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Volume 12, January

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Viruses, Volume 12, Issue 2 (February 2020) – 126 articles

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Open AccessArticle
A Hyper-Glycosylation of HBV Surface Antigen Correlates with HBsAg-Negativity at Immunosuppression-Driven HBV Reactivation in Vivo and Hinders HBsAg Recognition in Vitro
Viruses 2020, 12(2), 251; https://doi.org/10.3390/v12020251 (registering DOI) - 23 Feb 2020
Abstract
Immune-suppression driven Hepatitis B Virus (HBV)-reactivation poses serious concerns since it occurs in several clinical settings and can result in severe forms of hepatitis. Previous studies showed that HBV strains, circulating in patients with HBV-reactivation, are characterized by an enrichment of immune-escape mutations [...] Read more.
Immune-suppression driven Hepatitis B Virus (HBV)-reactivation poses serious concerns since it occurs in several clinical settings and can result in severe forms of hepatitis. Previous studies showed that HBV strains, circulating in patients with HBV-reactivation, are characterized by an enrichment of immune-escape mutations in HBV surface antigen (HBsAg). Here, we focused on specific immune-escape mutations associated with the acquisition of N-linked glycosylation sites in HBsAg (NLGSs). In particular, we investigated profiles of NLGSs in 47 patients with immunosuppression-driven HBV-reactivation and we evaluated their impact on HBsAg-antigenicity and HBV-replication in vitro. At HBV-reactivation, despite a median serum HBV-DNA of 6.7 [5.3–8.0] logIU/mL, 23.4% of patients remained HBsAg-negative. HBsAg-negativity at HBV-reactivation correlated with the presence of >1 additional NLGSs (p < 0.001). These NLGSs are located in the major hydrophilic region of HBsAg (known to be the target of antibodies) and resulted from the single mutation T115N, T117N, T123N, N114ins, and from the triple mutant S113N+T131N+M133T. In vitro, NLGSs strongly alter HBsAg antigenic properties and recognition by antibodies used in assays for HBsAg-quantification without affecting HBsAg-secretion and other parameters of HBV-replication. In conclusion, additional NLGSs correlate with HBsAg-negativity despite HBV-reactivation, and hamper HBsAg-antigenicity in vitro, supporting the role of NGSs in immune-escape and the importance of HBV-DNA for a proper diagnosis of HBV-reactivation. Full article
(This article belongs to the Special Issue Hepatitis B Virus Reactivation)
Open AccessReview
The Cajal Body in Plant-Virus Interactions
Viruses 2020, 12(2), 250; https://doi.org/10.3390/v12020250 (registering DOI) - 23 Feb 2020
Abstract
Cajal bodies (CBs) are nuclear membraneless bodies composed of proteins and RNA. Although it is known that CBs play a role in RNA metabolism and the formation of functional ribonucleoprotein (RNP) particles, the whole breadth of CB functions is far from being fully [...] Read more.
Cajal bodies (CBs) are nuclear membraneless bodies composed of proteins and RNA. Although it is known that CBs play a role in RNA metabolism and the formation of functional ribonucleoprotein (RNP) particles, the whole breadth of CB functions is far from being fully elucidated. In this short review, we will summarize and discuss the growing body of evidence pointing to an involvement of this subnuclear compartment in plant-virus interactions. Full article
(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)
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Open AccessArticle
Chicken eEF1α is a Critical Factor for the Polymerase Complex Activity of Very Virulent Infectious Bursal Disease Virus
Viruses 2020, 12(2), 249; https://doi.org/10.3390/v12020249 (registering DOI) - 23 Feb 2020
Abstract
Infectious bursal disease (IBD) is an immunosuppressive, highly contagious, and lethal disease of young chickens caused by IBD virus (IBDV). It results in huge economic loss to the poultry industry worldwide. Infection caused by very virulent IBDV (vvIBDV) strains results in high mortality [...] Read more.
Infectious bursal disease (IBD) is an immunosuppressive, highly contagious, and lethal disease of young chickens caused by IBD virus (IBDV). It results in huge economic loss to the poultry industry worldwide. Infection caused by very virulent IBDV (vvIBDV) strains results in high mortality in young chicken flocks. However, the replication characteristics of vvIBDV are not well studied. Publications have shown that virus protein 3 (VP3) binds to VP1 and viral double-stranded RNA, and together they form a ribonucleoprotein complex that plays a key role in virus replication. In this study, vvIBDV VP3 was used to identify host proteins potentially involved in modulating vvIBDV replication. Chicken eukaryotic translation elongation factor 1α (cheEF1α) was chosen to further investigate effects on vvIBDV replication. By small interfering RNA-mediated cheEF1α knockdown, we demonstrated the possibility of significantly reducing viral polymerase activity, with a subsequent reduction in virus yields. Conversely, over-expression of cheEF1α significantly increased viral polymerase activity and virus replication. Further study confirmed that cheEF1α interacted only with vvIBDV VP3 but not with attenuated IBDV (aIBDV) VP3. Furthermore, the amino acids at the N- and C-termini were important in the interaction between vvIBDV VP3 and cheEF1α. Domain III was essential for interactions between cheEF1α and vvIBDV VP3. In summary, cheEF1α enhances vvIBDV replication by promoting the activity of virus polymerase. Our study indicates cheEF1α is a potential target for limiting vvIBDV infection. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle
Substantial Antigenic Drift in the Hemagglutinin Protein of Swine Influenza A Viruses
Viruses 2020, 12(2), 248; https://doi.org/10.3390/v12020248 (registering DOI) - 23 Feb 2020
Abstract
The degree of antigenic drift in swine influenza A viruses (swIAV) has historically been regarded as minimal compared to that of human influenza A virus strains. However, as surveillance activities on swIAV have increased, more isolates have been characterized, revealing a high level [...] Read more.
The degree of antigenic drift in swine influenza A viruses (swIAV) has historically been regarded as minimal compared to that of human influenza A virus strains. However, as surveillance activities on swIAV have increased, more isolates have been characterized, revealing a high level of genetic and antigenic differences even within the same swIAV lineage. The objective of this study was to investigate the level of genetic drift in one enzootically infected swine herd over one year. Nasal swabs were collected monthly from sows (n = 4) and piglets (n = 40) in the farrowing unit, and from weaners (n = 20) in the nursery. Virus from 1–4 animals were sequenced per month. Analyses of the sequences revealed that the hemagglutinin (HA) gene was the main target for genetic drift with a substitution rate of 7.6 × 10−3 substitutions/site/year and evidence of positive selection. The majority of the mutations occurred in the globular head of the HA protein and in antigenic sites. The phylogenetic tree of the HA sequences displayed a pectinate typology, where only a single lineage persists and forms the ancestor for subsequent lineages. This was most likely caused by repeated selection of a single immune-escape variant, which subsequently became the founder of the next wave of infections. Full article
(This article belongs to the Section Animal Viruses)
Open AccessArticle
Tick-Borne Encephalitis Virus: An Emerging Ancient Zoonosis?
