed a transcriptome analysis of wild type HEK 293T cells and those with depleted DNA-PK subunits. The genes regulated by each subunit were defined and the genes that were mainly dependent on Ku subunits were selected. Among them, we identified transcription factors enhancing HIV-1 transcription, whose levels were downregulated in Ku-depleted cells. The study was supported by RFBR grant №18-04-00542 and RSF grant №17-14-01107. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 154 KiB

Open AccessAbstract

Influenza Virus Usurps an Interferon-Induced Translational Program to Promote Viral Replication

by Mitchell P. Ledwith, Vy Tran, Thiprampai Thamamongood, Christina A. Higgins, Shashank Tripathi, Max W. Chang, Christopher Benner, Adolfo García-Sastre, Martin Schwemmle, Adrianus C. M. Boon, Michael S. Diamond and Andrew Mehle

Proceedings 2020, 50(1), 134; https://doi.org/10.3390/proceedings2020050134 - 21 Jul 2020

Viewed by 1649

Abstract

Hosts mount prudently tuned responses to viral infection in an attempt to block nearly every step of the replication cycle. Viruses must adapt to replicate in this hostile antiviral cellular state. Interferon stimulation or pathogen challenge robustly induces expression of IFIT (interferon-induced proteins [...] Read more.

Hosts mount prudently tuned responses to viral infection in an attempt to block nearly every step of the replication cycle. Viruses must adapt to replicate in this hostile antiviral cellular state. Interferon stimulation or pathogen challenge robustly induces expression of IFIT (interferon-induced proteins with tetratricopeptide repeats) proteins. IFITs are a family of proteins that bind RNA and play antiviral roles during infection. Thus, we were surprised to identify the IFIT family as top candidate proviral host factors for influenza A virus (IAV) in a genome-wide CRISPR–Cas9 knockout screen. We validated the proviral activity of IFIT2 by showing that IFIT2-deficient cells support lower levels of IAV replication and exhibit defects in viral gene expression. The molecular functions of IFIT2, let alone how they are used by influenza virus, are unknown. Using CLIP-seq, we showed that IFIT2 binds directly to viral and cellular mRNAs in AU-rich regions largely in the 3’UTR, with a preference for a subset of interferon-stimulated mRNAs. IFIT2 also associates with actively translating ribosomes in infected cells to facilitate the translation of viral messages. IFIT2-responsive elements from an IAV mRNA were sufficient to confer translational enhancement to exogenous transcripts in cis. Conversely, mutation of these elements or the use of an IFIT2 RNA-binding mutant ablated stimulation of viral gene expression. Together, these data link the RNA-binding capability of IFIT2 to changes in translational efficiency of target viral mRNAs and the stimulation of viral replication. They establish a model for the normal function of IFIT2 as an antiviral protein affecting the post-transcriptional fate of cellular mRNAs and explain how influenza virus repurposes IFIT2 to support viral replication. Our work highlights a new node for the regulation of translation during interferon responses and highlights how canonical antiviral responses may be repurposed to support viral replication. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 170 KiB

Open AccessAbstract

The Characteristics of New SSB Proteins from Metagenomic Libraries and Their Use in Biotech Applications

by Olesia Werbowy, Aleksandra Stefańska-Kaźmierczak, Agata Jurczak-Kurek, Karolina Kwiatkowska-Semrau, Anna-Karina Kaczorowska, Magdalena Płotka, Olafur H. Fridjonsson, Gudmundur O. Hreggvidsson, Slawomir Dąbrowski and Tadeusz Kaczorowski

Proceedings 2020, 50(1), 135; https://doi.org/10.3390/proceedings2020050135 - 21 Jul 2020

Viewed by 1969

Abstract

Single-stranded DNA binding proteins (SSBs) bind to single-stranded DNA in a sequence-independent manner to prevent formation of secondary structures and protect DNA from nuclease degradation. These ubiquitous proteins are present in prokaryotes, eukaryotes, and viruses, and play a pivotal role in the following [...] Read more.

Single-stranded DNA binding proteins (SSBs) bind to single-stranded DNA in a sequence-independent manner to prevent formation of secondary structures and protect DNA from nuclease degradation. These ubiquitous proteins are present in prokaryotes, eukaryotes, and viruses, and play a pivotal role in the following major cellular processes: replication, recombination, and repair of genetic material. In DNA replication, SSB proteins specifically stimulate DNA polymerase, increase fidelity of DNA synthesis, assist the advance of DNA polymerase, and organize and stabilize replication forks. Here, we present our characterization of four SSB proteins of different origins. One of them was isolated from Clostridium sp. phage phiCP130 (SSB C1: 124 aa, Mr = 13,905). Three others (SSB M2: 136 aa, Mr = 15,009; SSB M3: 144 aa, Mr = 16,106; and SSB M5: 138 aa, Mr = 15,851) were isolated from metagenomics libraries. They show high similarity to SSB proteins from Caldanaerovirga acetigigens, Caldanaerobius fijiensis, and Fervidobacterium gondwanense. The recombinant proteins were overproduced in E. coli Rosetta (pRARE), except for SSB M5, which was overproduced in E. coli BL21. Proteins were purified using a metal-affinity chromatography as His-tagged fusion proteins. Electrophoretic mobility shift assay was used to examine their DNA binding activity with fluorescein-labeled oligonucleotide (dT40) used as a substrate. Thermal stability analysis revealed that they are stable at elevated temperatures, with the exception of SSB protein C1, which loses its activity above 65 °C. The other proteins are active at high temperatures, SSB M3 up to 85 °C, while SSB M2 and SSB M5 are active up to 98.7 °C. The subunit structure of proteins was analyzed by gel filtration on Superdex 75 column (AKTA). This allowed us to conclude that in solution, the analyzed proteins exist in oligomeric form, a feature which is characteristic of other SSB proteins. Purified SSB proteins were tested to improve specificity of PCR-based DNA amplification. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 134 KiB

