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Viruses, Volume 10, Issue 5 (May 2018)

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Cover Story (view full-size image) Plant virus-based vectors are valuable tools for recombinant gene expression and functional [...] Read more.
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Open AccessArticle Efficient Protein Expression and Virus-Induced Gene Silencing in Plants Using a Crinivirus-Derived Vector
Viruses 2018, 10(5), 216; https://doi.org/10.3390/v10050216
Received: 8 April 2018 / Revised: 19 April 2018 / Accepted: 21 April 2018 / Published: 24 April 2018
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Abstract
Plant virus-based vectors are valuable tools for recombinant gene expression and functional genomics for both basic and applied research. In this study, Lettuce infectious yellows virus (LIYV) of the genus Crinivirus was engineered into a virus vector that is applicable for efficient protein
[...] Read more.
Plant virus-based vectors are valuable tools for recombinant gene expression and functional genomics for both basic and applied research. In this study, Lettuce infectious yellows virus (LIYV) of the genus Crinivirus was engineered into a virus vector that is applicable for efficient protein expression and virus-induced gene silencing (VIGS) in plants. We examined gene replacement and “add a gene” strategies to develop LIYV-derived vectors for transient expression of the green fluorescent protein (GFP) reporter in Nicotiana benthamiana plants. The latter yielded higher GFP expression and was further examined by testing the effects of heterologous controller elements (CEs). A series of five vector constructs with progressively extended LIYV CP sgRNA CEs were tested, the longest CE gave the highest GFP expression but lower virus accumulation. The whitefly transmissibility of the optimized vector construct to other host plants, and the capability to accommodate and express a larger gene, a 1.8 kb β-glucuronidase (GUS) gene, were confirmed. Furthermore, the LIYV vector was also validated VIGS by silencing the endogenous gene, phytoene desaturase (PDS) in N. benthamiana plants, and the transgene GFP in N. benthamiana line 16c plants. Therefore, LIYV-derived vectors could provide a technical reference for developing vectors of other economically important criniviruses. Full article
(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)
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Open AccessArticle The Sequence of Two Bacteriophages with Hypermodified Bases Reveals Novel Phage-Host Interactions
Viruses 2018, 10(5), 217; https://doi.org/10.3390/v10050217
Received: 28 March 2018 / Revised: 19 April 2018 / Accepted: 20 April 2018 / Published: 24 April 2018
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Abstract
Bacteriophages SP-15 and ΦW-14 are members of the Myoviridae infecting Bacillus subtilis and Delftia (formerly Pseudomonas) acidovorans, respectively. What links them is that in both cases, approximately 50% of the thymine residues are replaced by hypermodified bases. The consequence of this
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Bacteriophages SP-15 and ΦW-14 are members of the Myoviridae infecting Bacillus subtilis and Delftia (formerly Pseudomonas) acidovorans, respectively. What links them is that in both cases, approximately 50% of the thymine residues are replaced by hypermodified bases. The consequence of this is that the physico-chemical properties of the DNA are radically altered (melting temperature (Tm), buoyant density and susceptibility to restriction endonucleases). Using 454 pyrosequencing technology, we sequenced the genomes of both viruses. Phage ΦW-14 possesses a 157-kb genome (56.3% GC) specifying 236 proteins, while SP-15 is larger at 222 kb (38.6 mol % G + C) and encodes 318 proteins. In both cases, the phages can be considered genomic singletons since they do not possess BLASTn homologs. While no obvious genes were identified as being responsible for the modified base in ΦW-14, SP-15 contains a cluster of genes obviously involved in carbohydrate metabolism. Full article
(This article belongs to the Special Issue Phage-Host Interactions)
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Open AccessArticle Relative Contribution of Cellular Complement Inhibitors CD59, CD46, and CD55 to Parainfluenza Virus 5 Inhibition of Complement-Mediated Neutralization
Viruses 2018, 10(5), 219; https://doi.org/10.3390/v10050219
Received: 10 April 2018 / Revised: 20 April 2018 / Accepted: 22 April 2018 / Published: 25 April 2018
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Abstract
The complement system is a part of the innate immune system that viruses need to face during infections. Many viruses incorporate cellular regulators of complement activation (RCA) to block complement pathways and our prior work has shown that Parainfluenza virus 5 (PIV5) incorporates
[...] Read more.
The complement system is a part of the innate immune system that viruses need to face during infections. Many viruses incorporate cellular regulators of complement activation (RCA) to block complement pathways and our prior work has shown that Parainfluenza virus 5 (PIV5) incorporates CD55 and CD46 to delay complement-mediated neutralization. In this paper, we tested the role of a third individual RCA inhibitor CD59 in PIV5 interactions with complement pathways. Using a cell line engineered to express CD59, we show that small levels of functional CD59 are associated with progeny PIV5, which is capable of blocking assembly of the C5b-C9 membrane attack complex (MAC). PIV5 containing CD59 (PIV5-CD59) showed increased resistance to complement-mediated neutralization in vitro comparing to PIV5 lacking regulators. Infection of A549 cells with PIV5 and RSV upregulated CD59 expression. TGF-beta treatment of PIV5-infected cells also increased cell surface CD59 expression and progeny virions were more resistant to complement-mediated neutralization. A comparison of individual viruses containing only CD55, CD46, or CD59 showed a potency of inhibiting complement-mediated neutralization, which followed a pattern of CD55 > CD46 > CD59. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle A New Approach to Assessing HSV-1 Recombination during Intercellular Spread
Viruses 2018, 10(5), 220; https://doi.org/10.3390/v10050220
Received: 9 March 2018 / Revised: 17 April 2018 / Accepted: 20 April 2018 / Published: 25 April 2018
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Abstract
The neuroinvasive Herpes simplex virus type 1 (HSV-1) utilizes intergenomic recombination in order to diversify viral populations. Research efforts to assess HSV-1 recombination are often complicated by the use of attenuating mutations, which differentiate viral progeny but unduly influence the replication and spread.
[...] Read more.
The neuroinvasive Herpes simplex virus type 1 (HSV-1) utilizes intergenomic recombination in order to diversify viral populations. Research efforts to assess HSV-1 recombination are often complicated by the use of attenuating mutations, which differentiate viral progeny but unduly influence the replication and spread. In this work, we generated viruses with markers that allowed for classification of viral progeny with limited attenuation of viral replication. We isolated viruses, harboring either a cyan (C) or yellow (Y) fluorescent protein (FP) expression cassette inserted in two different locations within the viral genome, in order to visually quantify the recombinant progeny based on plaque fluorescence. We found that the FP marked genomes had a limited negative affect on the viral replication and production of progeny virions. A co-infection of the two viruses resulted in recombinant progeny that was dependent on the multiplicity of infection and independent of the time post infection, at a rate that was similar to previous reports. The sequential passage of mixed viral populations revealed a limited change in the distribution of the parental and recombinant progeny. Interestingly, the neuroinvasive spread within neuronal cultures and an in vivo mouse model, revealed large, random shifts in the parental and recombinant distributions in viral populations. In conclusion, our approach highlights the utility of FP expressing viruses in order to provide new insights into mechanisms of HSV-1 recombination. Full article
(This article belongs to the Special Issue Viral Recombination: Ecology, Evolution and Pathogenesis)
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Open AccessArticle Within-Host Recombination in the Foot-and-Mouth Disease Virus Genome
Viruses 2018, 10(5), 221; https://doi.org/10.3390/v10050221
Received: 28 February 2018 / Revised: 23 April 2018 / Accepted: 23 April 2018 / Published: 25 April 2018
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Abstract
Recombination is one of the determinants of genetic diversity in the foot-and-mouth disease virus (FMDV). FMDV sequences have a mosaic structure caused by extensive intra- and inter-serotype recombination, with the exception of the capsid-encoding region. While these genome-wide patterns of broad-scale recombination are
[...] Read more.
Recombination is one of the determinants of genetic diversity in the foot-and-mouth disease virus (FMDV). FMDV sequences have a mosaic structure caused by extensive intra- and inter-serotype recombination, with the exception of the capsid-encoding region. While these genome-wide patterns of broad-scale recombination are well studied, not much is known about the patterns of recombination that may exist within infected hosts. In addition, detection of recombination among viruses evolving at the within-host level is challenging due to the similarity of the sequences and the limitations in differentiating recombination from point mutations. Here, we present the first analysis of recombination events between closely related FMDV sequences occurring within buffalo hosts. The detection of these events was made possible by the occurrence of co-infection of two viral swarms with about 1% nucleotide divergence. We found more than 15 recombination events, unequally distributed across eight samples from different animals. The distribution of these events along the FMDV genome was neither uniform nor related to the phylogenetic distribution of recombination breakpoints, suggesting a mismatch between within-host evolutionary pressures and long-term selection for infectivity and transmissibility. Full article
(This article belongs to the Special Issue Viral Recombination: Ecology, Evolution and Pathogenesis)
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Open AccessArticle Characterization of NS5A and NS5B Resistance-Associated Substitutions from Genotype 1 Hepatitis C Virus Infected Patients in a Portuguese Cohort
Viruses 2018, 10(5), 223; https://doi.org/10.3390/v10050223
Received: 11 April 2018 / Revised: 20 April 2018 / Accepted: 22 April 2018 / Published: 26 April 2018
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Abstract
This study is focused on the prevalent NS5 coding region resistance-associated substitutions (RASs) in DAA-naive genotype (GT)1 HCV-infected patients and their potential impact on success rates. Plasma RNA from 81 GT1 HCV-infected patients was extracted prior to an in-house nested RT-PCR of the
[...] Read more.
This study is focused on the prevalent NS5 coding region resistance-associated substitutions (RASs) in DAA-naive genotype (GT)1 HCV-infected patients and their potential impact on success rates. Plasma RNA from 81 GT1 HCV-infected patients was extracted prior to an in-house nested RT-PCR of the NS5 coding region, which is followed by Sanger population sequencing. NS5A RASs were present in 28.4% (23/81) of all GT1-infected patients with 9.9% (8/81) having the Y93C/H mutation. NS5B RASs showed a prevalence of 14.8% (12/81) and were only detected in GT1b. Overall 38.3% (31/81) of all GT1 HCV-infected patients presented baseline RASs. The obtained data supports the usefulness of resistance testing prior to treatment since a statistically significant association was found between treatment failure and the baseline presence of specific NS5 RASs known as Y93C/H (p = 0.04). Full article
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Open AccessArticle Anti-Inflammatory Effect of Baicalein on Polyinosinic–Polycytidylic Acid-Induced RAW 264.7 Mouse Macrophages
Viruses 2018, 10(5), 224; https://doi.org/10.3390/v10050224
Received: 8 March 2018 / Revised: 14 April 2018 / Accepted: 24 April 2018 / Published: 26 April 2018
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Abstract
Baicalein (3,3′,4′,5,6-pentahydroxyflavone) is a well-known antioxidant found in many plants, such as in the roots of Scutellaria baicalensis. In this study, we evaluate the inhibitory effect of baicalein on the inflammatory cascade in RAW 264.7 mouse macrophages induced by viral-like material. Experimental
[...] Read more.
Baicalein (3,3′,4′,5,6-pentahydroxyflavone) is a well-known antioxidant found in many plants, such as in the roots of Scutellaria baicalensis. In this study, we evaluate the inhibitory effect of baicalein on the inflammatory cascade in RAW 264.7 mouse macrophages induced by viral-like material. Experimental assays used in this study included Griess reagent assay for nitric oxide (NO) production, Fluo-4 assay for intracellular calcium release, multiplex cytokine assay, and quantitative real time RT-PCR assay. To induce inflammation, RAW 264.7 cells were treated with polyinosinic–polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA (dsRNA). Baicalein at concentrations up to 100 μM significantly inhibited the production of NO, IL-1α, IL-6, G-CSF, GM-CSF, VEGF, MCP-1, IP-10, LIX, and RANTES as well as calcium release in RAW 264.7 cells induced by poly I:C (50 µg/mL) (all p < 0.05). Baicalein at concentrations up to 50 μM also significantly inhibited mRNA expression of STAT1, STAT3, CHOP, and Fas in poly I:C-induced RAW 264.7 cells (p < 0.05). In conclusion, baicalein has anti-inflammatory effect in double-stranded RNA (dsRNA)-induced macrophages by inhibiting NO, cytokines, chemokines, and growth factors via the endoplasmic reticulum stress–CHOP/STAT pathway. Full article
(This article belongs to the Special Issue Cytokine Responses in Viral Infections)
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Open AccessArticle Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus
Viruses 2018, 10(5), 226; https://doi.org/10.3390/v10050226
Received: 21 March 2018 / Revised: 17 April 2018 / Accepted: 20 April 2018 / Published: 27 April 2018
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Abstract
Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact
[...] Read more.
Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3. Full article
(This article belongs to the Special Issue Advances in Alphavirus Research)
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Open AccessArticle Molecular Mechanisms Governing “Hair-Trigger” Induction of Shiga Toxin-Encoding Prophages
Viruses 2018, 10(5), 228; https://doi.org/10.3390/v10050228
Received: 16 March 2018 / Revised: 25 April 2018 / Accepted: 25 April 2018 / Published: 29 April 2018
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Abstract
Shiga toxin (Stx)-encoding E. coli (STEC) strains are responsible for sporadic outbreaks of food poisoning dating to 1982, when the first STEC strain, E. coli O157:H7, was isolated. Regardless of STEC serotype, the primary symptoms of STEC infections are caused by Stx that
[...] Read more.
Shiga toxin (Stx)-encoding E. coli (STEC) strains are responsible for sporadic outbreaks of food poisoning dating to 1982, when the first STEC strain, E. coli O157:H7, was isolated. Regardless of STEC serotype, the primary symptoms of STEC infections are caused by Stx that is synthesized from genes resident on lambdoid prophage present in STEC. Despite similar etiology, the severity of STEC-mediated disease varies by outbreak. However, it is unclear what modulates the severity of STEC-mediated disease. Stx production and release is controlled by lytic growth of the Stx-encoding bacteriophage, which in turn, is controlled by the phage repressor. Here, we confirm our earlier suggestion that the higher spontaneous induction frequency of Stx-encoding prophage is a consequence, in part, of lower intracellular repressor levels in STEC strains versus non-STEC strains. We also show that this lowered intracellular repressor concentration is a consequence of the utilization of alternative binding/regulatory strategies by the phage repressor. We suggest that a higher spontaneous induction frequency would lead to increased virulence. Full article
(This article belongs to the Special Issue Phage-Host Interactions)
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Open AccessArticle Comparative Pathogenesis of Asian and African-Lineage Zika Virus in Indian Rhesus Macaque’s and Development of a Non-Human Primate Model Suitable for the Evaluation of New Drugs and Vaccines
Viruses 2018, 10(5), 229; https://doi.org/10.3390/v10050229
Received: 6 April 2018 / Revised: 24 April 2018 / Accepted: 27 April 2018 / Published: 1 May 2018
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Abstract
The establishment of a well characterized non-human primate model of Zika virus (ZIKV) infection is critical for the development of medical interventions. In this study, challenging Indian rhesus macaques (IRMs) with ZIKV strains of the Asian lineage resulted in dose-dependent peak viral loads
[...] Read more.
The establishment of a well characterized non-human primate model of Zika virus (ZIKV) infection is critical for the development of medical interventions. In this study, challenging Indian rhesus macaques (IRMs) with ZIKV strains of the Asian lineage resulted in dose-dependent peak viral loads between days 2 and 5 post infection and a robust immune response which protected the animals from homologous and heterologous re-challenge. In contrast, viremia in IRMs challenged with an African lineage strain was below the assay’s lower limit of quantitation, and the immune response was insufficient to protect from re-challenge. These results corroborate previous observations but are contrary to reports using other African strains, obviating the need for additional studies to elucidate the variables contributing to the disparities. Nonetheless, the utility of an Asian lineage ZIKV IRM model for countermeasure development was verified by vaccinating animals with a formalin inactivated reference vaccine and demonstrating sterilizing immunity against a subsequent subcutaneous challenge. Full article
(This article belongs to the Special Issue New Advances on Zika Virus Research)
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Open AccessArticle An Immunodominant Region of the Envelope Glycoprotein of Small Ruminant Lentiviruses May Function as Decoy Antigen
Viruses 2018, 10(5), 231; https://doi.org/10.3390/v10050231
Received: 30 March 2018 / Revised: 28 April 2018 / Accepted: 30 April 2018 / Published: 2 May 2018
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Abstract
(1) Background: Small ruminant lentiviruses (SRLV) persist in infected goats that mount a strong humoral immune response characterized by low neutralizing titers. In this study, we characterized the antibody response to SU5, a variable, immunodominant epitope of the envelope glycoprotein of SRLV. We
[...] Read more.
(1) Background: Small ruminant lentiviruses (SRLV) persist in infected goats that mount a strong humoral immune response characterized by low neutralizing titers. In this study, we characterized the antibody response to SU5, a variable, immunodominant epitope of the envelope glycoprotein of SRLV. We tested the working hypothesis that the variability of SU5 reflects escape from neutralizing antibody. (2) Methods: Affinity purified anti-SU5 antibody were tested for their neutralizing activity to the homologous lentivirus. Virus culture supernatant—in native form or following sonication and filtration—was used to test the ability of free envelope glycoproteins to compete for binding in a SU5-peptide-ELISA. (3) Results: Anti-SU5 antibodies are not neutralizing, strongly suggesting that they do not bind intact viral particles. In contrast, shed envelope glycoproteins efficiently compete for binding in a SU5-ELISA, providing convincing evidence that the SU5 epitope is exposed only on shed envelope glycoproteins. (4) Conclusions: Our results show that the antibody engaging SU5 is not neutralizing and does not appear to bind to SU expressed at the surface of virus particles. We propose that SU5 is a potential decoy epitope exposed on shaded envelope glycoproteins, luring the humoral immune response in committing an original antigenic sin to a functionally irrelevant epitope. Full article
(This article belongs to the Special Issue Nonprimate Lentivirus)
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Open AccessArticle Involvement of Pro-Inflammatory Macrophages in Liver Pathology of Pirital Virus-Infected Syrian Hamsters
Viruses 2018, 10(5), 232; https://doi.org/10.3390/v10050232
Received: 18 January 2018 / Revised: 25 April 2018 / Accepted: 28 April 2018 / Published: 2 May 2018
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Abstract
New World arenaviruses cause fatal hemorrhagic disease in South America. Pirital virus (PIRV), a mammarenavirus hosted by Alston’s cotton rat (Sigmodon alstoni), causes a disease in Syrian golden hamsters (Mesocricetus auratus) (biosafety level-3, BSL-3) that has many pathologic similarities
[...] Read more.
New World arenaviruses cause fatal hemorrhagic disease in South America. Pirital virus (PIRV), a mammarenavirus hosted by Alston’s cotton rat (Sigmodon alstoni), causes a disease in Syrian golden hamsters (Mesocricetus auratus) (biosafety level-3, BSL-3) that has many pathologic similarities to the South American hemorrhagic fevers (BSL-4) and, thus, is considered among the best small-animal models for human arenavirus disease. Here, we extend in greater detail previously described clinical and pathological findings in Syrian hamsters and provide evidence for a pro-inflammatory macrophage response during PIRV infection. The liver was the principal target organ of the disease, and signs of Kupffer cell involvement were identified in mortally infected hamster histopathology data. Differential expression analysis of liver mRNA revealed signatures of the pro-inflammatory response, hematologic dysregulation, interferon pathway and other host response pathways, including 17 key transcripts that were also reported in two non-human primate (NHP) arenavirus liver-infection models, representing both Old and New World mammarenavirus infections. Although antigen presentation may differ among rodent and NHP species, key hemostatic and innate immune-response components showed expression parallels. Signatures of pro-inflammatory macrophage involvement in PIRV-infected livers included enrichment of Ifng, Nfkb2, Stat1, Irf1, Klf6, Il1b, Cxcl10, and Cxcl11 transcripts. Together, these data indicate that pro-inflammatory macrophage M1 responses likely contribute to the pathogenesis of acute PIRV infection. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle Attenuated Phenotype and Immunogenic Characteristics of a Mutated Herpes Simplex Virus 1 Strain in the Rhesus Macaque
Viruses 2018, 10(5), 234; https://doi.org/10.3390/v10050234
Received: 15 March 2018 / Revised: 28 April 2018 / Accepted: 28 April 2018 / Published: 2 May 2018
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Abstract
Herpes simplex virus type 1(HSV-1) presents a conundrum to public health worldwide because of its specific pathogenicity and clinical features. Some experimental vaccines, such as the recombinant viral glycoproteins, exhibit the viral immunogenicity of a host-specific immune response, but none of these has
[...] Read more.
Herpes simplex virus type 1(HSV-1) presents a conundrum to public health worldwide because of its specific pathogenicity and clinical features. Some experimental vaccines, such as the recombinant viral glycoproteins, exhibit the viral immunogenicity of a host-specific immune response, but none of these has achieved a valid epidemiological protective efficacy in the human population. In the present study, we constructed an attenuated HSV-1 strain M3 through the partial deletion of UL7, UL41, and the latency-associated transcript (LAT) using the CRISPR/Cas9 system. The mutant strain exhibited lowered infectivity and virulence in macaques. Neutralization testing and ELISpot detection of the specific T-cell responses confirmed the specific immunity induced by M3 immunization and this immunity defended against the challenges of the wild-type strain and restricted the entry of the wild-type strain into the trigeminal ganglion. These results in rhesus macaques demonstrated the potential of the attenuated vaccine for the prevention of HSV-1 in humans. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle Computational Analysis of the Interaction Energies between Amino Acid Residues of the Measles Virus Hemagglutinin and Its Receptors
Viruses 2018, 10(5), 236; https://doi.org/10.3390/v10050236
Received: 7 February 2018 / Revised: 27 April 2018 / Accepted: 30 April 2018 / Published: 3 May 2018
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Abstract
Measles virus (MV) causes an acute and highly devastating contagious disease in humans. Employing the crystal structures of three human receptors, signaling lymphocyte-activation molecule (SLAM), CD46, and Nectin-4, in complex with the measles virus hemagglutinin (MVH), we elucidated computationally the details of binding
[...] Read more.
Measles virus (MV) causes an acute and highly devastating contagious disease in humans. Employing the crystal structures of three human receptors, signaling lymphocyte-activation molecule (SLAM), CD46, and Nectin-4, in complex with the measles virus hemagglutinin (MVH), we elucidated computationally the details of binding energies between the amino acid residues of MVH and those of the receptors with an ab initio fragment molecular orbital (FMO) method. The calculated inter-fragment interaction energies (IFIEs) revealed a number of significantly interacting amino acid residues of MVH that played essential roles in binding to the receptors. As predicted from previously reported experiments, some important amino-acid residues of MVH were shown to be common but others were specific to interactions with the three receptors. Particularly, some of the (non-polar) hydrophobic residues of MVH were found to be attractively interacting with multiple receptors, thus indicating the importance of the hydrophobic pocket for intermolecular interactions (especially in the case of Nectin-4). In contrast, the electrostatic interactions tended to be used for specific molecular recognition. Furthermore, we carried out FMO calculations for in silico experiments of amino acid mutations, finding reasonable agreements with virological experiments concerning the substitution effect of residues. Thus, the present study demonstrates that the electron-correlated FMO method is a powerful tool to search exhaustively for amino acid residues that contribute to interactions with receptor molecules. It is also applicable for designing inhibitors of MVH and engineered MVs for cancer therapy. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle Phytohormone Signaling of the Resistance to Plum pox virus (PPV, Sharka Disease) Induced by Almond (Prunus dulcis (Miller) Webb) Grafting to Peach (P. persica L. Batsch)
Viruses 2018, 10(5), 238; https://doi.org/10.3390/v10050238
Received: 16 March 2018 / Revised: 19 April 2018 / Accepted: 2 May 2018 / Published: 3 May 2018
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Abstract
Plum pox virus (PPV, sharka) is a limiting factor for peach production, and no natural sources of resistance have been described. Recent studies, however, have demonstrated that grafting the almond cultivar “Garrigues” onto the “GF305” peach infected with Dideron-type (PPV-D) isolates progressively reduces
[...] Read more.