Viruses 2020, 12(2), 247; https://doi.org/10.3390/v12020247 (registering DOI) - 23 Feb 2020
Abstract
Tick-borne encephalitis (TBE) is one of the most important viral zoonosis transmitted by the bite of infected ticks. In this study, all tick-borne encephalitis virus (TBEV) E gene sequences available in GenBank as of June 2019 with known date of isolation (n [...] Read more.
Tick-borne encephalitis (TBE) is one of the most important viral zoonosis transmitted by the bite of infected ticks. In this study, all tick-borne encephalitis virus (TBEV) E gene sequences available in GenBank as of June 2019 with known date of isolation (n = 551) were analyzed. Simulation studies showed that a sample bias could significantly affect earlier studies, because small TBEV datasets (n = 50) produced non-overlapping intervals for evolutionary rate estimates. An apparent lack of a temporal signal in TBEV, in general, was found, precluding molecular clock analysis of all TBEV subtypes in one dataset. Within all subtypes and most of the smaller groups in these subtypes, there was evidence of many medium- and long-distance virus transfers. These multiple random events may play a key role in the virus spreading. For some groups, virus diversity within one territory was similar to diversity over the whole geographic range. This is best exemplified by the virus diversity observed in Switzerland or Czech Republic. These two countries yielded most of the known European subtype Eu3 subgroup sequences, and the diversity of viruses found within each of these small countries is comparable to that of the whole Eu3 subgroup, which is prevalent all over Central and Eastern Europe. Most of the deep tree nodes within all three established TBEV subtypes dated less than 300 years back. This could be explained by the recent emergence of most of the known TBEV diversity. Results of bioinformatics analysis presented here, together with multiple field findings, suggest that TBEV may be regarded as an emerging disease. Full article
(This article belongs to the Special Issue Emerging Arboviruses)
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Open AccessArticle
Updating the Quarantine Status of Prunus Infecting Viruses in Australia
Viruses 2020, 12(2), 246; https://doi.org/10.3390/v12020246 (registering DOI) - 23 Feb 2020
Abstract
One hundred Prunus trees, including almond (P. dulcis), apricot (P. armeniaca), nectarine (P. persica var. nucipersica), peach (P. persica), plum (P. domestica), purple leaf plum (P. cerasifera) and sweet cherry ( [...] Read more.
One hundred Prunus trees, including almond (P. dulcis), apricot (P. armeniaca), nectarine (P. persica var. nucipersica), peach (P. persica), plum (P. domestica), purple leaf plum (P. cerasifera) and sweet cherry (P. avium), were selected from growing regions Australia-wide and tested for the presence of 34 viruses and three viroids using species-specific reverse transcription-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) tests. In addition, the samples were tested using some virus family or genus-based RT-PCR tests. The following viruses were detected: Apple chlorotic leaf spot virus (ACLSV) (13/100), Apple mosaic virus (ApMV) (1/100), Cherry green ring mottle virus (CGRMV) (4/100), Cherry necrotic rusty mottle virus (CNRMV) (2/100), Cherry virus A (CVA) (14/100), Little cherry virus 2 (LChV2) (3/100), Plum bark necrosis stem pitting associated virus (PBNSPaV) (4/100), Prune dwarf virus (PDV) (3/100), Prunus necrotic ringspot virus (PNRSV) (52/100), Hop stunt viroid (HSVd) (9/100) and Peach latent mosaic viroid (PLMVd) (6/100). The results showed that PNRSV is widespread in Prunus trees in Australia. Metagenomic high-throughput sequencing (HTS) and bioinformatics analysis were used to characterise the genomes of some viruses that were detected by RT-PCR tests and Apricot latent virus (ApLV), Apricot vein clearing associated virus (AVCaV), Asian Prunus Virus 2 (APV2) and Nectarine stem pitting-associated virus (NSPaV) were also detected. This is the first report of ApLV, APV2, CGRMV, CNRNV, LChV1, LChV2, NSPaV and PBNSPaV occurring in Australia. It is also the first report of ASGV infecting Prunus species in Australia, although it is known to infect other plant species including pome fruit and citrus. Full article
(This article belongs to the Special Issue Plant Virus Epidemiology and Control)
Open AccessArticle
The Heat Shock Response in the Western Honey Bee (Apis mellifera) is Antiviral
Viruses 2020, 12(2), 245; https://doi.org/10.3390/v12020245 (registering DOI) - 22 Feb 2020
Viewed by 130
Abstract
Honey bees (Apis mellifera) are an agriculturally important pollinator species that live in easily managed social groups (i.e., colonies). Unfortunately, annual losses of honey bee colonies in many parts of the world have reached unsustainable levels. Multiple abiotic and biotic stressors, [...] Read more.
Honey bees (Apis mellifera) are an agriculturally important pollinator species that live in easily managed social groups (i.e., colonies). Unfortunately, annual losses of honey bee colonies in many parts of the world have reached unsustainable levels. Multiple abiotic and biotic stressors, including viruses, are associated with individual honey bee and colony mortality. Honey bees have evolved several antiviral defense mechanisms including conserved immune pathways (e.g., Toll, Imd, JAK/STAT) and dsRNA-triggered responses including RNA interference and a non-sequence specific dsRNA-mediated response. In addition, transcriptome analyses of virus-infected honey bees implicate an antiviral role of stress response pathways, including the heat shock response. Herein, we demonstrate that the heat shock response is antiviral in honey bees. Specifically, heat-shocked honey bees (i.e., 42 °C for 4 h) had reduced levels of the model virus, Sindbis-GFP, compared with bees maintained at a constant temperature. Virus-infection and/or heat shock resulted in differential expression of six heat shock protein encoding genes and three immune genes, many of which are positively correlated. The heat shock protein encoding and immune gene transcriptional responses observed in virus-infected bees were not completely recapitulated by administration of double stranded RNA (dsRNA), a virus-associated molecular pattern, indicating that additional virus–host interactions are involved in triggering antiviral stress response pathways. Full article
(This article belongs to the Special Issue Advances in Honey Bee Virus Research)
Open AccessCommunication
Systematic Comparison of Two Animal-to-Human Transmitted Human Coronaviruses: SARS-CoV-2 and SARS-CoV
Viruses 2020, 12(2), 244; https://doi.org/10.3390/v12020244 (registering DOI) - 22 Feb 2020
Viewed by 152
Abstract
After the outbreak of the severe acute respiratory syndrome (SARS) in the world in 2003, human coronaviruses (HCoVs) have been reported as pathogens that cause severe symptoms in respiratory tract infections. Recently, a new emerged HCoV isolated from the respiratory epithelium of unexplained [...] Read more.