Open AccessAbstract

Metabarcoding-Like Approach for High Throughput Detection and Identification of Viral Nucleic Acids

by Alina Matsvay, Daniel Kiselev, Andrey Ayginin, Ivan Abramov, Vladimir Dedkov, German Shipulin and Kamil Khafizov

Proceedings 2020, 50(1), 136; https://doi.org/10.3390/proceedings2020050136 - 22 Jul 2020

Viewed by 1872

Abstract

Next generation sequencing (NGS) technologies have greatly enhanced our ability to identify new viral pathogens in various types of biological samples. This approach has led to the discovery of new viruses and has revealed hidden associations of viromes with many diseases. However, unlike [...] Read more.

Next generation sequencing (NGS) technologies have greatly enhanced our ability to identify new viral pathogens in various types of biological samples. This approach has led to the discovery of new viruses and has revealed hidden associations of viromes with many diseases. However, unlike the 16S rRNA, which allows for bacterial detection by metabarcoding, the diversity and variability of viral genomes render the creation of universal oligonucleotides for targeting all known and novel viruses impossible. While wholegenome sequencing solves this problem, its efficiency is inadequate due to the high cost per sample and relatively low sensitivity. Furthermore, the existing approaches to designing oligonucleotides for targeted PCR enrichment are usually incomprehensive, being oriented at detecting a particular viral species or a genus based on the presumption of its presence in the sample. In this study, we developed a computational pipeline for designing genus-specific oligonucleotides that would simultaneously cover a multitude of known viruses from different taxonomic groups. This new tool was used to design an oligonucleotide panel for targeted enrichment of viral nucleic acids in different types of samples, and its applicability for the detection of multiple viral genera at once was demonstrated. Next, we created a custom protocol for NGS library preparation adapted to the new primer panel, which was tested together on a number of samples and proved highly efficient in pathogen detection and identification. Since a reliable algorithm for bioinformatic analysis is crucial for rapid classification of the sequences, in this work, we developed an NGSbased data analysis module and demonstrated its functionality both for detecting novel viruses and analyzing virome diversity. This work was supported by an RSF (Russian Science Foundation) grant (No. 17-74-20096). Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 113 KiB

Open AccessAbstract

Taking Advantage of Nature’s Benefits: Soluble and Stable Antigen Straight Out of the Pathogen

by Didier Clénet, Léna Clavier, Benoit Strobbe, Christel Le Bon, Manuela Zoonens and Aure Saulnier

Proceedings 2020, 50(1), 137; https://doi.org/10.3390/proceedings2020050137 - 23 Jul 2020

Viewed by 1483

Abstract

Integral membrane proteins (MP) exhibit specific tridimensional conformation and topology that define their various functions. Pathogen surface antigens, encompassing many MP, are at the forefront of the viral strategy which is broadly targeted by the host immune response. These antigens are present in [...] Read more.

Integral membrane proteins (MP) exhibit specific tridimensional conformation and topology that define their various functions. Pathogen surface antigens, encompassing many MP, are at the forefront of the viral strategy which is broadly targeted by the host immune response. These antigens are present in equilibrium under different oligomeric forms with distinctive epitopes, and to obtain them in a soluble form and/or stable constitutes a real risk. The solubilization of a full-length MP directly from a pathogen to rapidly obtain a native antigen mimicking the original conformation of the MP at the pathogen surface is the process development reported in this work. Rabies virus (RABV) was used as a model for this demonstration and its full-length glycoprotein (G) was stabilized in amphiphatic polymers (A8-35 amphipols). The stability of the soluble RABV-G was evaluated under various stress conditions (temperatures, agitation and light exposures) and a long-term stable RABV-G formulation, suitable for the freeze-drying process, was defined using a design of experiment approach. RABV-G/A8-35 in liquid form was shown to be antigenically stable at 5 °C and 25 °C for one month, and a dedicated kinetic model predicted its stability up to 1 year at 5 °C. To mitigate the RABV-G/A8-35 sensitivity to mechanical stress, a solid form of RABV-G/A8-35 and a freeze-drying process were considered, resulting in a 2-year thermally stable product at 5 °C, 25 °C and 37 °C. To the best of our knowledge, this is the first time that a natural full-length MP, extracted from a virus and trapped in amphipols, was kept antigenically stable in the long term, in a defined freeze-dried form out of any refrigerated storage conditions. These results described an easy process to obtain a pure, well conformed native-like antigen of interest, from a circulating pathogen which is of concern for diagnostic (quantification/characterization assays), therapeutic and vaccine strategies. After the physical characterization of the protein, the identification of RABV G/A8-35 neutralizing epitopes has been underway before in vivo testing. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 142 KiB