Plum pox virus (PPV, sharka) is a limiting factor for peach production, and no natural sources of resistance have been described. Recent studies, however, have demonstrated that grafting the almond cultivar “Garrigues” onto the “GF305” peach infected with Dideron-type (PPV-D) isolates progressively reduces disease symptoms and virus accumulation. Furthermore, grafting “Garrigues” onto “GF305” prior to PPV-D inoculation has been found to completely prevent virus infection, showing that resistance is constitutive and not induced by the virus. To unravel the phytohormone signaling of this mechanism, we analyzed the following phytohormones belonging to the principal hormone classes: the growth-related phytohormones cytokinin trans-zeatin (tZ) and the gibberellins GA3 and GA4; and the stress-related phytohormones ethylene acid precursor 1-aminocyclopropane-1-carboxylic acid (ACC), abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA). PPV inoculation produced a significant increase in GA3 and ABA in peach, and these imbalances were related to the presence of chlorosis symptoms. However, grafting “Garrigues” almond onto the PPV-inoculated “GF305” peach produced the opposite effect, reducing GA3 and ABA contents in parallel to the elimination of symptoms. Our results showed the significant implication of SA in this induced resistance in peach with an additional effect on tZ and JA concentrations. This SA-induced resistance based in the decrease in symptoms seems to be different from Systemic Acquired Resistance (SAR) and Induced Systemic Resistance (ISR), which are based in other reactions producing necrosis. Further studies are necessary, however, to validate these results against PPV-D isolates in the more aggressive Marcus-type (PPV-M) isolates. Full article
(This article belongs to the Special Issue Plant-Virus Interactions)
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Open AccessArticle Complete Nucleotide Sequence Analysis of a Novel Bacillus subtilis-Infecting Bacteriophage BSP10 and Its Effect on Poly-Gamma-Glutamic Acid Degradation
Viruses 2018, 10(5), 240; https://doi.org/10.3390/v10050240
Received: 17 April 2018 / Revised: 1 May 2018 / Accepted: 1 May 2018 / Published: 4 May 2018
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Abstract
While the harmful effects of lactic acid bacterial bacteriophages in the dairy industry are well-established, the importance of Bacillus subtilis-infecting bacteriophages on soybean fermentation is poorly-studied. In this study, we isolated a B. subtilis-infecting bacteriophage BSP10 from Meju (a brick of
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While the harmful effects of lactic acid bacterial bacteriophages in the dairy industry are well-established, the importance of Bacillus subtilis-infecting bacteriophages on soybean fermentation is poorly-studied. In this study, we isolated a B. subtilis-infecting bacteriophage BSP10 from Meju (a brick of dried fermented soybean) and further characterized it. This Myoviridae family bacteriophage exhibited a narrow host range against B. subtilis strains (17/52, 32.7%). The genome of bacteriophage BSP10 is 153,767 bp long with 236 open reading frames and 5 tRNAs. Comparative genomics (using dot plot, progressiveMauve alignment, heat-plot, and BLASTN) and phylogenetic analysis strongly suggest its incorporation as a new species in the Nit1virus genus. Furthermore, bacteriophage BSP10 was efficient in the growth inhibition of B. subtilis ATCC 15245 in liquid culture and in Cheonggukjang (a soybean fermented food) fermentation. Artificial contamination of as low as 102 PFU/g of bacteriophage BSP10 during Cheonggukjang fermentation significantly reduced bacterial numbers by up to 112 fold in comparison to the control (no bacteriophage). Moreover, for the first time, we experimentally proved that B. subtilis-infecting bacteriophage greatly enhanced poly-γ-glutamic acid degradation during soybean fermentation, which is likely to negatively affect the functionalities of Cheonggukjang. Full article
(This article belongs to the Section Bacterial Viruses)
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Open AccessArticle A CRISPR-Cas9-Based Toolkit for Fast and Precise In Vivo Genetic Engineering of Bacillus subtilis Phages
Viruses 2018, 10(5), 241; https://doi.org/10.3390/v10050241
Received: 6 April 2018 / Revised: 29 April 2018 / Accepted: 1 May 2018 / Published: 4 May 2018
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Abstract
Phages are currently under discussion as a solution for the antibiotic crisis, as they may cure diseases caused by multi-drug-resistant pathogens. However, knowledge of phage biology and genetics is limited, which impedes risk assessment of therapeutic applications. In order to enable advances in
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Phages are currently under discussion as a solution for the antibiotic crisis, as they may cure diseases caused by multi-drug-resistant pathogens. However, knowledge of phage biology and genetics is limited, which impedes risk assessment of therapeutic applications. In order to enable advances in phage genetic research, the aim of this work was to create a toolkit for simple and fast genetic engineering of phages recruiting Bacillus subtilis as host system. The model organism B. subtilis represents a non-pathogenic surrogate of its harmful relatives, such as Bacillus anthracis or Bacillus cereus. This toolkit comprises the application CutSPR, a bioinformatic tool for rapid primer design, and facilitates the cloning of specific CRISPR-Cas9-based mutagenesis plasmids. The employment of the prophage-free and super-competent B. subtilis TS01 strain enables an easy and fast introduction of specific constructs for in vivo phage mutagenesis. Clean gene deletions and a functional clean gene insertion into the genome of the model phage vB_BsuP-Goe1 served as proof of concept and demonstrate reliability and high efficiency. The here presented toolkit allows comprehensive investigation of the diverse phage genetic pool, a better understanding of phage biology, and safe phage applications. Full article
(This article belongs to the Section Bacterial Viruses)
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Open AccessCommunication Inhibition of v-rel-Induced Oncogenesis through microRNA Targeting
Viruses 2018, 10(5), 242; https://doi.org/10.3390/v10050242
Received: 27 March 2018 / Revised: 30 April 2018 / Accepted: 3 May 2018 / Published: 5 May 2018
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Abstract
Several studies have shown that microRNA-targeting is an effective strategy for the selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. However, the exploitation of microRNA-targeting for the inhibition of transformation by oncogenic viruses has not been studied. The v-
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Several studies have shown that microRNA-targeting is an effective strategy for the selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. However, the exploitation of microRNA-targeting for the inhibition of transformation by oncogenic viruses has not been studied. The v-rel oncoprotein encoded by reticuloendotheliosis virus T strain (Rev-T) is a member of the rel/NF-κB family of transcription factors capable of transforming primary chicken spleen and bone marrow cells. Here, by engineering the target sequence of endogenous microRNA miR-142 downstream of the v-rel gene in a Replication-Competent ALV (avian leukosis virus) long terminal repeat (LTR) with a splice acceptor (RCAS) vector and using a v-rel-induced transformation model of chicken embryonic splenocyte cultures, we show that hematopoietic-specific miR-142 can inhibit the v-rel-induced transformation, and that this inhibition effect is due to the silencing of v-rel expression. The data supports the idea that microRNA-targeting can be used to inhibit viral oncogene-induced oncogenesis. Full article
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Open AccessArticle Characterization of an N-Terminal Non-Core Domain of RAG1 Gene Disrupted Syrian Hamster Model Generated by CRISPR Cas9
Viruses 2018, 10(5), 243; https://doi.org/10.3390/v10050243
Received: 20 March 2018 / Revised: 25 April 2018 / Accepted: 3 May 2018 / Published: 6 May 2018
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Abstract
The accumulating evidence demonstrates that Syrian hamsters have advantages as models for various diseases. To develop a Syrian hamster (Mesocricetus auratus) model of human immunodeficiency caused by RAG1 gene mutations, we employed the CRISPR/Cas9 system and introduced an 86-nucleotide frameshift deletion
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The accumulating evidence demonstrates that Syrian hamsters have advantages as models for various diseases. To develop a Syrian hamster (Mesocricetus auratus) model of human immunodeficiency caused by RAG1 gene mutations, we employed the CRISPR/Cas9 system and introduced an 86-nucleotide frameshift deletion in the hamster RAG1 gene encoding part of the N-terminal non-core domain of RAG1. Histological and immunohistochemical analyses demonstrated that these hamsters (referred herein as RAG1-86nt hamsters) had atrophic spleen and thymus, and developed significantly less white pulp and were almost completely devoid of splenic lymphoid follicles. The RAG1-nt86 hamsters had barely detectable CD3+ and CD4+ T cells. The expression of B and T lymphocyte-specific genes (CD3γ and CD4 for T cell-specific) and (CD22 and FCMR for B cell-specific) was dramatically reduced, whereas the expression of macrophage-specific (CD68) and natural killer (NK) cell-specific (CD94 and KLRG1) marker genes was increased in the spleen of RAG1-nt86 hamsters compared to wildtype hamsters. Interestingly, despite the impaired development of B and T lymphocytes, the RAG1-86nt hamsters still developed neutralizing antibodies against human adenovirus type C6 (HAdV-C6) upon intranasal infection and were capable of clearing the infectious viruses, albeit with slower kinetics. Therefore, the RAG1-86nt hamster reported herein (similar to the hypomorphic RAG1 mutations in humans that cause Omenn syndrome), may provide a useful model for studying the pathogenesis of the specific RAG1-mutation-induced human immunodeficiency, the host immune response to adenovirus infection and other pathogens as well as for evaluation of cell and gene therapies for treatment of this subset of RAG1 mutation patients. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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Open AccessArticle An Ointment Consisting of the Phage Lysin LysGH15 and Apigenin for Decolonization of Methicillin-Resistant Staphylococcus aureus from Skin Wounds
Viruses 2018, 10(5), 244; https://doi.org/10.3390/v10050244
Received: 16 March 2018 / Revised: 3 May 2018 / Accepted: 4 May 2018 / Published: 6 May 2018
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Abstract
Staphylococcus aureus (S. aureus) is a common and dangerous pathogen that causes various infectious diseases. Skin damage, such as burn wounds, are at high risk of Staphylococcus aureus colonization and infection, which increases morbidity and mortality. The phage lysin LysGH15 exhibits
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Staphylococcus aureus (S. aureus) is a common and dangerous pathogen that causes various infectious diseases. Skin damage, such as burn wounds, are at high risk of Staphylococcus aureus colonization and infection, which increases morbidity and mortality. The phage lysin LysGH15 exhibits highly efficient lytic activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. Apigenin (api) significantly decreases haemolysis of rabbit erythrocytes caused by S. aureus and shows anti-inflammatory function. LysGH15 and api were added to Aquaphor to form an LysGH15-api-Aquaphor (LAA) ointment. The LAA ointment simultaneously exhibited bactericidal activity against S. aureus and inhibited haemolysis. In an LAA-treated mouse model of an MRSA-infected skin wound, the mean bacterial colony count decreased to approximately 102 CFU/mg at 18 h after treatment (and the bacteria became undetectable at 96 h), whereas the mean count in untreated mice was approximately 105 CFU/mg of tissue. The LAA ointment also reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IFN-γ) and accelerated wound healing in the mouse model. These data demonstrate the potential efficacy of a combination of LysGH15 and api for use as a topical antimicrobial agent against S. aureus. Full article
(This article belongs to the Special Issue Phage Lytic Enzymes and Their Applications)
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Open AccessArticle Transposition Behavior Revealed by High-Resolution Description of Pseudomonas Aeruginosa Saltovirus Integration Sites
Viruses 2018, 10(5), 245; https://doi.org/10.3390/v10050245
Received: 9 April 2018 / Revised: 27 April 2018 / Accepted: 4 May 2018 / Published: 7 May 2018
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Abstract
Transposable phages, also called saltoviruses, of which the Escherichia coli phage Mu is the reference, are temperate phages that multiply their genome through replicative transposition at multiple sites in their host chromosome. The viral genome is packaged together with host DNA at both
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Transposable phages, also called saltoviruses, of which the Escherichia coli phage Mu is the reference, are temperate phages that multiply their genome through replicative transposition at multiple sites in their host chromosome. The viral genome is packaged together with host DNA at both ends. In the present work, genome sequencing of three Pseudomonas aeruginosa transposable phages, HW12, 2P1, and Ab30, incidentally gave us access to the location of thousands of replicative integration sites and revealed the existence of a variable number of hotspots. Taking advantage of deep sequencing, we then designed an experiment to study 13,000,000 transposon integration sites of bacteriophage Ab30. The investigation revealed the presence of 42 transposition hotspots adjacent to bacterial interspersed mosaic elements (BIME) accounting for 5% of all transposition sites. The rest of the sites appeared widely distributed with the exception of coldspots associated with low G-C content segments, including the putative O-antigen biosynthesis cluster. Surprisingly, 0.4% of the transposition events occurred in a copy of the phage genome itself, indicating that the previously described immunity against such events is slightly leaky. This observation allowed drawing an image of the phage chromosome supercoiling into four loops. Full article
(This article belongs to the Special Issue Bacteriophage Genomes and Genomics: News from the Wild)
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Open AccessArticle A VP26-mNeonGreen Capsid Fusion HSV-2 Mutant Reactivates from Viral Latency in the Guinea Pig Genital Model with Normal Kinetics
Viruses 2018, 10(5), 246; https://doi.org/10.3390/v10050246
Received: 9 April 2018 / Revised: 1 May 2018 / Accepted: 3 May 2018 / Published: 8 May 2018
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Abstract
Fluorescent herpes simplex viruses (HSV) are invaluable tools for localizing virus in cells, permitting visualization of capsid trafficking and enhancing neuroanatomical research. Fluorescent viruses can also be used to study virus kinetics and reactivation in vivo. Such studies would be facilitated by fluorescent
[...] Read more.