After the outbreak of the severe acute respiratory syndrome (SARS) in the world in 2003, human coronaviruses (HCoVs) have been reported as pathogens that cause severe symptoms in respiratory tract infections. Recently, a new emerged HCoV isolated from the respiratory epithelium of unexplained pneumonia patients in the Wuhan seafood market caused a major disease outbreak and has been named the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus causes acute lung symptoms, leading to a condition that has been named as “coronavirus disease 2019” (COVID-19). The emergence of SARS-CoV-2 and of SARS-CoV caused widespread fear and concern and has threatened global health security. There are some similarities and differences in the epidemiology and clinical features between these two viruses and diseases that are caused by these viruses. The goal of this work is to systematically review and compare between SARS-CoV and SARS-CoV-2 in the context of their virus incubation, originations, diagnosis and treatment methods, genomic and proteomic sequences, and pathogenic mechanisms. Full article
(This article belongs to the Special Issue Pathogenesis of Human and Animal Coronaviruses)
Open AccessArticle
Letea Virus: Comparative Genomics and Phylogenetic Analysis of a Novel Reassortant Orbivirus Discovered in Grass Snakes (Natrix natrix)
Viruses 2020, 12(2), 243; https://doi.org/10.3390/v12020243 - 21 Feb 2020
Viewed by 173
Abstract
The discovery and characterization of novel arthropod-borne viruses provide valuable information on their genetic diversity, ecology, evolution and potential to threaten animal or public health. Arbovirus surveillance is not conducted regularly in Romania, being particularly very scarce in the remote and diverse areas [...] Read more.
The discovery and characterization of novel arthropod-borne viruses provide valuable information on their genetic diversity, ecology, evolution and potential to threaten animal or public health. Arbovirus surveillance is not conducted regularly in Romania, being particularly very scarce in the remote and diverse areas like the Danube Delta. Here we describe the detection and genetic characterization of a novel orbivirus (Reoviridae: Orbivirus) designated as Letea virus, which was found in grass snakes (Natrix natrix) during a metagenomic and metatranscriptomic survey conducted between 2014 and 2017. This virus is the first orbivirus discovered in reptiles. Phylogenetic analyses placed Letea virus as a highly divergent species in the Culicoides-/sand fly-borne orbivirus clade. Gene reassortment and intragenic recombination were detected in the majority of the nine Letea virus strains obtained, implying that these mechanisms play important roles in the evolution and diversification of the virus. However, the screening of arthropods, including Culicoides biting midges collected within the same surveillance program, tested negative for Letea virus infection and could not confirm the arthropod vector of the virus. The study provided complete genome sequences for nine Letea virus strains and new information about orbivirus diversity, host range, ecology and evolution. The phylogenetic associations warrant further screening of arthropods, as well as sustained surveillance efforts for elucidation of Letea virus natural cycle and possible implications for animal and human health. Full article
(This article belongs to the Section Animal Viruses)
Open AccessArticle
Measles Vaccines Designed for Enhanced CD8+ T Cell Activation
Viruses 2020, 12(2), 242; https://doi.org/10.3390/v12020242 - 21 Feb 2020
Viewed by 117
Abstract
Priming and activation of CD8+ T cell responses is crucial to achieve anti-viral and anti-tumor immunity. Live attenuated measles vaccine strains have been used successfully for immunization for decades and are currently investigated in trials of oncolytic virotherapy. The available reverse genetics [...] Read more.
Priming and activation of CD8+ T cell responses is crucial to achieve anti-viral and anti-tumor immunity. Live attenuated measles vaccine strains have been used successfully for immunization for decades and are currently investigated in trials of oncolytic virotherapy. The available reverse genetics systems allow for insertion of additional genes, including heterologous antigens. Here, we designed recombinant measles vaccine vectors for priming and activation of antigen-specific CD8+ T cells. For proof-of-concept, we used cytotoxic T lymphocyte (CTL) lines specific for the melanoma-associated differentiation antigen tyrosinase-related protein-2 (TRP-2), or the model antigen chicken ovalbumin (OVA), respectively. We generated recombinant measles vaccine vectors with TRP-2 and OVA epitope cassette variants for expression of the full-length antigen or the respective immunodominant CD8+ epitope, with additional variants mediating secretion or proteasomal degradation of the epitope. We show that these recombinant measles virus vectors mediate varying levels of MHC class I (MHC-I)-restricted epitope presentation, leading to activation of cognate CTLs, as indicated by secretion of interferon-gamma (IFNγ) in vitro. Importantly, the recombinant OVA vaccines also mediate priming of naïve OT-I CD8+ T cells by dendritic cells. While all vaccine variants can prime and activate cognate T cells, IFNγ release was enhanced using a secreted epitope variant and a variant with epitope strings targeted to the proteasome. The principles presented in this study will facilitate the design of recombinant vaccines to elicit CD8+ responses against pathogens and tumor antigens. Full article
Open AccessArticle
Identification of HIV-1 Envelope Mutations that Enhance Entry Using Macaque CD4 and CCR5
Viruses 2020, 12(2), 241; https://doi.org/10.3390/v12020241 - 21 Feb 2020
Viewed by 115
Abstract
Although Rhesus macaques are an important animal model for HIV-1 vaccine development research, most transmitted HIV-1 strains replicate poorly in macaque cells. A major genetic determinant of this species-specific restriction is a non-synonymous mutation in macaque CD4 that results in reduced HIV-1 Envelope [...] Read more.
Although Rhesus macaques are an important animal model for HIV-1 vaccine development research, most transmitted HIV-1 strains replicate poorly in macaque cells. A major genetic determinant of this species-specific restriction is a non-synonymous mutation in macaque CD4 that results in reduced HIV-1 Envelope (Env)-mediated viral entry compared to human CD4. Recent research efforts employing either laboratory evolution or structure-guided design strategies have uncovered several mutations in Env’s gp120 subunit that enhance binding of macaque CD4 by transmitted/founder HIV-1 viruses. In order to identify additional Env mutations that promote infection of macaque cells, we utilized deep mutational scanning to screen thousands of Env point mutants for those that enhance HIV-1 entry via macaque receptors. We identified many uncharacterized amino acid mutations in the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions of gp41 that increased entry into cells bearing macaque receptors up to 9-fold. Many of these mutations also modestly increased infection of cells bearing human CD4 and CCR5 (up to 1.5-fold). NHR/CHR mutations identified by deep mutational scanning that enhanced entry also increased sensitivity to neutralizing antibodies targeting the MPER epitope, and to inactivation by cold-incubation, suggesting that they promote sampling of an intermediate trimer conformation between closed and receptor bound states. Identification of this set of mutations can inform future macaque model studies, and also further our understanding of the relationship between Env structure and function. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle
Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376
Viruses 2020, 12(2), 240; https://doi.org/10.3390/v12020240 - 21 Feb 2020
Viewed by 105
Abstract
Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CL [...] Read more.
Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CLpro) has a conserved structure and catalytic mechanism and plays a key role during viral polyprotein processing, thus serving as an appealing antiviral drug target. Here, we report the anti-PEDV effect of the broad-spectrum inhibitor GC376 (targeting 3Cpro or 3CLpro of viruses in the picornavirus-like supercluster). GC376 was highly effective against the PEDV 3CLpro and exerted similar inhibitory effects on two PEDV strains. Furthermore, the structure of the PEDV 3CLpro in complex with GC376 was determined at 1.65 Å. We elucidated structural details and analyzed the differences between GC376 binding with the PEDV 3CLpro and GC376 binding with the transmissible gastroenteritis virus (TGEV) 3CLpro. Finally, we explored the substrate specificity of PEDV 3CLpro at the P2 site and analyzed the effects of Leu group modification in GC376 on inhibiting PEDV infection. This study helps us to understand better the PEDV 3CLpro substrate specificity, providing information on the optimization of GC376 for development as an antiviral therapeutic against coronaviruses. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle
Extracellular Vesicles from Human Papilloma Virus-Infected Cervical Cancer Cells Enhance HIV-1 Replication in Differentiated U1 Cell Line
Viruses 2020, 12(2), 239; https://doi.org/10.3390/v12020239 - 21 Feb 2020
Viewed by 109
Abstract
In the current study, we hypothesized that extracellular vesicles (EVs) secreted from human papilloma virus (HPV)-infected cervical cancer cells exacerbate human immunodeficiency virus (HIV)-1 replication in differentiated U1 cell line through an oxidative stress pathway. To test the hypothesis, we treated an HIV-1-infected [...] Read more.
In the current study, we hypothesized that extracellular vesicles (EVs) secreted from human papilloma virus (HPV)-infected cervical cancer cells exacerbate human immunodeficiency virus (HIV)-1 replication in differentiated U1 cell line through an oxidative stress pathway. To test the hypothesis, we treated an HIV-1-infected macrophage cell line (U1) with HPV-infected Caski cell culture supernatant (CCS). We observed a significant increase in HIV-1 replication, which was associated with an increase in the expression of cytochrome P450 (CYPs 1A1 and 2A6) in the CCS-treated U1 cells. Furthermore, we isolated EVs from CCS (CCS-EVs), which showed the presence of CYPs (1A1, 2A6), superoxide dismutase 1 (SOD1), and HPV oncoproteins HPV16 E6. CCS-EVs when exposed to the U1 cells also significantly increased HIV-1 replication. Treatment of antioxidant, CYP1A1 and CYP2A6 inhibitors, and chemodietary agents with antioxidant properties significantly reduced the CCS and CCS-EVs mediated HIV-1 replication in U1 cells. Altogether, we demonstrate that cervical cancer cells exacerbate HIV-1 replication in differentiated U1 cell line via transferring CYPs and HPV oncoproteins through EVs. We also show that the viral replication occurs via CYP and oxidative stress pathways, and the viral replication is also reduced by chemodietary agents. This study provides important information regarding biological interactions between HPV and HIV-1 via EVs leading to enhanced HIV-1 replication. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle
pUC18-CpG Is an Effective Adjuvant for a Duck Tembusu Virus Inactivated Vaccine
Viruses 2020, 12(2), 238; https://doi.org/10.3390/v12020238 - 20 Feb 2020
Viewed by 124
Abstract
Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus responsible for massive economic losses in the duck industry. However, commercially inactivated DTMUV vaccines have been ineffective at inducing protective immunity in ducks. The widely used adjuvant cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) reportedly improve humoral [...] Read more.
Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus responsible for massive economic losses in the duck industry. However, commercially inactivated DTMUV vaccines have been ineffective at inducing protective immunity in ducks. The widely used adjuvant cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) reportedly improve humoral and cellular immunities in animal models. However, its effectiveness in DTMUV vaccines requires validation. Here, we assessed the protective efficacy of pUC18-CpG as an adjuvant in an inactivated live DTMUV vaccine in ducks. Our results revealed that the serum hemagglutination inhibition (HI) antibody titers, positive rates of anti-DTMUV antibodies, the concentration of serum cytokines, and protection efficacy were significantly increased in ducks immunized with pUC18-CpG compared to that in the control group. Moreover, ducks immunized with a full vaccine dose containing a half dose of antigen supplemented with 40 μg of pUC18-CpG exhibited the most potent responses. This study suggests that pUC18-CpG is a promising adjuvant against DTMUV, which might prove effective in treating other viral diseases in waterfowl. Full article
(This article belongs to the Section Antivirals & Vaccines)
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Open AccessArticle
In Vivo Characterization of a Bank Vole-Derived Cowpox Virus Isolate in Natural Hosts and the Rat Model
Viruses 2020, 12(2), 237; https://doi.org/10.3390/v12020237 - 20 Feb 2020
Viewed by 119
Abstract
Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the Poxviridae family and is endemic in western Eurasia. Based on seroprevalence studies in different voles from continental Europe and UK, voles are suspected to be the major reservoir host. Recently, a CPXV was [...] Read more.
Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the Poxviridae family and is endemic in western Eurasia. Based on seroprevalence studies in different voles from continental Europe and UK, voles are suspected to be the major reservoir host. Recently, a CPXV was isolated from a bank vole (Myodes glareolus) in Germany that showed a high genetic similarity to another isolate originating from a Cotton-top tamarin (Saguinus oedipus). Here we characterize this first bank vole-derived CPXV isolate in comparison to the related tamarin-derived isolate. Both isolates grouped genetically within the provisionally called CPXV-like 3 clade. Previous phylogenetic analysis indicated that CPXV is polyphyletic and CPXV-like 3 clade represents probably a different species if categorized by the rules used for other orthopoxviruses. Experimental infection studies with bank voles, common voles (Microtus arvalis) and Wistar rats showed very clear differences. The bank vole isolate was avirulent in both common voles and Wistar rats with seroconversion seen only in the rats. In contrast, inoculated bank voles exhibited viral shedding and seroconversion for both tested CPXV isolates. In addition, bank voles infected with the tamarin-derived isolate experienced a marked weight loss. Our findings allow for the conclusion that CPXV isolates might differ in their replication capacity in different vole species and rats depending on their original host. Moreover, the results indicate host-specific differences concerning CPXV-specific virulence. Further experiments are needed to identify individual virulence and host factors involved in the susceptibility and outcome of CPXV-infections in the different reservoir hosts. Full article
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Open AccessReview
Cell Lines for Honey Bee Virus Research
Viruses 2020, 12(2), 236; https://doi.org/10.3390/v12020236 - 20 Feb 2020
Viewed by 149
Abstract
With ongoing colony losses driven in part by the Varroa mite and the associated exacerbation of the virus load, there is an urgent need to protect honey bees (Apis mellifera) from fatal levels of virus infection and from the non-target effects [...] Read more.