Open AccessAbstract

Zika Virus Diversity Is Maintained during Transmission from Placenta to Fetal Periphery but Restricted in Fetal Brains

by Alyssa B. Evans, Clayton W. Winkler, Sarah L. Anzick, Stacy M. Ricklefs, Dan E. Sturdevant and Karin E. Peterson

Proceedings 2020, 50(1), 138; https://doi.org/10.3390/proceedings2020050138 - 26 Jul 2020

Viewed by 1311

Abstract

Since emerging in French Polynesia and Brazil in the 2010s, Zika virus (ZIKV) has been associated with fetal congenital disease. Previous studies have compared ancestral and epidemic ZIKV strains to identify strain differences that may contribute to vertical transmission and fetal disease. However, [...] Read more.

Since emerging in French Polynesia and Brazil in the 2010s, Zika virus (ZIKV) has been associated with fetal congenital disease. Previous studies have compared ancestral and epidemic ZIKV strains to identify strain differences that may contribute to vertical transmission and fetal disease. However, within-host ZIKV variation during vertical transmission has not been well studied. Here, we used the established anti-interferon-treated Rag1−/− mouse model of ZIKV vertical transmission to compare ZIKV populations in matched placentas, fetal bodies, and fetal brains via RNASeq. ZIKV transmission from the placenta to the fetal periphery involved a loose population bottleneck. There was a restriction in the amount of virus entering the fetus from the placenta but not in ZIKV diversity, as the ZIKV population structures were similar in placentas and fetal bodies. In contrast, ZIKV transmission from the fetal periphery to the brain involved a sharp reduction in ZIKV diversity. All fetal brain ZIKV populations were comprised of either one of two variants as largely homogenous populations. In most cases, the predominant variant present in the fetal brain was also the majority variant present in the placenta. However, in two of ten fetal brains, the predominant ZIKV variants were undetectable in the matched placental ZIKV population, suggesting possible evidence of selection for certain variants during ZIKV transmission to fetal brains. Thus, certain variants may influence ZIKV’s ability to enter the fetal brain and cause disease. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 147 KiB

Open AccessAbstract

A Novel System to Study Dengue Virus Replication Organelle Formation Independent from Viral RNA Replication

by Berati Cerikan, Sarah Goellner, Christopher John Neufeldt, Uta Haselmann, Mirko Cortese and Ralf Bartenschlager

Proceedings 2020, 50(1), 139; https://doi.org/10.3390/proceedings2020050139 - 7 Aug 2020

Viewed by 1676

Abstract

Positive-strand RNA viruses, such as dengue virus (DENV), induce the extensive rearrangement of intracellular membranes that serve as a scaffold for the assembly of the viral replication machinery. In the case of DENV, the main endomembrane ultrastructure produced in infected cells consists of [...] Read more.

Positive-strand RNA viruses, such as dengue virus (DENV), induce the extensive rearrangement of intracellular membranes that serve as a scaffold for the assembly of the viral replication machinery. In the case of DENV, the main endomembrane ultrastructure produced in infected cells consists of invaginations of the endoplasmic reticulum, designated vesicle packets (VPs), which are the assumed sites of viral RNA replication. VPs are observed as arrays of vesicles surrounded by an outer membrane, the formation of which is induced by the viral nonstructural proteins, presumably in conjunction with specific host factors. However, little is known about the mechanisms governing VP formation, which is mainly due to the lack of a replication-independent system supporting the biogenesis of these membranous structures. Here we describe an expression-based, viral RNA replication-independent, DENV polyprotein system, designated as pIRO (plasmid-induced replication organelle), which is sufficient to induce VP formation. We show that VPs induced by pIRO expression are morphologically indistinguishable from those found in infected cells, suggesting that DENV replication organelle formation does not require RNA replication. We conclude that the pIRO system is a novel and valuable tool that can be used to dissect the mechanisms underlying DENV replication organelle formation. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 156 KiB

Open AccessAbstract

4D Analyses Show That Replication Compartments Are Clonal Factories in Which Epstein–Barr Viral DNA Amplification Is Coordinated

by Thejaswi Nagaraju, Arthur Sugden and Bill Sugden

Proceedings 2020, 50(1), 140; https://doi.org/10.3390/proceedings2020050140 - 7 Aug 2020

Viewed by 1323

Abstract

Most DNA viruses must amplify their DNA to form new viral particles. To kickstart their DNA amplification, herpesviruses alter the host cell cycle dynamics by halting G1/S progression. Soon after, the viruses begin amplifying their DNA and halt any detectable cellular DNA synthesis. [...] Read more.