Fluorescent herpes simplex viruses (HSV) are invaluable tools for localizing virus in cells, permitting visualization of capsid trafficking and enhancing neuroanatomical research. Fluorescent viruses can also be used to study virus kinetics and reactivation in vivo. Such studies would be facilitated by fluorescent herpes simplex virus recombinants that exhibit wild-type kinetics of replication and reactivation and that are genetically stable. We engineered an HSV-2 strain expressing the fluorescent mNeonGreen protein as a fusion with the VP26 capsid protein. This virus has normal replication and in vivo recurrence phenotypes, providing an essential improved tool for further study of HSV-2 infection. Full article
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Open AccessCommunication Multiplex Detection of Aspergillus fumigatus Mycoviruses
Viruses 2018, 10(5), 247; https://doi.org/10.3390/v10050247
Received: 5 April 2018 / Revised: 5 May 2018 / Accepted: 6 May 2018 / Published: 8 May 2018
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Abstract
Mycoviruses are viruses that naturally infect and replicate in fungi. They are widespread in all major fungal groups including plant and animal pathogenic fungi. Several dsRNA mycoviruses have been reported in Aspergillus fumigatus. Multiplex polymerase chain reaction (PCR) amplification is a version
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Mycoviruses are viruses that naturally infect and replicate in fungi. They are widespread in all major fungal groups including plant and animal pathogenic fungi. Several dsRNA mycoviruses have been reported in Aspergillus fumigatus. Multiplex polymerase chain reaction (PCR) amplification is a version of PCR that enables amplification of different targets simultaneously. This technique has been widely used for detection and differentiation of viruses especially plant viruses such as those which infect tobacco, potato and garlic. For rapid detection, multiplex RT-PCR was developed to screen new isolates for the presence of A. fumigatus mycoviruses. Aspergillus fumigatus chrysovirus (AfuCV), Aspergillus fumigatus partitivirus (AfuPV-1), and Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) dsRNAs were amplified in separate reactions using a mixture of multiplex primer pairs. It was demonstrated that in the presence of a single infection, primer pair mixtures only amplify the corresponding single virus infection. Mixed infections using dual or triple combinations of dsRNA viruses were also amplified simultaneously using multiplex RT-PCR. Up until now, methods for the rapid detection of Aspergillus mycoviruses have been restricted to small scale dsRNA extraction approaches which are laborious and for large numbers of samples not as sensitive as RT-PCR. The multiplex RT-PCR assay developed here will be useful for studies on determining the incidence of A. fumigatus mycoviruses. This is the first report on multiplex detection of A. fumigatus mycoviruses. Full article
(This article belongs to the Special Issue Mycoviruses)
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Open AccessArticle Molecular and Clinical Characterization of Chikungunya Virus Infections in Southeast Mexico
Viruses 2018, 10(5), 248; https://doi.org/10.3390/v10050248
Received: 23 March 2018 / Revised: 29 April 2018 / Accepted: 30 April 2018 / Published: 9 May 2018
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Abstract
Chikungunya fever is an arthropod-borne infection caused by Chikungunya virus (CHIKV). Even though clinical features of Chikungunya fever in the Mexican population have been described before, there is no detailed information. The aim of this study was to perform a full description of
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Chikungunya fever is an arthropod-borne infection caused by Chikungunya virus (CHIKV). Even though clinical features of Chikungunya fever in the Mexican population have been described before, there is no detailed information. The aim of this study was to perform a full description of the clinical features in confirmed Chikungunya-infected patients and describe the molecular epidemiology of CHIKV. We evaluated febrile patients who sought medical assistance in Tapachula, Chiapas, Mexico, from June through July 2015. Infection was confirmed with molecular and serological methods. Viruses were isolated and the E1 gene was sequenced. Phylogeny reconstruction was inferred using maximum-likelihood and maximum clade credibility approaches. We studied 52 patients with confirmed CHIKV infection. They were more likely to have wrist, metacarpophalangeal, and knee arthralgia. Two combinations of clinical features were obtained to differentiate between Chikungunya fever and acute undifferentiated febrile illness. We obtained 10 CHIKV E1 sequences that grouped with the Asian lineage. Seven strains diverged from the formerly reported. Patients infected with the divergent CHIKV strains showed a broader spectrum of clinical manifestations. We defined the complete clinical features of Chikungunya fever in patients from Southeastern Mexico. Our results demonstrate co-circulation of different CHIKV strains in the state of Chiapas. Full article
(This article belongs to the Special Issue 6th Pan-American Dengue Research Network Meeting)
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Open AccessArticle Thiol-Activated Hydrogen Sulfide Donors Antiviral and Anti-Inflammatory Activity in Respiratory Syncytial Virus Infection
Viruses 2018, 10(5), 249; https://doi.org/10.3390/v10050249
Received: 12 March 2018 / Revised: 8 May 2018 / Accepted: 8 May 2018 / Published: 10 May 2018
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Abstract
We have recently shown that endogenous hydrogen sulfide (H2S), an important cellular gaseous mediator, exerts an antiviral and anti-inflammatory activity in vitro and in vivo, and that exogenous H2S delivered via the synthetic H2S-releasing compound GYY4137 also
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We have recently shown that endogenous hydrogen sulfide (H2S), an important cellular gaseous mediator, exerts an antiviral and anti-inflammatory activity in vitro and in vivo, and that exogenous H2S delivered via the synthetic H2S-releasing compound GYY4137 also has similar properties. In this study, we sought to extend our findings to a novel class of H2S donors, thiol-activated gem-dithiol-based (TAGDDs). In an in vitro model of human respiratory syncytial virus (RSV) infection, TAGDD-1 treatment significantly reduced viral replication, even when added up to six hours after infection. Using a mouse model of RSV infection, intranasal delivery of TAGDD-1 to infected mice significantly reduced viral replication and lung inflammation, markedly improving clinical disease parameters and pulmonary dysfunction, compared to vehicle treated controls. Overall our results indicate that this novel synthetic class of H2S-releasing compounds exerts antiviral and anti-inflammatory activity in the context of RSV infection and represents a potential novel pharmacological approach to ameliorate viral-induced lung disease. Full article
(This article belongs to the Section Antivirals & Vaccines)
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Open AccessArticle Clostridium perfringens Virulent Bacteriophage CPS2 and Its Thermostable Endolysin LysCPS2
Viruses 2018, 10(5), 251; https://doi.org/10.3390/v10050251
Received: 4 April 2018 / Revised: 10 May 2018 / Accepted: 11 May 2018 / Published: 11 May 2018
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Abstract
Clostridium perfringens is one of the most common causes of food-borne illness. The increasing prevalence of multidrug-resistant bacteria requires the development of alternatives to typical antimicrobial treatments. Here, we isolated and characterized a C. perfringens-specific virulent bacteriophage CPS2 from chicken feces. The
[...] Read more.
Clostridium perfringens is one of the most common causes of food-borne illness. The increasing prevalence of multidrug-resistant bacteria requires the development of alternatives to typical antimicrobial treatments. Here, we isolated and characterized a C. perfringens-specific virulent bacteriophage CPS2 from chicken feces. The CPS2 phage contains a 17,961 bp double-stranded DNA genome with 25 putative ORFs, and belongs to the Picovirinae, subfamily of Podoviridae. Bioinformatic analysis of the CPS2 genome revealed a putative endolysin, LysCPS2, which is homologous to the endolysin of Clostridium phage phiZP2 and phiCP7R. The enzyme showed strong lytic activity against C. perfringens with optimum conditions at pH 7.5–10, 25–65 °C, and over a broad range of NaCl concentrations. Interestingly, LysCPS2 was found to be highly thermostable, with up to 30% of its lytic activity remaining after 10 min of incubation at 95 °C. The cell wall binding domain in the C-terminal region of LysCPS2 showed a binding spectrum specific to C. perfringens strains. This is the first report to characterize highly thermostable endolysin isolated from virulent C. perfringens bacteriophage. The enzyme can be used as an alternative biocontrol and detection agent against C. perfringens. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessArticle Genome Sequences of Akhmeta Virus, an Early Divergent Old World Orthopoxvirus
Viruses 2018, 10(5), 252; https://doi.org/10.3390/v10050252
Received: 18 April 2018 / Revised: 8 May 2018 / Accepted: 11 May 2018 / Published: 12 May 2018
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Abstract
Annotated whole genome sequences of three isolates of the Akhmeta virus (AKMV), a novel species of orthopoxvirus (OPXV), isolated from the Akhmeta and Vani regions of the country Georgia, are presented and discussed. The AKMV genome is similar in genomic content and structure
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Annotated whole genome sequences of three isolates of the Akhmeta virus (AKMV), a novel species of orthopoxvirus (OPXV), isolated from the Akhmeta and Vani regions of the country Georgia, are presented and discussed. The AKMV genome is similar in genomic content and structure to that of the cowpox virus (CPXV), but a lower sequence identity was found between AKMV and Old World OPXVs than between other known species of Old World OPXVs. Phylogenetic analysis showed that AKMV diverged prior to other Old World OPXV. AKMV isolates formed a monophyletic clade in the OPXV phylogeny, yet the sequence variability between AKMV isolates was higher than between the monkeypox virus strains in the Congo basin and West Africa. An AKMV isolate from Vani contained approximately six kb sequence in the left terminal region that shared a higher similarity with CPXV than with other AKMV isolates, whereas the rest of the genome was most similar to AKMV, suggesting recombination between AKMV and CPXV in a region containing several host range and virulence genes. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle Detection of Specific ZIKV IgM in Travelers Using a Multiplexed Flavivirus Microsphere Immunoassay
Viruses 2018, 10(5), 253; https://doi.org/10.3390/v10050253
Received: 28 March 2018 / Revised: 30 April 2018 / Accepted: 10 May 2018 / Published: 12 May 2018
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Abstract
Zika virus (ZIKV) has spread widely in the Pacific and recently throughout the Americas. Unless detected by RT-PCR, confirming an acute ZIKV infection can be challenging. We developed and validated a multiplexed flavivirus immunoglobulin M (IgM) microsphere immunoassay (flaviMIA) which can differentiate ZIKV-specific
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Zika virus (ZIKV) has spread widely in the Pacific and recently throughout the Americas. Unless detected by RT-PCR, confirming an acute ZIKV infection can be challenging. We developed and validated a multiplexed flavivirus immunoglobulin M (IgM) microsphere immunoassay (flaviMIA) which can differentiate ZIKV-specific IgM from that due to other flavivirus infections in humans. The flaviMIA bound 12 inactivated flavivirus antigens, including those from ZIKV and yellow fever virus (YFV), to distinct anti-flavivirus antibody coupled beads. These beads were used to interrogate sera from patients with suspected ZIKV infection following travel to relevant countries. FlaviMIA results were validated by comparison to the ZIKV plaque reduction neutralization test (PRNT). The results highlight the complexity of serological ZIKV diagnosis, particularly in patients previously exposed to or vaccinated against other flaviviruses. We confirmed 99 patients with ZIKV infection by a combination of RT-PCR and serology. Importantly, ZIKV antibodies could be discriminated from those ascribed to other flavivirus infections. Serological results were sometimes confounded by the presence of pre-existing antibodies attributed to previous flavivirus infection or vaccination. Where RT-PCR results were negative, testing of appropriately timed paired sera was necessary to demonstrate seroconversion or differentiation of recent from past infection with or exposure to ZIKV. Full article
(This article belongs to the Special Issue New Advances on Zika Virus Research)
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Open AccessArticle Two Novel Hypovirulence-Associated Mycoviruses in the Phytopathogenic Fungus Botrytis cinerea: Molecular Characterization and Suppression of Infection Cushion Formation
Viruses 2018, 10(5), 254; https://doi.org/10.3390/v10050254
Received: 13 April 2018 / Revised: 6 May 2018 / Accepted: 9 May 2018 / Published: 13 May 2018
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Abstract
Botrytis cinerea is a necrotrophic fungus causing disease on many important agricultural crops. Two novel mycoviruses, namely Botrytis cinerea hypovirus 1 (BcHV1) and Botrytis cinerea fusarivirus 1 (BcFV1), were fully sequenced. The genome of BcHV1 is 10,214 nt long excluding a poly-A tail
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Botrytis cinerea is a necrotrophic fungus causing disease on many important agricultural crops. Two novel mycoviruses, namely Botrytis cinerea hypovirus 1 (BcHV1) and Botrytis cinerea fusarivirus 1 (BcFV1), were fully sequenced. The genome of BcHV1 is 10,214 nt long excluding a poly-A tail and possesses one large open reading frame (ORF) encoding a polyprotein possessing several conserved domains including RNA-dependent RNA polymerase (RdRp), showing homology to hypovirus-encoded polyproteins. Phylogenetic analysis indicated that BcHV1 may belong to the proposed genus Betahypovirus in the viral family Hypoviridae. The genome of BcFV1 is 8411 nt in length excluding the poly A tail and theoretically processes two major ORFs, namely ORF1 and ORF2. The larger ORF1 encoded polypeptide contains protein domains of an RdRp and a viral helicase, whereas the function of smaller ORF2 remains unknown. The BcFV1 was phylogenetically clustered with other fusariviruses forming an independent branch, indicating BcFV1 was a member in Fusariviridae. Both BcHV1 and BcFV1 were capable of being transmitted horizontally through hyphal anastomosis. Infection by BcHV1 alone caused attenuated virulence without affecting mycelial growth, significantly inhibited infection cushion (IC) formation, and altered expression of several IC-formation-associated genes. However, wound inoculation could fully rescue the virulence phenotype of the BcHV1 infected isolate. These results indicate the BcHV1-associated hypovirulence is caused by the viral influence on IC-formation-associated pathways. Full article