With ongoing colony losses driven in part by the Varroa mite and the associated exacerbation of the virus load, there is an urgent need to protect honey bees (Apis mellifera) from fatal levels of virus infection and from the non-target effects of insecticides used in agricultural settings. A continuously replicating cell line derived from the honey bee would provide a valuable tool for the study of molecular mechanisms of virus–host interaction, for the screening of antiviral agents for potential use within the hive, and for the assessment of the risk of current and candidate insecticides to the honey bee. However, the establishment of a continuously replicating honey bee cell line has proved challenging. Here, we provide an overview of attempts to establish primary and continuously replicating hymenopteran cell lines, methods (including recent results) of establishing honey bee cell lines, challenges associated with the presence of latent viruses (especially Deformed wing virus) in established cell lines and methods to establish virus-free cell lines. We also describe the potential use of honey bee cell lines in conjunction with infectious clones of honey bee viruses for examination of fundamental virology. Full article
(This article belongs to the Special Issue Advances in Honey Bee Virus Research)
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Open AccessReview
Bacteriophages for Chronic Wound Treatment: from Traditional to Novel Delivery Systems
Viruses 2020, 12(2), 235; https://doi.org/10.3390/v12020235 - 20 Feb 2020
Viewed by 152
Abstract
The treatment and management of chronic wounds presents a massive financial burden for global health care systems, with significant and disturbing consequences for the patients affected. These wounds remain challenging to treat, reduce the patients’ life quality, and are responsible for a high [...] Read more.
The treatment and management of chronic wounds presents a massive financial burden for global health care systems, with significant and disturbing consequences for the patients affected. These wounds remain challenging to treat, reduce the patients’ life quality, and are responsible for a high percentage of limb amputations and many premature deaths. The presence of bacterial biofilms hampers chronic wound therapy due to the high tolerance of biofilm cells to many first- and second-line antibiotics. Due to the appearance of antibiotic-resistant and multidrug-resistant pathogens in these types of wounds, the research for alternative and complementary therapeutic approaches has increased. Bacteriophage (phage) therapy, discovered in the early 1900s, has been revived in the last few decades due to its antibacterial efficacy against antibiotic-resistant clinical isolates. Its use in the treatment of non-healing wounds has shown promising outcomes. In this review, we focus on the societal problems of chronic wounds, describe both the history and ongoing clinical trials of chronic wound-related treatments, and also outline experiments carried out for efficacy evaluation with different phage-host systems using in vitro, ex vivo, and in vivo animal models. We also describe the modern and most recent delivery systems developed for the incorporation of phages for species-targeted antibacterial control while protecting them upon exposure to harsh conditions, increasing the shelf life and facilitating storage of phage-based products. In this review, we also highlight the advances in phage therapy regulation. Full article
(This article belongs to the Special Issue Bacteriophages and Biofilms)
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Open AccessArticle
Field Trial Vaccination against Cowpox in Two Alpaca Herds
Viruses 2020, 12(2), 234; https://doi.org/10.3390/v12020234 - 20 Feb 2020
Viewed by 119
Abstract
In Europe, cowpox virus (CPXV) infection in South American camelids occurs as a so-called spill-over infection. Although infected animals generally have a mild form of the disease and survive, cases of fatal generalised CPXV infection have also been described. Prevention by prophylactic vaccination [...] Read more.
In Europe, cowpox virus (CPXV) infection in South American camelids occurs as a so-called spill-over infection. Although infected animals generally have a mild form of the disease and survive, cases of fatal generalised CPXV infection have also been described. Prevention by prophylactic vaccination is the only way to protect animals from disease. In the present study, modified vaccinia virus Ankara (MVA) vaccine, which has been successfully used in many animal species, was used in a prime-boost vaccination regimen in two alpaca herds with a history of CPXV infection. The focus of the study was the prevention of further clinical cases, and to determine the safety and immunogenicity of the MVA vaccine in alpacas. The MVA vaccine was well tolerated and safe in the 94 animals vaccinated. An indirect immunofluorescence assay (IFA) using MVA as an antigen showed that the seroprevalence of antibody after booster vaccination was 81.3% in herd I and 91.7% in herd II. Detectable antibody titres declined to 15.6% in herd I and 45.8% in herd II over a 12-month period after booster vaccination. Animals could be divided into four groups based on individual antibody titres determined over one year: Group 1 consisted of 19.3% of animals that were seropositive until the end of the trial period; Group 2 consisted of 58.0% of animals that were seropositive after booster vaccination, but seronegative one year later; Group 3 consisted of 14.7% of animals that were not seropositive at any time point; and Group 4 consisted of 7.9% of animals that were seropositive after initial immunisation, seronegative six months later, but seropositive or intermediate in IFA one year after immunisation, likely because of natural exposure. In new-born crias born to MVA-vaccinated mares, specific maternal antibodies were detected in 50.0% of animals up to 14 weeks of age. Our results confirm that MVA vaccination is a feasible tool for the prevention of CPXV disease in alpacas. Long-term studies are needed to verify future vaccination regimen in CPXV affected herds. Full article
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Open AccessArticle
TCR Repertoire Characterization for T Cells Expanded in Response to hRSV Infection in Mice Immunized with a Recombinant BCG Vaccine
Viruses 2020, 12(2), 233; https://doi.org/10.3390/v12020233 - 20 Feb 2020
Viewed by 116
Abstract
T cells play an essential role in the immune response against the human respiratory syncytial virus (hRSV). It has been described that both CD4+ and CD8+ T cells can contribute to the clearance of the virus during an infection. However, for [...] Read more.