Most DNA viruses must amplify their DNA to form new viral particles. To kickstart their DNA amplification, herpesviruses alter the host cell cycle dynamics by halting G1/S progression. Soon after, the viruses begin amplifying their DNA and halt any detectable cellular DNA synthesis. Viral DNA amplification takes place in specialized regions of the cell known as replication compartments. The genesis and maturation of replication compartments are not well understood. While replication compartments can only be visualized via microscopy, examining DNA synthetic events requires ensemble approaches. We have therefore exploited single-cell assays, including live-cell imaging, fluorescence in situ hybridization (FISH), and EdU-pulse labeling, in combination with computational simulations and ensemble approaches, to study the role of replication compartments in the DNA amplification of the Epstein–Barr virus (EBV). FISH revealed that each replication compartment initially contained a single DNA molecule which did not travel between compartments. DNA amplification lasted for 13–14 h in single cells, as shown by live cell imaging. Replication compartments eventually grew to occupy 30% of the nucleus, which itself grew by 50%. We found that early in the lytic phase, the availability of DNA templates limited DNA synthesis, while late in the lytic phase, the majority of viral DNA molecules no longer served as templates, which correlated with a drop in the levels of the replication protein. The eventual decline in DNA synthesis did not result from encapsidation; only 1–2% of the viral DNA was encapsidated. The levels of viral DNA synthesis in each compartment were similar. Therefore, the number of compartments determined the total amount of DNA synthesized and, consequently, the levels of amplified DNA. This finding was predicted by computational simulations of the amplification of the two distinct EBV derived replicons that we studied. Our results establish that replication compartments represent clonal factories for DNA amplification that are regulated coordinately during the lytic phase. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 130 KiB

Open AccessAbstract

Functional and Structural Characterization of Novel Insect-Restricted Negev Viruses and Their Interaction with Host Cells

by Alexander Rouvinski

Proceedings 2020, 50(1), 141; https://doi.org/10.3390/proceedings2020050141 - 10 Aug 2020

Viewed by 1469

Abstract

Many viruses transmitted by blood-sucking arthropods have emerged worldwide and cause serious human and animal diseases. Arthropod-borne (arbo-)viruses possess a peculiar capacity to replicate in very different hosts, such as mosquitoes and mammals (e.g., dengue and chikungunya). However, the molecular mechanisms that enable [...] Read more.

Many viruses transmitted by blood-sucking arthropods have emerged worldwide and cause serious human and animal diseases. Arthropod-borne (arbo-)viruses possess a peculiar capacity to replicate in very different hosts, such as mosquitoes and mammals (e.g., dengue and chikungunya). However, the molecular mechanisms that enable arboviruses to enter and replicate in very different hosts—such as arthropods, mammals, and birds—remain highly enigmatic. Insect-restricted viruses provide a reference point regarding this issue, since they lack the strategies of infecting vertebrate hosts and are viewed as evolutionary precursors of arboviruses. Negev viruses comprise a recently discovered insect-restricted taxon exhibiting worldwide distribution among blood sucking arthropods. Negev viruses are enveloped, positive sense, nonsegmented RNA viruses. The virus genome is ca. 10 kb long and contains three ORFs, with the longest ORF1 (7 kb) encoding putative replicative enzymes. ORF2 (1.2 kb) and ORF3 (0.7 kb) do not have any clear homologs and are predicted to encode membrane proteins. We explored the functional and structural aspects of Negev viruses and the role of their membrane glycoproteins during Negev virus entry into the mosquito cells. We employ biochemistry, structural biology, and microscopy to reveal mechanisms of Negev virion organization, and its entry into host cells followed by fusion with the host membranes to allow subsequent genome delivery and replication. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 137 KiB

Open AccessAbstract

Unique Features of Immunity within the Immunoglobulin Heavy Chain Locus of Egyptian Rousette Bats

by Maggie L. Bartlett, Peter A. Larson, Mariano Sanchez-Lockhart and Gustavo Palacios

Proceedings 2020, 50(1), 142; https://doi.org/10.3390/proceedings2020050142 - 11 Aug 2020

Viewed by 1443

Abstract

Marburg virus (MARV) presents with a hemorrhagic fever in primates but asymptomatically in its known reservoir, the Egyptian rousette bat (Rousettus aegyptiacus, ERB). Understanding the biological mechanisms that explain these differential outcomes could be used to develop efficient therapeutics [...] Read more.