(This article belongs to the Special Issue Mycoviruses)
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Open AccessArticle Time-Course Microarray Analysis Reveals Differences between Transcriptional Changes in Tomato Leaves Triggered by Mild and Severe Variants of Potato Spindle Tuber Viroid
Viruses 2018, 10(5), 257; https://doi.org/10.3390/v10050257
Received: 25 April 2018 / Revised: 9 May 2018 / Accepted: 12 May 2018 / Published: 15 May 2018
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Abstract
Viroids are small non-capsidated non-coding RNA replicons that utilize host factors for efficient propagation and spread through the entire plant. They can incite specific disease symptoms in susceptible plants. To better understand viroid-plant interactions, we employed microarray analysis to observe the changes of
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Viroids are small non-capsidated non-coding RNA replicons that utilize host factors for efficient propagation and spread through the entire plant. They can incite specific disease symptoms in susceptible plants. To better understand viroid-plant interactions, we employed microarray analysis to observe the changes of gene expression in “Rutgers” tomato leaves in response to the mild (M) and severe (S23) variants of potato spindle tuber viroid (PSTVd). The changes were analyzed over a time course of viroid infection development: (i) the pre-symptomatic stage; (ii) early symptoms; (iii) full spectrum of symptoms and (iv) the so-called ‘recovery’ stage, when stem regrowth was observed in severely affected plants. Gene expression profiles differed depending on stage of infection and variant. In S23-infected plants, the expression of over 3000 genes was affected, while M-infected plants showed 3-fold fewer differentially expressed genes, only 20% of which were specific to the M variant. The differentially expressed genes included many genes related to stress; defense; hormone metabolism and signaling; photosynthesis and chloroplasts; cell wall; RNA regulation, processing and binding; protein metabolism and modification and others. The expression levels of several genes were confirmed by nCounter analysis. Full article
(This article belongs to the Special Issue Viroid-2018: International Conference on Viroids and Viroid-Like RNAs)
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Open AccessArticle CRISPR/Cas9 Mutagenesis of UL21 in Multiple Strains of Herpes Simplex Virus Reveals Differential Requirements for pUL21 in Viral Replication
Viruses 2018, 10(5), 258; https://doi.org/10.3390/v10050258
Received: 26 April 2018 / Revised: 10 May 2018 / Accepted: 13 May 2018 / Published: 15 May 2018
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Abstract
Studies from multiple laboratories using different strains or species of herpes simplex virus (HSV) with deletions in UL21 have yielded conflicting results regarding the necessity of pUL21 in HSV infection. To resolve this discrepancy, we utilized CRISPR/Cas9 mutagenesis to isolate pUL21 deficient viruses
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Studies from multiple laboratories using different strains or species of herpes simplex virus (HSV) with deletions in UL21 have yielded conflicting results regarding the necessity of pUL21 in HSV infection. To resolve this discrepancy, we utilized CRISPR/Cas9 mutagenesis to isolate pUL21 deficient viruses in multiple HSV backgrounds, and performed a side-by-side comparison of the cell-to-cell spread and replication phenotypes of these viruses. These analyses confirmed previous studies implicating the involvement of pUL21 in cell-to-cell spread of HSV. Cell-to-cell spread of HSV-2 was more greatly affected by the lack of pUL21 than HSV-1, and strain-specific differences in the requirement for pUL21 in cell-to-cell spread were also noted. HSV-2 strain 186 lacking pUL21 was particularly crippled in both cell-to-cell spread and viral replication in non-complementing cells, in comparison to other HSV strains lacking pUL21, suggesting that the strict requirement for pUL21 by strain 186 may not be representative of the HSV-2 species as a whole. This work highlights CRISPR/Cas9 technology as a useful tool for rapidly constructing deletion mutants of alphaherpesviruses, regardless of background strain, and should find great utility whenever strain-specific differences need to be investigated. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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Open AccessArticle Zika Virus Induces Autophagy in Human Umbilical Vein Endothelial Cells
Viruses 2018, 10(5), 259; https://doi.org/10.3390/v10050259
Received: 27 March 2018 / Revised: 8 May 2018 / Accepted: 9 May 2018 / Published: 15 May 2018
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Abstract
Autophagy is a common strategy for cell protection; however, some viruses can in turn adopt cellular autophagy to promote viral replication. Zika virus (ZIKV) is the pathogen that causes Zika viral disease, and it is a mosquito-borne virus. However, its pathogenesis, especially the
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Autophagy is a common strategy for cell protection; however, some viruses can in turn adopt cellular autophagy to promote viral replication. Zika virus (ZIKV) is the pathogen that causes Zika viral disease, and it is a mosquito-borne virus. However, its pathogenesis, especially the interaction between ZIKV and target cells during the early stages of infection, is still unclear. In this study, we demonstrate that infecting human umbilical vein endothelial cells (HUVEC) with ZIKV triggers cellular autophagy. We observed both an increase in the conversion of LC3-I to LC3-II and increased accumulation of fluorescent cells with LC3 dots, which are considered to be the two key indicators of autophagy. The ratio of LC3-II/GAPDH in each group was significantly increased at different times after ZIKV infection at different MOIs, indicating that the production of lipidated LC3-II increased. Moreover, both the ratio of LC3-II/GAPDH and the expression of viral NS3 protein increased with increasing time of viral infection. The expression level of p62 decreased gradually from 12 h post-infection. Expression profile of double fluorescent protein labelling LC3 indicated that the autophagy induced by ZIKV infection was a complete process. We further investigated the role of autophagy in ZIKV replication. We demonstrated that either the treatment with inhibitors of autophagosomes formation or short hairpin RNA targeting the Beclin-1 gene, which is critical for the formation of autophagosomes, significantly reduced viral production. Taken together, our results indicate that ZIKV infection induces autophagy of HUVEC, and inhibition of ZIKV-induced autophagy restrains viral replication. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle Blackcurrant Leaf Chlorosis Associated Virus: Evidence of the Presence of Circular RNA during Infections
Viruses 2018, 10(5), 260; https://doi.org/10.3390/v10050260
Received: 30 March 2018 / Revised: 27 April 2018 / Accepted: 11 May 2018 / Published: 15 May 2018
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Abstract
Blackcurrant leaf chlorosis associated virus (BCLCaV) was detected recently by next-generation sequencing (NGS) and a new and distinct species in the genus Idaeovirus was proposed. Analysis of NGS-derived paired-end reads revealed the existence of bridge reads encompassing the 3′-terminus and 5′-terminus of RNA-2
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Blackcurrant leaf chlorosis associated virus (BCLCaV) was detected recently by next-generation sequencing (NGS) and a new and distinct species in the genus Idaeovirus was proposed. Analysis of NGS-derived paired-end reads revealed the existence of bridge reads encompassing the 3′-terminus and 5′-terminus of RNA-2 or RNA-3 of BCLCaV. The full RNA-2 or RNA-3 could be amplified using outward facing or abutting primers; also, RNA-2/RNA-3 could be detected even after three consecutive RNase R enzyme treatments, with denaturation at 95 °C preceding each digestion. Evidence was obtained indicating that there are circular forms of BCLCaV RNA-2 and RNA-3. Full article
(This article belongs to the Special Issue Fruit Tree Viruses and Viroids)
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Open AccessArticle Gp120 V5 Is Targeted by the First Wave of Sequential Neutralizing Antibodies in SHIVSF162P3N-Infected Rhesus Macaques
Viruses 2018, 10(5), 262; https://doi.org/10.3390/v10050262
Received: 23 April 2018 / Revised: 11 May 2018 / Accepted: 14 May 2018 / Published: 16 May 2018
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Abstract
Simian-human immunodeficiency virus (SHIV) infection provides a relevant animal model to study HIV-1 neutralization breadth. With previously identified SHIVSF162P3N infected rhesus macaques that did or did not develop neutralization breadth, we characterized the transmitted/founder viruses and initial autologous/homologous neutralizing antibodies in these
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Simian-human immunodeficiency virus (SHIV) infection provides a relevant animal model to study HIV-1 neutralization breadth. With previously identified SHIVSF162P3N infected rhesus macaques that did or did not develop neutralization breadth, we characterized the transmitted/founder viruses and initial autologous/homologous neutralizing antibodies in these animals. The plasma viral load and blood CD4 count did not distinguish macaques with and without breadth, and only one tested homologous envelope clone revealed a trend for macaques with breadth to favor an early homologous response. In two macaques with breadth, GB40 and FF69, infected with uncloned SHIVSF162P3N, multiple viral variants were transmitted, and the transmitted variants were not equal in neutralization sensitivity. The targets of initial autologous neutralizing antibodies, arising between 10 and 20 weeks post infection, were mapped to N462 glycan and G460a in gp120 V5 in GB40 and FF69, respectively. Although it is unclear whether these targets are related to later neutralization breadth development, the G460a target but not N462 glycan appeared more common in macaques with breadth than those without. Longitudinal plasmas revealed 2–3 sequential waves of neutralizing antibodies in macaques with breadth, implicating that 3 sequential envelope variants, if not more, may be required for the broadening of HIV-1 neutralizing antibodies. Full article
(This article belongs to the Special Issue HIV Vaccines)
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Open AccessArticle Encapsidated Host Factors in Alphavirus Particles Influence Midgut Infection of Aedes aegypti
Viruses 2018, 10(5), 263; https://doi.org/10.3390/v10050263
Received: 10 April 2018 / Revised: 8 May 2018 / Accepted: 11 May 2018 / Published: 16 May 2018
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Abstract
Transmission of mosquito-borne viruses requires the efficient infection of both a permissive vertebrate host and a competent mosquito vector. The infectivity of Sindbis virus (SINV), the type species of the Alphavirus genus, is influenced by both the original and new host cell. We
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Transmission of mosquito-borne viruses requires the efficient infection of both a permissive vertebrate host and a competent mosquito vector. The infectivity of Sindbis virus (SINV), the type species of the Alphavirus genus, is influenced by both the original and new host cell. We have shown that infection of vertebrate cells by SINV, chikungunya virus (CHIKV), and Ross River virus (RRV) produces two subpopulations of virus particles separable based on density. In contrast, a single population of viral particles is produced by mosquito cells. Previous studies demonstrated that the denser vertebrate-derived particles and the mosquito-derived particles contain components of the small subunit of the host cell ribosome, whereas the less dense vertebrate-derived particles do not. Infection of mice with RRV showed that both particle subpopulations are produced in an infected vertebrate, but in a tissue specific manner with serum containing only the less dense version of the virus particles. Previous infectivity studies using SINV particles have shown that the denser particles (SINVHeavy) and mosquito derived particles SINVC6/36 are significantly more infectious in vertebrate cells than the less dense vertebrate derived particles (SINVLight). The current study shows that SINVLight particles, initiate the infection of the mosquito midgut more efficiently than SINVHeavy particles and that this enhanced infectivity is associated with an exacerbated immune response to SINVLight infection in midgut tissues. The enhanced infection of SINVLight is specific to the midgut as intrathoracically injected virus do not exhibit the same fitness advantage. Together, our data indicate a biologically significant role for the SINVLight subpopulation in the efficient transmission from infected vertebrates to the mosquito vector. Full article
(This article belongs to the Special Issue Advances in Alphavirus Research)
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Open AccessArticle Strain-Specific Antagonism of the Human H1N1 Influenza A Virus against Equine Tetherin
Viruses 2018, 10(5), 264; https://doi.org/10.3390/v10050264
Received: 21 March 2018 / Revised: 11 May 2018 / Accepted: 14 May 2018 / Published: 16 May 2018
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Abstract
Tetherin/BST-2/CD317 is an interferon-induced host restriction factor that can block the budding of enveloped viruses by tethering them to the cell surface. Many viruses use certain proteins to counteract restriction by tetherin from their natural hosts, but not from other species. The influenza
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Tetherin/BST-2/CD317 is an interferon-induced host restriction factor that can block the budding of enveloped viruses by tethering them to the cell surface. Many viruses use certain proteins to counteract restriction by tetherin from their natural hosts, but not from other species. The influenza A virus (FLUAV) has a wide range of subtypes with different host tropisms. Human tetherin (huTHN) has been reported to restrict only specific FLUAV strains and the viral hemagglutinin (HA) and neuraminidase (NA) genes determine the sensitivity to huTHN. Whether tetherins from other hosts can block human FLUAV is still unknown. Here, we evaluate the impact of equine tetherin (eqTHN) and huTHN on the replication of A/Sichuan/1/2009 (H1N1) and A/equine/Xinjiang/1/2007 (H3N8) strains. Our results show that eqTHN had higher restriction activity towards both viruses, and its shorter cytoplasmic tail contributed to that activity. We further demonstrated that HA and NA of A/Hamburg/4/2009 (H1N1) could counteract eqTHN. Notably, our results indicate that four amino acids, 13T and 49L of HA and 32T and 80V of NA, were involved in blocking the restriction activity of eqTHN. These findings reveal interspecies restriction by eqTHN towards FLUAV, and the role of the HA and NA proteins in overcoming this restriction. Full article
(This article belongs to the Special Issue What’s New with Flu?)