T cells play an essential role in the immune response against the human respiratory syncytial virus (hRSV). It has been described that both CD4+ and CD8+ T cells can contribute to the clearance of the virus during an infection. However, for some individuals, such an immune response can lead to an exacerbated and detrimental inflammatory response with high recruitment of neutrophils to the lungs. The receptor of most T cells is a heterodimer consisting of α and β chains (αβTCR) that upon antigen engagement induces the activation of these cells. The αβTCR molecule displays a broad sequence diversity that defines the T cell repertoire of an individual. In our laboratory, a recombinant Bacille Calmette–Guérin (BCG) vaccine expressing the nucleoprotein (N) of hRSV (rBCG-N-hRSV) was developed. Such a vaccine induces T cells with a Th1 polarized phenotype that promote the clearance of hRSV infection without causing inflammatory lung damage. Importantly, as part of this work, the T cell receptor (TCR) repertoire of T cells expanded after hRSV infection in naïve and rBCG-N-hRSV-immunized mice was characterized. A more diverse TCR repertoire was observed in the lungs from rBCG-N-hRSV-immunized as compared to unimmunized hRSV-infected mice, suggesting that vaccination with the recombinant rBCG-N-hRSV vaccine triggers the expansion of T cell populations that recognize more viral epitopes. Furthermore, differential expansion of certain TCRVβ chains was found for hRSV infection (TCRVβ+8.3 and TCRVβ+5.1,5.2) as compared to rBCG-N-hRSV vaccination (TCRVβ+11 and TCRVβ+12). Our findings contribute to better understanding the T cell response during hRSV infection, as well as the functioning of a vaccine that induces a protective T cell immunity against this virus. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle
Characterization of Experimental Oro-Nasal Inoculation of Seba’s Short-Tailed Bats (Carollia perspicillata) with Bat Influenza A Virus H18N11
Viruses 2020, 12(2), 232; https://doi.org/10.3390/v12020232 - 19 Feb 2020
Viewed by 152
Abstract
In 2012 and 2013, the genomic sequences of two novel influenza A virus (IAV) subtypes, designated H17N10 and H18N11, were identified via next-generation sequencing in the feces of the little yellow-shouldered fruit bat (Sturnira lilium) and the flat-faced fruit-eating bat ( [...] Read more.
In 2012 and 2013, the genomic sequences of two novel influenza A virus (IAV) subtypes, designated H17N10 and H18N11, were identified via next-generation sequencing in the feces of the little yellow-shouldered fruit bat (Sturnira lilium) and the flat-faced fruit-eating bat (Artibeus planirostris), respectively. The pathogenesis caused by these viruses in their respective host species is currently insufficiently understood, which is primarily due to the inability to obtain and keep these bat species under appropriate environmental and biosafety conditions. Seba’s short-tailed bats (Carollia perspicillata), in contrast, are close relatives and a natural H18N11 reservoir species, with the advantage of established animal husbandry conditions in academic research. To study viral pathogenesis in more detail, we here oro-nasally inoculated Seba’s short-tailed bats with the bat IAV H18N11 subtype. Following inoculation, bats appeared clinically healthy, but the histologic examination of tissues revealed a mild necrotizing rhinitis. Consistently, IAV-matrix protein and H18-RNA positive cells were seen in lesioned respiratory and olfactory nasal epithelia, as well as in intestinal tissues. A RT-qPCR analysis confirmed viral replication in the conchae and intestines as well as the presence of viral RNA in the excreted feces, without horizontal transmission to naïve contact animals. Moreover, all inoculated animals seroconverted with low titers of neutralizing antibodies. Full article
(This article belongs to the Section Animal Viruses)
Open AccessCommunication
Molecular Characterization of a Novel Ourmia-Like Virus Infecting Phoma matteucciicola
Viruses 2020, 12(2), 231; https://doi.org/10.3390/v12020231 - 19 Feb 2020
Viewed by 137
Abstract
Here, we report a novel (+) ssRNA mycovirus, Phoma matteucciicola ourmia-like virus 1 (PmOLV1), isolated from Phoma matteucciicola strain LG915-1. The genome of PmOLV1 was 2603 nucleotides long and contained a single open reading frame (ORF), which could be translated into a product [...] Read more.
Here, we report a novel (+) ssRNA mycovirus, Phoma matteucciicola ourmia-like virus 1 (PmOLV1), isolated from Phoma matteucciicola strain LG915-1. The genome of PmOLV1 was 2603 nucleotides long and contained a single open reading frame (ORF), which could be translated into a product of RNA-dependent RNA polymerase (RdRp) by both standard and mitochondrial genetic codons. Cellular fractionation assay indicated that PmOLV1 RNAs are likely more enriched in mitochondria than in cytoplasm. Phylogenetic analysis indicated that PmOLV1 is a new member of the genus Penoulivirus (recently proposed) within the family Botourmiaviridae. Full article
(This article belongs to the collection Mycoviruses)
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Open AccessArticle
Differential Accumulation of Innate- and Adaptive-Immune-Response-Derived Transcripts during Antagonism between Papaya Ringspot Virus and Papaya Mosaic Virus
Viruses 2020, 12(2), 230; https://doi.org/10.3390/v12020230 - 19 Feb 2020
Viewed by 181
Abstract
Papaya ringspot virus (PRSV), a common potyvirus infecting papaya plants worldwide, can lead to either antagonism or synergism in mixed infections with Papaya mosaic virus (PapMV), a potexvirus. These two unrelated viruses produce antagonism or synergism depending on their order of infection in [...] Read more.
Papaya ringspot virus (PRSV), a common potyvirus infecting papaya plants worldwide, can lead to either antagonism or synergism in mixed infections with Papaya mosaic virus (PapMV), a potexvirus. These two unrelated viruses produce antagonism or synergism depending on their order of infection in the plant. When PRSV is inoculated first or at the same time as PapMV, the viral interaction is synergistic. However, an antagonistic response is observed when PapMV is inoculated before PRSV. In the antagonistic condition, PRSV is deterred from the plant and its drastic effects are overcome. Here, we examine differences in gene expression by high-throughput RNA sequencing, focused on immune system pathways. We present the transcriptomic expression of single and mixed inoculations of PRSV and PapMV leading to synergism and antagonism. Upregulation of dominant and hormone-mediated resistance transcripts suggests that the innate immune system participates in synergism. In antagonism, in addition to innate immunity, upregulation of RNA interference-mediated resistance transcripts suggests that adaptive immunity is involved. Full article
(This article belongs to the Special Issue The Complexity of the Potyviral Interaction Network)
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Open AccessArticle
Novel Lentivirus-Based Method for Rapid Selection of Inhibitory Nanobody against PRRSV
Viruses 2020, 12(2), 229; https://doi.org/10.3390/v12020229 - 19 Feb 2020
Viewed by 157
Abstract
The emergence and re-emergence of porcine reproductive and respiratory syndrome virus (PRRSV) has resulted in huge economic losses for the swine industry. Current vaccines are of limited efficacy against endemic circulating PRRSV variants. New strategies against PRRSV infection are in urgent need. Here, [...] Read more.