Marburg virus (MARV) presents with a hemorrhagic fever in primates but asymptomatically in its known reservoir, the Egyptian rousette bat (Rousettus aegyptiacus, ERB). Understanding the biological mechanisms that explain these differential outcomes could be used to develop efficient therapeutics against MARV disease in humans. Since one of the antiviral mechanisms to control viruses is the humoral response, we hypothesize that the B cell repertoire is unique to primates and contributes to the ERB’s ability to overcome MARV infection. Immunoglobulin (Ig) heavy and light chains undergo DNA rearrangement to generate a diverse repertoire. To be able to study B cell rearrangement, the accurate annotation of the Ig heavy chain (IGH) locus is needed. We implemented three complementary strategies to describe and annotate the IGH locus of ERBs. First, we identified and annotated genes at the IGH locus, utilizing the previously described genome and transcriptome of the ERB our group created in collaboration with the CDC and the University of Boston. Second, we sequenced the specific IgM transcriptome of B cells from ERB peripheral blood mononuclear cells (PBMCs), to confirm or identify new IGH germline genes. Third, we generated bacterial artificial chromosome (BAC) libraries to confirm and improve the layout of the IGH locus. We were able to resolve misassemblies of these regions and identify multiple gene expansions unique to ERBs that may contribute to their ability to generate B cell diversity and control infections. We found an expansion of genes associated with protection from various viruses in humans, differential expression of ERB isotypes across tissues, and two functional IgE genes. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 172 KiB

Open AccessAbstract

Induction of the Type I IFN Response by Human Metapneumovirus Lacking SH, G, or M2.2 Expression

by Kevin Groen, Stefan van Nieuwkoop, Ron Fouchier and Bernadette van den Hoogen

Proceedings 2020, 50(1), 143; https://doi.org/10.3390/proceedings2020050143 - 11 Aug 2020

Viewed by 1316

Abstract

The human metapneumovirus (HMPV), a member of the Pneumoviridae family, is a major cause of respiratory illness, primarily in young children, the elderly, and immunocompromised individuals. Having a fundamental understanding of the viral evasion of innate immune responses is crucial for the rational [...] Read more.

The human metapneumovirus (HMPV), a member of the Pneumoviridae family, is a major cause of respiratory illness, primarily in young children, the elderly, and immunocompromised individuals. Having a fundamental understanding of the viral evasion of innate immune responses is crucial for the rational design of antiviral therapies. Several studies have reported on how HMPV subverts innate immune responses, with roles for SH, G, and M2.2 proteins. However, these studies often conflict. It has also been reported that eliminating the M2.2 ORF results in insertions and deletions around the M2.2 ORF, which could result in an M2.2-independent interaction with the immune system. We aimed to investigate how HMPV interacts with the innate immune response. Therefore, recombinant viruses lacking M2.2, SH, or G protein expression were generated either by deletion or by ablation of protein expression through mutations. Phenotypic analysis revealed that viruses lacking M2.2 expression are attenuated on interferon-competent A549 cells, but not on interferon-deficient cells. Deletion of ORFs compared to ablation of expression through mutations did not result in differences in replication kinetics. Viruses lacking M2.2 expression induced interferon-ẞ protein production, indicating interferon-antagonistic functions of the M2.2 protein, as previously reported. Phenotypic analysis of A549 cells knocked out for RIG-I, MAVS, and PKR revealed the role of RIG-I in the immune response towards HMPV. Next-generation sequencing analysis of viruses lacking M2.2 expression but not G or SH expression showed hypermutation throughout the virus genome. The hypermutation patterns suggest a role for adenosine deaminase acting on RNA (ADAR) editing. We addressed the question of whether RIG-I activation by viruses lacking M2.2 expression is due to hypermutated genomes or the absence of M2.2 as an interferon antagonist. Additionally, we investigated the role of ADAR in HMPV infection. We present our data on the possible influence of ADAR in HMPV infection by next-generation sequencing of viral stocks in cell knockdowns of ADAR generated by CRISPR-interference. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 142 KiB

Open AccessAbstract

Characterization of Glycoproteins from Insect-Specific Goutanap and Negev Viruses

by Kathakali Das, Miri Stolovich-Rain, Leora Gidon, Sujata Kumari, Tomer Schlosser, Manoj Kumar, Reuven Wiener and Alexander Rouvinski

Proceedings 2020, 50(1), 145; https://doi.org/10.3390/proceedings2020050145 - 17 Aug 2020

Viewed by 1354

Abstract

Negeviruses (NVs) are a recently discovered taxon of enveloped, positive sense, single-stranded RNA viruses, infecting blood-sucking insects. While classical arthropod-borne (arbo)viruses like dengue and Chikungunya infect both insects and vertebrates, NVs are restricted to insects and do not have any known vertebrate host [...] Read more.