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Open AccessArticle Inhibition of Human Immunodeficiency Virus Type 1 Entry by a Keggin Polyoxometalate
Viruses 2018, 10(5), 265; https://doi.org/10.3390/v10050265
Received: 20 March 2018 / Revised: 11 May 2018 / Accepted: 14 May 2018 / Published: 16 May 2018
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Abstract
Here, we report the anti-human immunodeficiency virus (HIV) potency and underlying mechanisms of a Keggin polyoxometalate (PT-1, K6HPTi2W10O40). Our findings showed that PT-1 exhibited highly potent effects against a diverse group of HIV type 1
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Here, we report the anti-human immunodeficiency virus (HIV) potency and underlying mechanisms of a Keggin polyoxometalate (PT-1, K6HPTi2W10O40). Our findings showed that PT-1 exhibited highly potent effects against a diverse group of HIV type 1 (HIV-1) strains and displayed low cytotoxicity and genotoxicity. The time-addition assay revealed that PT-1 acted at an early stage of infection, and these findings were supported by the observation that PT-1 had more potency against Env-pseudotyped virus than vesicular stomatitis virus glycoprotein (VSVG) pseudotyped virus. Surface plasmon resonance binding assays and flow cytometry analysis showed that PT-1 blocked the gp120 binding site in the CD4 receptor. Moreover, PT-1 bound directly to gp41 NHR (N36 peptide), thereby interrupting the core bundle formation of gp41. In conclusion, our data suggested that PT-1 may be developed as a new anti-HIV-1 agent through its effects on entry inhibition. Full article
(This article belongs to the Section Antivirals & Vaccines)
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Open AccessArticle Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line
Viruses 2018, 10(5), 266; https://doi.org/10.3390/v10050266
Received: 3 May 2018 / Revised: 14 May 2018 / Accepted: 14 May 2018 / Published: 16 May 2018
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Abstract
Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with
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Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with ectromelia virus (ECTV). We used two different cell lines and a combination of immunofluorescence techniques, confocal and electron microscopy, and flow cytometry to address subcellular changes following infection. Early in infection of L929 fibroblasts and RAW 264.7 macrophages, mitochondria gathered around viral factories. Later, the mitochondrial network became fragmented, forming punctate mitochondria that co-localized with the progeny virions. ECTV-co-localized mitochondria associated with the cytoskeleton components. Mitochondrial membrane potential, mitochondrial fission–fusion, mitochondrial mass, and generation of reactive oxygen species (ROS) were severely altered later in ECTV infection leading to damage of mitochondria. These results suggest an important role of mitochondria in supplying energy for virus replication and morphogenesis. Presumably, mitochondria participate in transport of viral particles inside and outside of the cell and/or they are a source of membranes for viral envelope formation. We speculate that the observed changes in the mitochondrial network organization and physiology in ECTV-infected cells provide suitable conditions for viral replication and morphogenesis. Full article
(This article belongs to the Special Issue Cytoskeleton in Virus Infections)
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Open AccessArticle Paradoxical Effect of Chloroquine Treatment in Enhancing Chikungunya Virus Infection
Viruses 2018, 10(5), 268; https://doi.org/10.3390/v10050268
Received: 25 April 2018 / Revised: 11 May 2018 / Accepted: 12 May 2018 / Published: 17 May 2018
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Abstract
Since 2005, Chikungunya virus (CHIKV) re-emerged and caused numerous outbreaks in the world, and finally, was introduced into the Americas in 2013. The lack of CHIKV-specific therapies has led to the use of non-specific drugs. Chloroquine, which is commonly used to treat febrile
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Since 2005, Chikungunya virus (CHIKV) re-emerged and caused numerous outbreaks in the world, and finally, was introduced into the Americas in 2013. The lack of CHIKV-specific therapies has led to the use of non-specific drugs. Chloroquine, which is commonly used to treat febrile illnesses in the tropics, has been shown to inhibit CHIKV replication in vitro. To assess the in vivo effect of chloroquine, two complementary studies were performed: (i) a prophylactic study in a non-human primate model (NHP); and (ii) a curative study “CuraChik”, which was performed during the Reunion Island outbreak in 2006 in a human cohort. Clinical, biological, and immunological data were compared between treated and placebo groups. Acute CHIKV infection was exacerbated in NHPs treated with prophylactic administration of chloroquine. These NHPs displayed a higher viremia and slower viral clearance (p < 0.003). Magnitude of viremia was correlated to the type I IFN response (Rho = 0.8, p < 0.001) and severe lymphopenia (Rho = 0.8, p < 0.0001), while treatment led to a delay in both CHIKV-specific cellular and IgM responses (p < 0.02 and p = 0.04, respectively). In humans, chloroquine treatment did not affect viremia or clinical parameters during the acute stage of the disease (D1 to D14), but affected the levels of C-reactive Protein (CRP), IFNα, IL-6, and MCP1 over time (D1 to D16). Importantly, no positive effect could be detected on prevalence of persistent arthralgia at Day 300. Although inhibitory in vitro, chloroquine as a prophylactic treatment in NHPs enhances CHIKV replication and delays cellular and humoral response. In patients, curative chloroquine treatment during the acute phase decreases the levels of key cytokines, and thus may delay adaptive immune responses, as observed in NHPs, without any suppressive effect on peripheral viral load. Full article
(This article belongs to the Special Issue Advances in Alphavirus Research)
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Open AccessCommunication A Novel Hepadnavirus Identified in an Immunocompromised Domestic Cat in Australia
Viruses 2018, 10(5), 269; https://doi.org/10.3390/v10050269
Received: 27 April 2018 / Revised: 10 May 2018 / Accepted: 14 May 2018 / Published: 17 May 2018
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Abstract
High-throughput transcriptome sequencing allows for the unbiased detection of viruses in host tissues. The application of this technique to immunosuppressed animals facilitates the detection of viruses that might otherwise be excluded or contained in immunocompetent individuals. To identify potential viral pathogens infecting domestic
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High-throughput transcriptome sequencing allows for the unbiased detection of viruses in host tissues. The application of this technique to immunosuppressed animals facilitates the detection of viruses that might otherwise be excluded or contained in immunocompetent individuals. To identify potential viral pathogens infecting domestic cats we performed high-throughput transcriptome sequencing of tissues from cats infected with feline immunodeficiency virus (FIV). A novel member of the Hepadnaviridae, tentatively named domestic cat hepadnavirus, was discovered in a lymphoma sample and its complete 3187 bp genome characterized. Phylogenetic analysis placed the domestic cat hepadnavirus as a divergent member of mammalian orthohepadnaviruses that exhibits no close relationship to any other virus. DNA extracted from whole blood from pet cats was positive for the novel hepadnavirus by PCR in 6 of 60 (10%) FIV-infected cats and 2 of 63 (3.2%) FIV-uninfected cats. The higher prevalence of hepadnavirus viraemia detected in FIV-infected cats mirrors that seen in human immunodeficiency virus-infected humans coinfected with hepatitis B virus. In summary, we report the first hepadnavirus infection in a carnivore and the first in a companion animal. The natural history, epidemiology and pathogenic potential of domestic cat hepadnavirus merits additional investigation. Full article
(This article belongs to the Special Issue Emerging Viruses)
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Open AccessArticle Avian Influenza Virus Subtype H9N2 Affects Intestinal Microbiota, Barrier Structure Injury, and Inflammatory Intestinal Disease in the Chicken Ileum
Viruses 2018, 10(5), 270; https://doi.org/10.3390/v10050270
Received: 28 April 2018 / Revised: 14 May 2018 / Accepted: 15 May 2018 / Published: 18 May 2018
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Abstract
Avian influenza virus subtype H9N2 (H9N2 AIV) has caused significant losses to the poultry industry due to the high mortality associated with secondary infections attributable to E. coli. This study tries to address the underlying secondary mechanisms after H9N2 AIV infection. Initially,
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Avian influenza virus subtype H9N2 (H9N2 AIV) has caused significant losses to the poultry industry due to the high mortality associated with secondary infections attributable to E. coli. This study tries to address the underlying secondary mechanisms after H9N2 AIV infection. Initially, nine day-old specific pathogen-free chickens were assigned to control (uninfected) and H9N2-infected groups, respectively. Using Illumina sequencing, histological examination, and quantitative real-time PCR, it was found that H9N2 AIV caused intestinal microbiota disorder, injury, and inflammatory damage to the intestinal mucosa. Notably, the genera Escherichia, especially E. coli, significantly increased (p < 0.01) at five days post-infection (dpi), while Lactobacillus, Enterococcus, and other probiotic organisms were significantly reduced (p < 0.01). Simultaneously, the mRNA expression of tight junction proteins (ZO-1, claudin 3, and occludin), TFF2, and Muc2 were significantly reduced (p < 0.01), indicating the destruction of the intestinal epithelial cell tight junctions and the damage of mucin layer construction. Moreover, the mRNA expression of proinflammatory cytokines IFN-γ, IL-22, IFN-α, and IL-17A in intestinal epithelial cells were significantly upregulated, resulting in the inflammatory response and intestinal injury. Our findings may provide a theoretical basis for observed gastroenteritis-like symptoms such as diarrhea and secondary E. coli infection following H9N2 AIV infection. Full article
(This article belongs to the Special Issue Animal Models for Viral Diseases)
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Open AccessArticle Synergistic Viral Replication of Marek’s Disease Virus and Avian Leukosis Virus Subgroup J is Responsible for the Enhanced Pathogenicity in the Superinfection of Chickens
Viruses 2018, 10(5), 271; https://doi.org/10.3390/v10050271
Received: 31 March 2018 / Revised: 9 May 2018 / Accepted: 15 May 2018 / Published: 18 May 2018
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Abstract
Superinfection of Marek’s disease virus (MDV) and avian leukosis virus subgroup J (ALV-J) causes lethal neoplasia and death in chickens. However, whether there is synergism between the two viruses in viral replication and pathogenicity has remained elusive. In this study, we found that
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Superinfection of Marek’s disease virus (MDV) and avian leukosis virus subgroup J (ALV-J) causes lethal neoplasia and death in chickens. However, whether there is synergism between the two viruses in viral replication and pathogenicity has remained elusive. In this study, we found that the superinfection of MDV and ALV-J increased the viral replication of the two viruses in RNA and protein level, and synergistically promoted the expression of IL-10, IL-6, and TGF-β in chicken embryo fibroblasts (CEF). Moreover, MDV and ALV-J protein expression in dual-infected cells detected by confocal laser scanning microscope appeared earlier in the cytoplasm and the nucleus, and caused more severe cytopathy than single infection, suggesting that synergistically increased MDV and ALV-J viral-protein biosynthesis is responsible for the severe cytopathy. In vivo, compared to the single virus infected chickens, the mortality and tumor formation rates increased significantly in MDV and ALV-J dual-infected chickens. Viral loads of MDV and ALV-J in tissues of dual-infected chickens were significantly higher than those of single-infected chickens. Histopathology observation showed that more severe inflammation and tumor cells metastases were present in dual-infected chickens. In the present study, we concluded that synergistic viral replication of MDV and ALV-J is responsible for the enhanced pathogenicity in superinfection of chickens. Full article
(This article belongs to the Special Issue Animal Models for Viral Diseases)
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Open AccessArticle Adaptive Mutations in Influenza A/California/07/2009 Enhance Polymerase Activity and Infectious Virion Production
Viruses 2018, 10(5), 272; https://doi.org/10.3390/v10050272
Received: 16 March 2018 / Revised: 15 May 2018 / Accepted: 15 May 2018 / Published: 18 May 2018
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Abstract
Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Here, we report the adaptation
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Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (also known as CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering three PB1 and PA mutations into the parental CA/07 strain, we demonstrated that these mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation. Full article
(This article belongs to the Special Issue What’s New with Flu?)