The emergence and re-emergence of porcine reproductive and respiratory syndrome virus (PRRSV) has resulted in huge economic losses for the swine industry. Current vaccines are of limited efficacy against endemic circulating PRRSV variants. New strategies against PRRSV infection are in urgent need. Here, a nanobody library in Marc-145 cells is constructed for antiviral nanobodies. Nanobody encoding sequences from two non-immunized llamas were cloned to generate a pseudotyped lentiviral library. Several candidates were selected from survival cells post-PRRSV inoculation and further characterized. Nb9 was identified with strong antiviral activity. Moreover, Nb9 exerted antiviral activity via its interaction with PRRSV viral proteins, as revealed by immunofluorescence assay and Western blot. Taken together, the novel function-based screen of the lentivirus nanobody library, instead of the conventional affinity-based screen, offers an alternative strategy for antiviral reagents against PRRSV and other pathogens. Full article
(This article belongs to the Section Antivirals & Vaccines)
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Open AccessArticle
A Novel Role for PX, a Structural Protein of Fowl Adenovirus Serotype 4 (FAdV4), as an Apoptosis-Inducer in Leghorn Male Hepatocellular Cell
Viruses 2020, 12(2), 228; https://doi.org/10.3390/v12020228 - 18 Feb 2020
Viewed by 157
Abstract
Hydropericardium-Hepatitis Syndrome (HHS) caused by Fowl Adenovirus Serotype 4 (FAdV4) infection is a severe threat to the poultry industry worldwide, especially in China since 2015. Recent studies show that FAdV4 induces liver injury through apoptosis. However, the underlying molecular mechanism is still unclear. [...] Read more.
Hydropericardium-Hepatitis Syndrome (HHS) caused by Fowl Adenovirus Serotype 4 (FAdV4) infection is a severe threat to the poultry industry worldwide, especially in China since 2015. Recent studies show that FAdV4 induces liver injury through apoptosis. However, the underlying molecular mechanism is still unclear. We report here that FAdV4 infection caused apoptosis in Leghorn male hepatocellular (LMH) cells and that PX, a structural protein of FAdV4, acted as a major viral factor inducing apoptosis. Furthermore, the nuclear localization of PX is determined by the R/K regions of PX and required for PX-induced apoptosis. Moreover, alanines 11 and 129 of PX are crucial to PX-induced apoptosis. Inhibition of FAdV4-induced apoptosis by caspase inhibitors retarded viral replication, suggesting that PX serves as a virulence factor for FAdV4 infection, which may further our understandings of the pathogenesis of FAdV4 infection. Full article
(This article belongs to the Special Issue Adenovirus Pathogenesis)
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Open AccessArticle
The Major Capsid Protein, VP1, of the Mouse Polyomavirus Stimulates the Activity of Tubulin Acetyltransferase 1 by Microtubule Stabilization
Viruses 2020, 12(2), 227; https://doi.org/10.3390/v12020227 - 18 Feb 2020
Viewed by 164
Abstract
Viruses have evolved mechanisms to manipulate microtubules (MTs) for the efficient realization of their replication programs. Studying the mechanisms of replication of mouse polyomavirus (MPyV), we observed previously that in the late phase of infection, a considerable amount of the main structural protein, [...] Read more.
Viruses have evolved mechanisms to manipulate microtubules (MTs) for the efficient realization of their replication programs. Studying the mechanisms of replication of mouse polyomavirus (MPyV), we observed previously that in the late phase of infection, a considerable amount of the main structural protein, VP1, remains in the cytoplasm associated with hyperacetylated microtubules. VP1–microtubule interactions resulted in blocking the cell cycle in the G2/M phase. We are interested in the mechanism leading to microtubule hyperacetylation and stabilization and the roles of tubulin acetyltransferase 1 (αTAT1) and deacetylase histone deacetylase 6 (HDAC6) and VP1 in this mechanism. Therefore, HDAC6 inhibition assays, αTAT1 knock out cell infections, in situ cell fractionation, and confocal and TIRF microscopy were used. The experiments revealed that the direct interaction of isolated microtubules and VP1 results in MT stabilization and a restriction of their dynamics. VP1 leads to an increase in polymerized tubulin in cells, thus favoring αTAT1 activity. The acetylation status of MTs did not affect MPyV infection. However, the stabilization of MTs by VP1 in the late phase of infection may compensate for the previously described cytoskeleton destabilization by MPyV early gene products and is important for the observed inhibition of the G2→M transition of infected cells to prolong the S phase. Full article
(This article belongs to the Section Animal Viruses)
Open AccessArticle
Analytical Performance of the RIDASCREEN® Hantavirus Puumala IgG/IgM ELISA Assay
Viruses 2020, 12(2), 226; https://doi.org/10.3390/v12020226 - 18 Feb 2020
Viewed by 151
Abstract
The National Reference Center for Hantavirus in Belgium is currently using the Hantavirus IgM/IgG ELISA Progen kit (Heidelberg, Germany) for the detection of the most prevalent Hantavirus in Western Europe, Puumala virus (PUUV). Two commercially available PUUV kits were compared: Progen and RIDASCREEN [...] Read more.
The National Reference Center for Hantavirus in Belgium is currently using the Hantavirus IgM/IgG ELISA Progen kit (Heidelberg, Germany) for the detection of the most prevalent Hantavirus in Western Europe, Puumala virus (PUUV). Two commercially available PUUV kits were compared: Progen and RIDASCREEN® Hantavirus Puumala IgM/IgG ELISA assay (Darmstadt, Germany). Methods: The sensitivity was evaluated with a panel of 68 samples from patients with an acute infection (n = 44) or a past infection (n = 24). Specificity was evaluated with a panel of 62 samples from patients with potentially false borderline results (n = 7) (no seroconversion), seronegative samples (n = 25) and potentially cross reacting samples (n = 30). Discordances were resolved by immunoblot. Substantial agreement was calculated using Cohen kappa coefficient. Results: The RIDASCREEN® kit showed a higher specificity (IgM: 94.3%; IgG: 94.4%) than the Progen kit (IgM: 77.0% IgG: 93.0%). The sensitivity for IgM ELISA was 100% for both assays. IgG sensitivity was, respectively, 98.3% and 100% for Progen and RIDASCREEN®. A Cohen kappa coefficient of 0.76 and 0.90 was found between Puumala IgM and IgG, respectively. Conclusions: This study showed a higher specificity for the RIDASCREEN® kit than the Progen kit, while the sensitivity was as good as for the Progen kit. Full article
(This article belongs to the Section Antivirals & Vaccines)
Open AccessArticle
An Oleanolic Acid Derivative Inhibits Hemagglutinin-Mediated Entry of Influenza A Virus
Viruses 2020, 12(2), 225; https://doi.org/10.3390/v12020225 - 18 Feb 2020
Viewed by 156
Abstract
Influenza A viruses (IAV) have been a major public health threat worldwide, and options for antiviral therapy become increasingly limited with the emergence of drug-resisting virus strains. New and effective anti-IAV drugs, especially for highly pathogenic influenza, with different modes of action, are [...] Read more.