Negeviruses (NVs) are a recently discovered taxon of enveloped, positive sense, single-stranded RNA viruses, infecting blood-sucking insects. While classical arthropod-borne (arbo)viruses like dengue and Chikungunya infect both insects and vertebrates, NVs are restricted to insects and do not have any known vertebrate host and are thus classified as insect-restricted viruses. Previous works have predicted a structure consisting of three ORFs, the first with homologous regions to RNA-dependent RNA polymerase, helicase, and methyl transferases in plant viruses. On the contrary, ORF2 and ORF3 do not have homologs and are predicted to encode membrane glycoproteins. Their structures, functions, and significance remain vague. We focus on the characterization of the viral proteins, structural organization of the virion, and the principles of their interaction with the host cell. We purified the virion particles of Negev virus produced in mosquito cells and identified its structural components. In addition, we cloned and overexpressed ORF2 and ORF3 of Negeviruses. Furthermore, we defined and successfully produced and purified recombinant ORF2. Subsequent characterization using gel filtration, ion exchange, and MALS techniques revealed that the ORF2 of Negeviruses exhibit different higher order assembly patterns: dimerization and multimerization in a concentration- and pH-dependent manner that correspond to their biological role. We combine biochemical, structural and cell biology techniques to unravel mechanisms of Negev virus interaction with the host cell. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 138 KiB

Open AccessAbstract

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

by Shuo Zhang, Harish N. Ramanathan, Florian Douam, Katrina B. Mar, Jinhong Chang, Priscilla L. Yang, John W. Schoggins, Alexander Ploss and Brett D. Lindenbach

Proceedings 2020, 50(1), 147; https://doi.org/10.3390/proceedings2020050147 - 27 Aug 2020

Viewed by 1412

Abstract

Flaviviruses are enveloped, arthropod-borne, positive-strand RNA viruses that cause significant human disease. While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We recently developed a sensitive, [...] Read more.

Flaviviruses are enveloped, arthropod-borne, positive-strand RNA viruses that cause significant human disease. While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We recently developed a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter to quantitively monitor the translation of incoming virus particle-delivered genomes. We validated that viral gene expression can be neutralized by YFV-specific antisera and requires known pathways of flavivirus entry; however, as expected, gene expression from the defective reporter virus was insensitive to a small molecule inhibitor of YFV RNA replication. The initial round of viral gene expression was also shown to require: (i) cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur, and (ii) valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large macromolecular complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 158 KiB

Open AccessAbstract

A Mycovirus Mediates the Virulence of an Insect-Killing Fungus against the Malaria Mosquito Vector

by Andre N. Pitaluga, Charalampos Filippou, Josephine Blakiston, Robert H.A. Coutts, George K. Christophides and Ioly Kotta-Loizou

Proceedings 2020, 50(1), 148; https://doi.org/10.3390/proceedings2020050148 - 27 Aug 2020

Cited by 2 | Viewed by 2171

Abstract

The cosmopolitan insect-pathogenic fungus and popular biocontrol agent Beauveria bassiana can be used to control Anopheles mosquito populations and restrict the spread of malaria, the deadliest vector-borne infectious disease in the world caused by the protozoan parasite Plasmodium. Here, we establish that [...] Read more.

The cosmopolitan insect-pathogenic fungus and popular biocontrol agent Beauveria bassiana can be used to control Anopheles mosquito populations and restrict the spread of malaria, the deadliest vector-borne infectious disease in the world caused by the protozoan parasite Plasmodium. Here, we establish that infection with a double-stranded (ds)RNA mycovirus, Beauveria bassiana polymycovirus (BbPmV)-1, significantly reduces B. bassiana virulence against A. coluzzii, the main vector of malaria. The BbPmV-1-mediated hypovirulence can be at least partially attributed to slow fungal growth on the mosquitos. Analysis of the dual next-generation sequencing of the B. bassiana and A. coluzzii transcriptomes provided insight into the molecular mechanisms of the BbPmV-1-mediated effects. BbPmV-1-free B. bassiana has a wide impact on the A. coluzzii transcriptome, affecting immunity and metabolism, and led to the identification of novel immune response proteins. BbPmV-1 regulates the gene expression profile of its fungal host, directing the use of available resources towards sporulation and suppressing the mosquito immune system. Additionally, BbPmV-1-infected and -free B. bassiana strains differentially modulate mosquito gut microbiota; the former reduces the bacterial genus Elizabethkingia and the latter Serratia. Co-transfection of mosquitos with B. bassiana and P. berghei revealed a reduction of ookinetes in the presence of BbPmV-1, potentially due to the upregulation of a mycotoxin. Finally, BbPmV-1-mediated hypovirulence is at least partially dependent on the A. coluzzii RNAi pathway, and silencing of the dicer-2 gene restores virulence. Taken together, our data clearly demonstrate the crucial role of mycovirus infection in mediating B. bassiana virulence against A. coluzzii and suggest that BbPmV-1 protects A. coluzzii from B. bassiana, the mosquito’s own immune system, potentially harmful gut microbiota, and Plasmodium parasites. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 140 KiB

Open AccessAbstract

A Genome-Wide CRISPR Activation Screen Identifies Genes Involved in Protection from Zika Virus Infection

by Anna Dukhovny, Kevin Lamkiewicz, Qian Chen, Markus Fricke, Nabila Jabrane-Ferrat, Manja Marz, Jae U. Jung and Ella Hava Sklan

Proceedings 2020, 50(1), 149; https://doi.org/10.3390/proceedings2020050149 - 27 Aug 2020

Cited by 2 | Viewed by 1565

Abstract

Zika virus (ZIKV) is an arthropod-borne emerging pathogen causing febrile illness. ZIKV is associated with the Guillain–Barré syndrome and other neurological complications. The vertical transmission of ZIKV can cause fetus demise, stillbirths or severe congenital abnormalities and neurological complications. There is still no [...] Read more.