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Open AccessArticle Following Acute Encephalitis, Semliki Forest Virus is Undetectable in the Brain by Infectivity Assays but Functional Virus RNA Capable of Generating Infectious Virus Persists for Life
Viruses 2018, 10(5), 273; https://doi.org/10.3390/v10050273
Received: 25 April 2018 / Revised: 14 May 2018 / Accepted: 17 May 2018 / Published: 18 May 2018
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Abstract
Alphaviruses are mosquito-transmitted RNA viruses which generally cause acute disease including mild febrile illness, rash, arthralgia, myalgia and more severely, encephalitis. In the mouse, peripheral infection with Semliki Forest virus (SFV) results in encephalitis. With non-virulent strains, infectious virus is detectable in the
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Alphaviruses are mosquito-transmitted RNA viruses which generally cause acute disease including mild febrile illness, rash, arthralgia, myalgia and more severely, encephalitis. In the mouse, peripheral infection with Semliki Forest virus (SFV) results in encephalitis. With non-virulent strains, infectious virus is detectable in the brain, by standard infectivity assays, for around ten days. As we have shown previously, in severe combined immunodeficient (SCID) mice, infectious virus is detectable for months in the brain. Here we show that in MHC-II-/- mice, with no functional CD4 T-cells, infectious virus is also detectable in the brain for long periods. In contrast, in the brains of CD8-/- mice, virus RNA persists but infectious virus is not detectable. In SCID mice infected with SFV, repeated intraperitoneal administration of anti-SFV immune serum rapidly reduced the titer of infectious virus in the brain to undetectable, however virus RNA persisted. Repeated intraperitoneal passive transfer of immune serum resulted in maintenance of brain virus RNA, with no detectable infectious virus, for several weeks. When passive antibody transfer was stopped, antibody levels declined and infectious virus was again detectable in the brain. In aged immunocompetent mice, previously infected with SFV, immunosuppression of antibody responses many months after initial infection also resulted in renewed ability to detect infectious virus in the brain. In summary, antiviral antibodies control and determine whether infectious virus is detectable in the brain but immune responses cannot clear this infection from the brain. Functional virus RNA capable of generating infectious virus persists and if antibody levels decline, infectious virus is again detectable. Full article
(This article belongs to the Special Issue Advances in Alphavirus Research)
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Open AccessArticle The Occurrence of a Commercial Npro and Erns Double Mutant BVDV-1 Live-Vaccine Strain in Newborn Calves
Viruses 2018, 10(5), 274; https://doi.org/10.3390/v10050274
Received: 25 April 2018 / Revised: 15 May 2018 / Accepted: 17 May 2018 / Published: 19 May 2018
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Abstract
The major source for the spread of bovine viral diarrhea virus (BVDV) are in-utero infected, immunotolerant, persistently infected (PI) animals since they shed enormous amounts of viruses throughout their lives. During the sequence-based virus typing of diagnostic ear notch samples performed in the
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The major source for the spread of bovine viral diarrhea virus (BVDV) are in-utero infected, immunotolerant, persistently infected (PI) animals since they shed enormous amounts of viruses throughout their lives. During the sequence-based virus typing of diagnostic ear notch samples performed in the context of the obligatory German BVDV eradication program, the commercial Npro and Erns double mutant BVDV-1 live-vaccine strain KE-9 was detected in seven newborn calves; their mothers were immunized in the first trimester of gestation. Six calves either succumbed or were culled immediately, but the one remaining animal was closely monitored for six months. The viral RNA was detected in the skin sample taken in its first and fifth week of life, but the virus could not be isolated. Further skin biopsies that were taken at monthly intervals as well as every serum and urine sample, nasal, oral, and rectal swabs taken weekly tested BVDV negative. However, neutralizing titers against BVDV-1 remained at a consistently high level. To further control for virus shedding, a BVDV antibody and antigen negative calf was co-housed which remained negative throughout the study. The missing viremia, a lack of excretion of infectious virus and negative follow-up skin samples combined with consistently high antibody titers speak against the induction of the classical persistent infection by vaccination with recombinant KE-9 during gestation. We, therefore, suggest that the epidemiological impact of the RNA/antigen positivity for an extended period in the skin is very low. The detection of live-vaccine viruses in skin biopsies mainly represents a diagnostic issue in countries that implemented ear notch-based control programs; and KE9-specific RT-PCRs or sequence analysis can be used to identify these animals and avoid culling measures. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessArticle Discovery and Biochemical Characterization of PlyP56, PlyN74, and PlyTB40—Bacillus Specific Endolysins
Viruses 2018, 10(5), 276; https://doi.org/10.3390/v10050276
Received: 30 April 2018 / Revised: 15 May 2018 / Accepted: 17 May 2018 / Published: 21 May 2018
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Abstract
Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a
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Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0–8.0), over a broad temperature range (4–55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies. Full article
(This article belongs to the Special Issue Phage Lytic Enzymes and Their Applications)
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Open AccessReview Framing the Future with Bacteriophages in Agriculture
Viruses 2018, 10(5), 218; https://doi.org/10.3390/v10050218
Received: 27 March 2018 / Revised: 20 April 2018 / Accepted: 22 April 2018 / Published: 25 April 2018
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Abstract
The ability of agriculture to continually provide food to a growing world population is of crucial importance. Bacterial diseases of plants and animals have continually reduced production since the advent of crop cultivation and animal husbandry practices. Antibiotics have been used extensively to
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The ability of agriculture to continually provide food to a growing world population is of crucial importance. Bacterial diseases of plants and animals have continually reduced production since the advent of crop cultivation and animal husbandry practices. Antibiotics have been used extensively to mitigate these losses. The rise of antimicrobial resistant (AMR) bacteria, however, together with consumers’ calls for antibiotic-free products, presents problems that threaten sustainable agriculture. Bacteriophages (phages) are proposed as bacterial population control alternatives to antibiotics. Their unique properties make them highly promising but challenging antimicrobials. The use of phages in agriculture also presents a number of unique challenges. This mini-review summarizes recent development and perspectives of phages used as antimicrobial agents in plant and animal agriculture at the farm level. The main pathogens and their adjoining phage therapies are discussed. Full article
(This article belongs to the Special Issue Hurdles for Phage Therapy (PT) to Become a Reality)
Open AccessReview A Systematic Review of the Natural Virome of Anopheles Mosquitoes
Viruses 2018, 10(5), 222; https://doi.org/10.3390/v10050222
Received: 7 April 2018 / Revised: 20 April 2018 / Accepted: 21 April 2018 / Published: 25 April 2018
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Abstract
Anopheles mosquitoes are vectors of human malaria, but they also harbor viruses, collectively termed the virome. The Anopheles virome is relatively poorly studied, and the number and function of viruses are unknown. Only the o’nyong-nyong arbovirus (ONNV) is known to be consistently transmitted
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Anopheles mosquitoes are vectors of human malaria, but they also harbor viruses, collectively termed the virome. The Anopheles virome is relatively poorly studied, and the number and function of viruses are unknown. Only the o’nyong-nyong arbovirus (ONNV) is known to be consistently transmitted to vertebrates by Anopheles mosquitoes. A systematic literature review searched four databases: PubMed, Web of Science, Scopus, and Lissa. In addition, online and print resources were searched manually. The searches yielded 259 records. After screening for eligibility criteria, we found at least 51 viruses reported in Anopheles, including viruses with potential to cause febrile disease if transmitted to humans or other vertebrates. Studies to date have not provided evidence that Anopheles consistently transmit and maintain arboviruses other than ONNV. However, anthropophilic Anopheles vectors of malaria are constantly exposed to arboviruses in human bloodmeals. It is possible that in malaria-endemic zones, febrile symptoms may be commonly misdiagnosed. It is also possible that anophelines may be inherently less competent arbovirus vectors than culicines, but if true, the biological basis would warrant further study. This systematic review contributes a context to characterize the biology, knowledge gaps, and potential public health risk of Anopheles viruses. Full article
(This article belongs to the Section Insect Viruses)
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Open AccessReview Paneth Cells during Viral Infection and Pathogenesis
Viruses 2018, 10(5), 225; https://doi.org/10.3390/v10050225
Received: 12 March 2018 / Revised: 17 April 2018 / Accepted: 24 April 2018 / Published: 26 April 2018
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Abstract
Paneth cells are major secretory cells located in the crypts of Lieberkühn in the small intestine. Our understanding of the diverse roles that Paneth cells play in homeostasis and disease has grown substantially since their discovery over a hundred years ago. Classically, Paneth
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Paneth cells are major secretory cells located in the crypts of Lieberkühn in the small intestine. Our understanding of the diverse roles that Paneth cells play in homeostasis and disease has grown substantially since their discovery over a hundred years ago. Classically, Paneth cells have been characterized as a significant source of antimicrobial peptides and proteins important in host defense and shaping the composition of the commensal microbiota. More recently, Paneth cells have been shown to supply key developmental and homeostatic signals to intestinal stem cells in the crypt base. Paneth cell dysfunction leading to dysbiosis and a compromised epithelial barrier have been implicated in the etiology of Crohn’s disease and susceptibility to enteric bacterial infection. Our understanding of the impact of Paneth cells on viral infection is incomplete. Enteric α-defensins, produced by Paneth cells, can directly alter viral infection. In addition, α-defensins and other antimicrobial Paneth cell products may modulate viral infection indirectly by impacting the microbiome. Here, we discuss recent insights into Paneth cell biology, models to study their function, and the impact, both direct and indirect, of Paneth cells on enteric viral infection. Full article
(This article belongs to the Special Issue Viruses–Bacteria Interactions in the Gut)
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Open AccessReview Epigenetic Modulation of CD8+ T Cell Function in Lentivirus Infections: A Review
Viruses 2018, 10(5), 227; https://doi.org/10.3390/v10050227
Received: 22 March 2018 / Revised: 23 April 2018 / Accepted: 24 April 2018 / Published: 28 April 2018
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Abstract
CD8+ T cells are critical for controlling viremia during human immunodeficiency virus (HIV) infection. These cells produce cytolytic factors and antiviral cytokines that eliminate virally- infected cells. During the chronic phase of HIV infection, CD8+ T cells progressively lose their proliferative
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CD8+ T cells are critical for controlling viremia during human immunodeficiency virus (HIV) infection. These cells produce cytolytic factors and antiviral cytokines that eliminate virally- infected cells. During the chronic phase of HIV infection, CD8+ T cells progressively lose their proliferative capacity and antiviral functions. These dysfunctional cells are unable to clear the productively infected and reactivated cells, representing a roadblock in HIV cure. Therefore, mechanisms to understand CD8+ T cell dysfunction and strategies to boost CD8+ T cell function need to be investigated. Using the feline immunodeficiency virus (FIV) model for lentiviral persistence, we have demonstrated that CD8+ T cells exhibit epigenetic changes such as DNA demethylation during the course of infection as compared to uninfected cats. We have also demonstrated that lentivirus-activated CD4+CD25+ T regulatory cells induce forkhead box P3 (Foxp3) expression in virus-specific CD8+ T cell targets, which binds the interleukin (IL)-2, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ promoters in these CD8+ T cells. Finally, we have reported that epigenetic modulation reduces Foxp3 binding to these promoter regions. This review compares and contrasts our current understanding of CD8+ T cell epigenetics and mechanisms of lymphocyte suppression during the course of lentiviral infection for two animal models, FIV and simian immunodeficiency virus (SIV). Full article
(This article belongs to the Special Issue Nonprimate Lentivirus)
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Open AccessReview Defense Mechanisms against Viral Infection in Drosophila: RNAi and Non-RNAi
Viruses 2018, 10(5), 230; https://doi.org/10.3390/v10050230
Received: 11 March 2018 / Revised: 20 April 2018 / Accepted: 27 April 2018 / Published: 1 May 2018
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Abstract
RNAi is considered a major antiviral defense mechanism in insects, but its relative importance as compared to other antiviral pathways has not been evaluated comprehensively. Here, it is attempted to give an overview of the antiviral defense mechanisms in Drosophila that involve both
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RNAi is considered a major antiviral defense mechanism in insects, but its relative importance as compared to other antiviral pathways has not been evaluated comprehensively. Here, it is attempted to give an overview of the antiviral defense mechanisms in Drosophila that involve both RNAi and non-RNAi. While RNAi is considered important in most viral infections, many other pathways can exist that confer antiviral resistance. It is noted that very few direct recognition mechanisms of virus infections have been identified in Drosophila and that the activation of immune pathways may be accomplished indirectly through cell damage incurred by viral replication. In several cases, protection against viral infection can be obtained in RNAi mutants by non-RNAi mechanisms, confirming the variability of the RNAi defense mechanism according to the type of infection and the physiological status of the host. This analysis is aimed at more systematically investigating the relative contribution of RNAi in the antiviral response and more specifically, to ask whether RNAi efficiency is affected when other defense mechanisms predominate. While Drosophila can function as a useful model, this issue may be more critical for economically important insects that are either controlled (agricultural pests and vectors of diseases) or protected from parasite infection (beneficial insects as bees) by RNAi products. Full article
(This article belongs to the Special Issue Antiviral Defense in Invertebrates)
Open AccessReview Probing Molecular Insights into Zika Virus–Host Interactions
Viruses 2018, 10(5), 233; https://doi.org/10.3390/v10050233
Received: 5 April 2018 / Revised: 26 April 2018 / Accepted: 28 April 2018 / Published: 2 May 2018
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Abstract
The recent Zika virus (ZIKV) outbreak in the Americas surprised all of us because of its rapid spread and association with neurologic disorders including fetal microcephaly, brain and ocular anomalies, and Guillain–Barré syndrome. In response to this global health crisis, unprecedented and world-wide