Influenza A viruses (IAV) have been a major public health threat worldwide, and options for antiviral therapy become increasingly limited with the emergence of drug-resisting virus strains. New and effective anti-IAV drugs, especially for highly pathogenic influenza, with different modes of action, are urgently needed. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. In this study, we show that OA-10, a newly synthesized triterpene out of 11 oleanane-type derivatives, exhibited significant antiviral activity against four different subtypes of IAV (H1N1, H5N1, H9N2 and H3N2) replications in A549 cell cultures with EC50 ranging from 6.7 to 19.6 μM and a negligible cytotoxicity (CC50 > 640 μM). It inhibited acid-induced hemolysis in a dose-dependent manner, with an IC50 of 26 µM, and had a weak inhibition on the adsorption of H5 HA to chicken erythrocytes at higher concentrations (≥40 µM). Surface plasmon resonance (SPR) analysis showed that OA-10 interacted with HA in a dose-dependent manner with the equilibrium dissociation constants (KD) of the interaction of 2.98 × 10−12 M. Computer-aided molecular docking analysis suggested that OA-10 might bind to the cavity in HA stem region which is known to undergo significant rearrangement during membrane fusion. Our results demonstrate that OA-10 inhibits H5N1 IAV replication mainly by blocking the conformational changes of HA2 subunit required for virus fusion with endosomal membrane. These findings suggest that OA-10 could serve as a lead for further development of novel virus entry inhibitors to prevent and treat IAV infections. Full article
(This article belongs to the Special Issue Antiviral Agents)
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Open AccessArticle
Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein
Viruses 2020, 12(2), 224; https://doi.org/10.3390/v12020224 - 18 Feb 2020
Viewed by 191
Abstract
Sacbrood virus (SBV) of honey bees is a picornavirus in the genus Iflavirus. Given its relatively small and simple genome structure, single positive-strand RNA with only one ORF, cloning the full genomic sequence is not difficult. However, adding nonsynonymous mutations to the [...] Read more.
Sacbrood virus (SBV) of honey bees is a picornavirus in the genus Iflavirus. Given its relatively small and simple genome structure, single positive-strand RNA with only one ORF, cloning the full genomic sequence is not difficult. However, adding nonsynonymous mutations to the bee iflavirus clone is difficult because of the lack of information about the viral protein processes. Furthermore, the addition of a reporter gene to the clones has never been accomplished. In preliminary trials, we found that the site between 3′ untranslated region (UTR) and poly(A) can retain added sequences. We added enhanced green fluorescent protein (EGFP) expression at this site, creating a SBV clone with an expression tag that does not affect virus genes. An intergenic region internal ribosome entry site (IRES) from Black queen cell virus (BQCV) was inserted to initiate EGFP expression. The SBV-IRES-EGFP clone successfully infected Apis cerana and Apis mellifera, and in A. cerana larvae, it was isolated and passaged using oral inoculation. The inoculated larvae had higher mortality and the dead larvae showed sacbrood symptoms. The added IRES-EGFP remained in the clone through multiple passages and expressed the expected EGFP in all infected bees. We demonstrated the ability to add gene sequences in the site between 3′-UTR and poly(A) in SBV and the potential to do so in other bee iflaviruses; however, further investigations of the mechanisms are needed. A clone with a desired protein expression reporter will be a valuable tool in bee virus studies. Full article
(This article belongs to the Special Issue Advances in Honey Bee Virus Research)
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Open AccessArticle
Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study
Viruses 2020, 12(2), 223; https://doi.org/10.3390/v12020223 - 17 Feb 2020
Viewed by 193
Abstract
Virus-like particles (VLPs) have emerged as a powerful scaffold for antigen presentation and delivery strategies. Compared to single protein-based therapeutics, quality assessment requires a higher degree of refinement due to the structure of VLPs and their similar properties to extracellular vesicles (EVs). Advances [...] Read more.
Virus-like particles (VLPs) have emerged as a powerful scaffold for antigen presentation and delivery strategies. Compared to single protein-based therapeutics, quality assessment requires a higher degree of refinement due to the structure of VLPs and their similar properties to extracellular vesicles (EVs). Advances in the field of nanotechnology with single particle and high-resolution analysis techniques provide appealing approaches to VLP characterization. In this study, six different biophysical methods have been assessed for the characterization of HIV-1-based VLPs produced in mammalian and insect cell platforms. Sample preparation and equipment set-up were optimized for the six strategies evaluated. Electron Microscopy (EM) disclosed the presence of several types of EVs within VLP preparations and cryogenic transmission electron microscopy (cryo-TEM) resulted in the best technique to resolve the VLP ultrastructure. The use of super-resolution fluorescence microscopy (SRFM), nanoparticle tracking analysis (NTA) and flow virometry enabled the high throughput quantification of VLPs. Interestingly, differences in the determination of nanoparticle concentration were observed between techniques. Moreover, NTA and flow virometry allowed the quantification of both EVs and VLPs within the same experiment while analyzing particle size distribution (PSD), simultaneously. These results provide new insights into the use of different analytical tools to monitor the production of nanoparticle-based biologicals and their associated contaminants. Full article
(This article belongs to the Special Issue Virus-Like Particle Vaccines)
Open AccessCase Report
Late-Relapsing Hepatitis after Yellow Fever
Viruses 2020, 12(2), 222; https://doi.org/10.3390/v12020222 - 17 Feb 2020
Viewed by 263
Abstract
One patient presented hyporexia, asthenia, adynamia, and jaundice two months after acute yellow fever (YF) onset; plus laboratory tests indicating hepatic cytolysis and a rebound of alanine and aspartate transaminases, and total and direct bilirubin levels. Laboratory tests discarded autoimmune hepatitis, inflammatory or [...] Read more.
One patient presented hyporexia, asthenia, adynamia, and jaundice two months after acute yellow fever (YF) onset; plus laboratory tests indicating hepatic cytolysis and a rebound of alanine and aspartate transaminases, and total and direct bilirubin levels. Laboratory tests discarded autoimmune hepatitis, inflammatory or metabolic liver disease, and new infections caused by hepatotropic agents. Anti-YFV IgM, IgG and neutralizing antibodies were detected in different times, but no viremia. A liver biopsy was collected three months after YF onset and tested positive for YFV antigens and wild-type YFV-RNA (364 RNA-copies/gram/liver). Transaminases and bilirubin levels remained elevated for five months, and the arresting of symptoms persisted for six months after the acute YF onset. Several serum chemokines, cytokines, and growth factors were measured. A similar immune response profile was observed in the earlier phases of the disease, followed by more pronounced changes in the later stages, when transaminases levels returned to normal. The results indicated viral persistence in the liver and continual liver cell damage three months after YF onset and reinforced the need for extended follow-ups of YF patients. Further studies to investigate the role of possible viral persistence and the immune response causing relapsing hepatitis following YF are also necessary. Full article
(This article belongs to the Special Issue Emerging Viruses: Surveillance, Prevention, Evolution and Control)
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