Zika virus (ZIKV) is an arthropod-borne emerging pathogen causing febrile illness. ZIKV is associated with the Guillain–Barré syndrome and other neurological complications. The vertical transmission of ZIKV can cause fetus demise, stillbirths or severe congenital abnormalities and neurological complications. There is still no vaccine or specific treatment for ZIKV infection. To identify the host factors that can rescue cells from ZIKV infection, we used a genome-scale CRISPR activation screen. Our highly ranking hits included a short list of interferon-stimulated genes (ISGs) previously reported to have antiviral activity. Validation of the screen results highlighted interferon lambda 2 (IFN-lamda2) and interferon alpha-inducible protein 6 (IFI6) as genes providing high levels of protection from ZIKV infection. The activation of these genes had an effect at an early stage in the viral infection. In addition, infected cells expressing single guide RNAs (sgRNAs) for both of these genes displayed lower levels of cell death than did the controls. Furthermore, the identified genes were significantly induced in ZIKV-infected placenta explants. These results highlighted a set of ISGs directly relevant for rescuing cells from ZIKV infection or its associated cell death, thus substantiating CRISPR activation screens as a valid tool for identifying host factors impeding pathogen infection. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

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2 pages, 153 KiB

Open AccessExtended Abstract

Identification of Novel Determinants of Neutralization Epitope Shielding for Hepatitis C Virus in Vitro

by Garazi Peña Alzua, Anne Finne Pihl, Anna Falden Offersgaard, Rodrigo Velázquez-Moctezuma, Elias Honeroed Augestad, Ulrik Fahnøe, Christian Kjaerulff Mathiesen, Mansun Law, Jannick Prentoe, Jens Bukh and Judith Margarete Gottwein

Proceedings 2020, 50(1), 5; https://doi.org/10.3390/proceedings2020050005 - 2 Jun 2020

Viewed by 1446

Abstract

Epitope shielding is suggested as an important mechanism mediating the escape of hepatitis C virus (HCV) from host-neutralizing antibodies (nAb). [...]
Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

2 pages, 196 KiB

Open AccessExtended Abstract

CRISPR/Cas9 Editing of Viral Receptors and Biotechnological Approach to Host Resistance

by Jiří Hejnar, Anna Koslová, Pavel Trefil, Jiří Plachý, Markéta Reinišová, Dana Kučerová, Jitka Mucksová and Jiří Kalina

Proceedings 2020, 50(1), 22; https://doi.org/10.3390/proceedings2020050022 - 8 Jun 2020

Viewed by 1127

Abstract

Avian sarcoma and leukosis virus (ASLV), diversified into seven phylogenetically relative
subgroups (A, B, C, D, E, J, and K), present as either exogenous or endogenous viruses in domestic
chicken. [...] Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 154 KiB

Open AccessExtended Abstract

African Swine Fever Virus Multigene Family Genes Inhibit the Type-I Interferon Response by Acting on the NFκB and IRF3 Signalling Pathways at the Transcription Factor Level or below

by Samuel Connell, Ana Reis, Anusyah Rathakrishnan, Sarah Gilbert and Linda Dixon

Proceedings 2020, 50(1), 57; https://doi.org/10.3390/proceedings2020050057 - 15 Jun 2020

Viewed by 1342

Abstract

African Swine Fever Virus (ASFV) is a haemorrhagic infection of swine, which routinely
displays 100% lethality. [...] Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 132 KiB

Open AccessProceeding Paper

Knock Out of Cell Death Pathway Components Results in Differential Caspase Expression in Response to HCV Infection

by Hannah L. Wallace, Lingyan Wang, Cassandra Davidson, Vipin Chelakkot, Michael Grant, John Pezacki, Kensuke Hirasawa and Rodney S. Russell

Proceedings 2020, 50(1), 144; https://doi.org/10.3390/proceedings2020050144 - 13 Aug 2020

Viewed by 1534

Abstract

Introduction: Pyroptosis (inflammatory programmed cell death) is induced after the activation of an inflammasome, ultimately resulting in pore formation and cell lysis. One factor in the pathology associated with chronic hepatitis C virus (HCV) infection is non-inflammatory caspase-3-mediated apoptosis. Our lab has found [...] Read more.

Introduction: Pyroptosis (inflammatory programmed cell death) is induced after the activation of an inflammasome, ultimately resulting in pore formation and cell lysis. One factor in the pathology associated with chronic hepatitis C virus (HCV) infection is non-inflammatory caspase-3-mediated apoptosis. Our lab has found both apoptosis and pyroptosis occurring in HCV-infected Huh-7.5 cells. In the context of some viral infections, pyroptosis is beneficial to the virus; for others, pyroptosis is believed to represent an innate antiviral response. This study aimed to test the effects of knocking out components of the inflammasome pathway on caspase activation in HCV-infected cells. Methods: FAM-FLICA (Carboxyfluorescein - Fluorochrome Inhibitor of Caspases) probes or antibodies were used to visualize active caspase-1 and active caspase-3 in vitro. Huh-7.5 cells with components of the pyroptotic or apoptotic pathways knocked out (NLRP3, GSDM-D or caspase-3) were used to determine the effects of their absence on the virus and caspase activation using confocal microscopy and flow cytometry. Results: Increased levels of caspase-1 were consistently observed in HCV-infected cells compared to those in uninfected cells, and these levels increased with subsequent days post-infection. The inhibition of inflammasome activation using knock out cell lines induced the differential activation of caspase-1 and caspase-3, with the inhibition of pyroptosis, resulting in a trend towards greater expression of caspase-3, indicative of apoptosis. The inhibition of NLRP3 did not fully stop caspase-1 activation, but it was decreased. The flow cytometry results revealed a small sub-set of cells positive for both caspase-1 and caspase-3. Conclusions: These data confirm the occurrence of pyroptosis in HCV-infected cells and demonstrate the involvement of the NLRP3 inflammasome, although other inflammasome sensors might be involved. Since the inhibition of one cell death pathway resulted in the increased activation of the other, along with the presence of double-positive cells, there may be cross-talk between apoptotic and pyroptotic pathways; the role of this cross-talk during infection remains to be elucidated. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 137 KiB

Open AccessProceeding Paper

MicroRNAs Are Predicted to Control the Ubiquitin/Proteasome System in Carica papaya Plants Infected by the Papaya Meleira Virus Complex

by Tathiana Ferreira Sá Antunes, Silas Pessini Rodrigues and Patricia Machado Bueno Fernandes

Proceedings 2020, 50(1), 146; https://doi.org/10.3390/proceedings2020050146 - 19 Aug 2020

Viewed by 1589

Abstract

Papaya sticky disease (PSD) is a severe disease that can destroy papaya trees. PSD is associated with a complex formed between a toti-like virus, Papaya meleira virus (PMeV), and an umbra-like virus—Papaya meleira virus 2 (PMeV2). PSD symptoms only appear after flowering, indicating [...] Read more.

Papaya sticky disease (PSD) is a severe disease that can destroy papaya trees. PSD is associated with a complex formed between a toti-like virus, Papaya meleira virus (PMeV), and an umbra-like virus—Papaya meleira virus 2 (PMeV2). PSD symptoms only appear after flowering, indicating that at the pre-flowering stage, there is a host stress response associated with tolerance to sticky disease symptoms. Transcriptomic and proteomic analyses of symptomatic plants revealed the modulation of protein turnover, suggesting the involvement of the ubiquitin/proteasome system (UPS) in this pathosystem. In parallel, the analysis of microRNAs modulated during the infection showed that microRNAs predicted to target UPS genes were specially altered. This study aimed to evaluate the importance of UPS for C. papaya–PMeV complex interaction by revisiting transcriptomic and proteomic datasets obtained from infected plants at different developmental phases. In the referred datasets, 1074 transcripts and 80 proteins were related to the UPS pathway. Among the 42 UPS-related genes responsive to PSD, 22 were detected at the transcript level and 21 at the protein level. In addition, the microRNAs predicted to target UPS-related genes were identified, especially those altered during papaya infection by PMeV complex. A total of 106 miRNAs assigned to 33 miRNA families and targeting 146 gene transcripts were found. Among them, 22 miRNAs were predicted to target four genes (U-box domain-containing protein, protein with BTB/POZ domains, 26S proteasome regulatory complex subunit PSMD10, and zinc finger C2H2 type domain) that were observed to be modulated at the transcript level at the pre-flowering stage and one gene (ubiquitin binding domain protein) modulated at the protein level at the post-flowering stage. Experimental evidence supports the idea that key miRNAs were especially relevant in controlling UPS during C. papaya response to the PMeV complex. The miRNA expression and the consequent reduction in transcripts levels could result in increased PMeV complex tolerance in C. papaya. The results presented here add to the knowledge on UPS involvement during virus infection in plants. Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

1 pages, 165 KiB

Open AccessRetraction

RETRACTED: Middleton, B.; Cosgrove, S.M. Isolation and Elimination of Latent and Productive Herpes Simplex Virus from the Sacral and Trigeminal Ganglions. Proceedings 2020, 50, 92

by Proceedings Editorial Office

Proceedings 2020, 50(1), 150; https://doi.org/10.3390/proceedings2020050150 - 13 Jan 2021

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Abstract

The Proceedings Editorial Office has been made aware that there is fake affiliation information. [...] Full article

(This article belongs to the Proceedings of Viruses 2020—Novel Concepts in Virology)

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