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The recent Zika virus (ZIKV) outbreak in the Americas surprised all of us because of its rapid spread and association with neurologic disorders including fetal microcephaly, brain and ocular anomalies, and Guillain–Barré syndrome. In response to this global health crisis, unprecedented and world-wide efforts are taking place to study the ZIKV-related human diseases. Much has been learned about this virus in the areas of epidemiology, genetic diversity, protein structures, and clinical manifestations, such as consequences of ZIKV infection on fetal brain development. However, progress on understanding the molecular mechanism underlying ZIKV-associated neurologic disorders remains elusive. To date, we still lack a good understanding of; (1) what virologic factors are involved in the ZIKV-associated human diseases; (2) which ZIKV protein(s) contributes to the enhanced viral pathogenicity; and (3) how do the newly adapted and pandemic ZIKV strains alter their interactions with the host cells leading to neurologic defects? The goal of this review is to explore the molecular insights into the ZIKV–host interactions with an emphasis on host cell receptor usage for viral entry, cell innate immunity to ZIKV, and the ability of ZIKV to subvert antiviral responses and to cause cytopathic effects. We hope this literature review will inspire additional molecular studies focusing on ZIKV–host Interactions. Full article
(This article belongs to the Special Issue New Advances on Zika Virus Research)
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Open AccessReview Current Strategies for Inhibition of Chikungunya Infection
Viruses 2018, 10(5), 235; https://doi.org/10.3390/v10050235
Received: 16 March 2018 / Revised: 7 April 2018 / Accepted: 8 April 2018 / Published: 3 May 2018
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Abstract
Increasing incidences of Chikungunya virus (CHIKV) infection and co-infections with Dengue/Zika virus have highlighted the urgency for CHIKV management. Failure in developing effective vaccines or specific antivirals has fuelled further research. This review discusses updated strategies of CHIKV inhibition and provides possible future
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Increasing incidences of Chikungunya virus (CHIKV) infection and co-infections with Dengue/Zika virus have highlighted the urgency for CHIKV management. Failure in developing effective vaccines or specific antivirals has fuelled further research. This review discusses updated strategies of CHIKV inhibition and provides possible future directions. In addition, it analyzes advances in CHIKV lifecycle, drug-target development, and potential hits obtained by in silico and experimental methods. Molecules identified with anti-CHIKV properties using traditional/rational drug design and their potential to succeed in subsequent stages of drug development have also been discussed. Possibilities of repurposing existing drugs based on their in vitro findings have also been elucidated. Probable modes of interference of these compounds at various stages of infection, including entry and replication, have been highlighted. The use of host factors as targets to identify antivirals against CHIKV has been addressed. While most of the earlier antivirals were effective in the early phases of the CHIKV life cycle, this review is also focused on drug candidates that are effective at multiple stages of its life cycle. Since most of these antivirals require validation in preclinical and clinical models, the challenges regarding this have been discussed and will provide critical information for further research. Full article
(This article belongs to the Special Issue Advances in Alphavirus Research)
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Open AccessReview The Role of the Polymeric Immunoglobulin Receptor and Secretory Immunoglobulins during Mucosal Infection and Immunity
Viruses 2018, 10(5), 237; https://doi.org/10.3390/v10050237
Received: 6 April 2018 / Revised: 27 April 2018 / Accepted: 30 April 2018 / Published: 3 May 2018
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Abstract
The gastrointestinal tract houses millions of microbes, and thus has evolved several host defense mechanisms to keep them at bay, and prevent their entry into the host. One such mucosal surface defense is the secretion of secretory immunoglobulins (SIg). Secretion of SIg depends
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The gastrointestinal tract houses millions of microbes, and thus has evolved several host defense mechanisms to keep them at bay, and prevent their entry into the host. One such mucosal surface defense is the secretion of secretory immunoglobulins (SIg). Secretion of SIg depends on the polymeric immunoglobulin receptor (pIgR), which transports polymeric Ig (IgA or IgM) from the basolateral surface of the epithelium to the apical side. Upon reaching the luminal side, a portion of pIgR, called secretory component (SC) is cleaved off to release Ig, forming SIg. Through antigen-specific and non-specific binding, SIg can modulate microbial communities and pathogenic microbes via several mechanisms: agglutination and exclusion from the epithelial surface, neutralization, or via host immunity and complement activation. Given the crucial role of SIg as a microbial scavenger, some pathogens also evolved ways to modulate and utilize pIgR and SIg to facilitate infection. This review will cover the regulation of the pIgR/SIg cycle, mechanisms of SIg-mediated mucosal protection as well as pathogen utilization of SIg. Full article
(This article belongs to the Special Issue Viruses–Bacteria Interactions in the Gut)
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Open AccessReview Single Viruses on the Fluorescence Microscope: Imaging Molecular Mobility, Interactions and Structure Sheds New Light on Viral Replication
Viruses 2018, 10(5), 250; https://doi.org/10.3390/v10050250
Received: 16 March 2018 / Revised: 24 April 2018 / Accepted: 4 May 2018 / Published: 10 May 2018
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Abstract
Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility
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Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. Here, we review different single-molecule sensitive fluorescence methods that we employ routinely to investigate viruses. We provide a brief overview of the microscopy hardware needed and discuss the different methods and their application. In particular, we review how we applied (i) single-molecule Förster resonance energy transfer (smFRET) to probe the subviral human immunodeficiency virus (HIV-1) integrase (IN) quaternary structure; (ii) single particle tracking to study interactions of the simian virus 40 with membranes; (iii) 3D confocal microscopy and smFRET to quantify the HIV-1 pre-integration complex content and quaternary structure; (iv) image correlation spectroscopy to quantify the cytosolic HIV-1 Gag assembly, and finally; (v) super-resolution microscopy to characterize the interaction of HIV-1 with tetherin during assembly. We hope this review is an incentive for setting up and applying similar single-virus imaging studies in daily virology practice. Full article
(This article belongs to the Special Issue Application of Advanced Imaging to the Study of Virus Replication)
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Open AccessReview A Review of the Ongoing Research on Zika Virus Treatment
Viruses 2018, 10(5), 255; https://doi.org/10.3390/v10050255
Received: 2 February 2018 / Revised: 11 April 2018 / Accepted: 14 April 2018 / Published: 14 May 2018
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Abstract
The Zika fever is an arboviral disease resulting from the infection with Zika virus (ZIKV). The virus is transmitted to humans by the bite of Aedes mosquitos, mainly Aedes aegypti and Aedes albopictus. ZIKV has been detected for decades in African and
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The Zika fever is an arboviral disease resulting from the infection with Zika virus (ZIKV). The virus is transmitted to humans by the bite of Aedes mosquitos, mainly Aedes aegypti and Aedes albopictus. ZIKV has been detected for decades in African and Asian regions and, since 2007, has spread to other continents; among them, infections are most reported in the Americas. This can be explained by the presence of vectors in highly populated and tropical regions where people are susceptible to contamination. ZIKV has been considered by the World Health Organization a serious public health problem because of the increasing number of cases of congenital malformation and neurological disorders related to its infection, such as microcephaly, Guillain–Barré syndrome, meningoencephalitis, and myelitis. There is no vaccine or specific antiviral against ZIKV. The infection is best prevented by avoiding mosquito bite, and the treatment of infected patients is palliative. In this context, the search for efficient antivirals is necessary but remains challenging. Here, we aim to review the molecules that have been described to interfere with ZIKV life cycle and discuss their potential use in ZIKV therapy. Full article
(This article belongs to the Section Antivirals & Vaccines)
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Open AccessReview Properties and Functions of Feline Immunodeficiency Virus Gag Domains in Virion Assembly and Budding
Viruses 2018, 10(5), 261; https://doi.org/10.3390/v10050261
Received: 5 April 2018 / Revised: 13 May 2018 / Accepted: 14 May 2018 / Published: 16 May 2018
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Abstract
Feline immunodeficiency virus (FIV) is an important cat pathogen worldwide whose biological and pathophysiological properties resemble those of human immunodeficiency virus type 1 (HIV-1). Therefore, the study of FIV not only benefits its natural host but is also useful for the development of
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Feline immunodeficiency virus (FIV) is an important cat pathogen worldwide whose biological and pathophysiological properties resemble those of human immunodeficiency virus type 1 (HIV-1). Therefore, the study of FIV not only benefits its natural host but is also useful for the development of antiviral strategies directed against HIV-1 infections in humans. FIV assembly results from the multimerization of a single but complex viral polypeptide, the Gag precursor. In this review, we will first give an overview of the current knowledge of the proteins encoded by the FIV pol, env, rev, vif, and orf-A genes, and then we will describe and discuss in detail the critical roles that each of the FIV Gag domains plays in virion morphogenesis. Since retroviral assembly is an attractive target for therapeutic interventions, gaining a better understanding of this process is highly desirable. Full article
(This article belongs to the Special Issue Nonprimate Lentivirus)
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Open AccessReview Drug Delivery Strategies for Antivirals against Hepatitis B Virus
Viruses 2018, 10(5), 267; https://doi.org/10.3390/v10050267
Received: 21 April 2018 / Revised: 4 May 2018 / Accepted: 8 May 2018 / Published: 17 May 2018
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Abstract
Chronic hepatitis B virus (HBV) infection poses a significant health challenge due to associated morbidity and mortality from cirrhosis and hepatocellular cancer that eventually results in the breakdown of liver functionality. Nanotechnology has the potential to play a pivotal role in reducing viral
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Chronic hepatitis B virus (HBV) infection poses a significant health challenge due to associated morbidity and mortality from cirrhosis and hepatocellular cancer that eventually results in the breakdown of liver functionality. Nanotechnology has the potential to play a pivotal role in reducing viral load levels and drug-resistant HBV through drug targeting, thus reducing the rate of evolution of the disease. Apart from tissue targeting, intracellular delivery of a wide range of drugs is necessary to exert a therapeutic action in the affected organelles. This review encompasses the strategies and techniques that have been utilized to target the HBV-infected nuclei in liver hepatocytes, with a significant look at the new insights and most recent advances in drug carriers and their role in anti-HBV therapy. Full article
(This article belongs to the Section Antivirals & Vaccines)
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Open AccessReview Live-Cell Imaging of Early Steps of Single HIV-1 Infection
Viruses 2018, 10(5), 275; https://doi.org/10.3390/v10050275
Received: 25 April 2018 / Revised: 15 May 2018 / Accepted: 18 May 2018 / Published: 19 May 2018
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Abstract
Live-cell imaging of single HIV-1 entry offers a unique opportunity to delineate the spatio-temporal regulation of infection. Novel virus labeling and imaging approaches enable the visualization of key steps of HIV-1 entry leading to nuclear import, integration into the host genome, and viral
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Live-cell imaging of single HIV-1 entry offers a unique opportunity to delineate the spatio-temporal regulation of infection. Novel virus labeling and imaging approaches enable the visualization of key steps of HIV-1 entry leading to nuclear import, integration into the host genome, and viral protein expression. Here, we discuss single virus imaging strategies, focusing on live-cell imaging of single virus fusion and productive uncoating that culminates in HIV-1 infection. Full article
(This article belongs to the Special Issue Application of Advanced Imaging to the Study of Virus Replication)
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Open AccessReview Lessons Learned in Developing a Commercial FIV Vaccine: The Immunity Required for an Effective HIV-1 Vaccine
Viruses 2018, 10(5), 277; https://doi.org/10.3390/v10050277
Received: 30 March 2018 / Revised: 8 May 2018 / Accepted: 20 May 2018 / Published: 22 May 2018
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Abstract
The feline immunodeficiency virus (FIV) vaccine called Fel-O-Vax® FIV is the first commercial FIV vaccine released worldwide for the use in domestic cats against global FIV subtypes (A–E). This vaccine consists of inactivated dual-subtype (A plus D) FIV-infected cells, whereas its prototype
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The feline immunodeficiency virus (FIV) vaccine called Fel-O-Vax® FIV is the first commercial FIV vaccine released worldwide for the use in domestic cats against global FIV subtypes (A–E). This vaccine consists of inactivated dual-subtype (A plus D) FIV-infected cells, whereas its prototype vaccine consists of inactivated dual-subtype whole viruses. Both vaccines in experimental trials conferred moderate-to-substantial protection against heterologous strains from homologous and heterologous subtypes. Importantly, a recent case-control field study of Fel-O-Vax-vaccinated cats with outdoor access and ≥3 years of annual vaccine boost, resulted in a vaccine efficacy of 56% in Australia where subtype-A viruses prevail. Remarkably, this protection rate is far better than the protection rate of 31.2% observed in the best HIV-1 vaccine (RV144) trial. Current review describes the findings from the commercial and prototype vaccine trials and compares their immune correlates of protection. The studies described in this review demonstrate the overarching importance of ant-FIV T-cell immunity more than anti-FIV antibody immunity in affording protection. Thus, future efforts in developing the next generation FIV vaccine and the first effective HIV-1 vaccine should consider incorporating highly conserved protective T-cell epitopes together with the conserved protective B-cell epitopes, but without inducing adverse factors that eliminate efficacy. Full article
(This article belongs to the Special Issue Nonprimate Lentivirus)

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Open AccessBrief Report Upregulation of the Chemokine Receptor CCR2B in Epstein‒Barr Virus-Positive Burkitt Lymphoma Cell Lines with the Latency III Program
Viruses 2018, 10(5), 239; https://doi.org/10.3390/v10050239
Received: 9 April 2018 / Revised: 28 April 2018 / Accepted: 29 April 2018 / Published: 3 May 2018
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Abstract
CCR2 is the cognate receptor to the chemokine CCL2. CCR2–CCL2 signaling mediates cancer progression and metastasis dissemination. However, the role of CCR2–CCL2 signaling in pathogenesis of B-cell malignancies is not clear. Previously, we showed that CCR2B was upregulated in ex vivo peripheral blood
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CCR2 is the cognate receptor to the chemokine CCL2. CCR2–CCL2 signaling mediates cancer progression and metastasis dissemination. However, the role of CCR2–CCL2 signaling in pathogenesis of B-cell malignancies is not clear. Previously, we showed that CCR2B was upregulated in ex vivo peripheral blood B cells upon Epstein‒Barr virus (EBV) infection and in established lymphoblastoid cell lines with the EBV latency III program. EBV latency III is associated with B-cell lymphomas in immunosuppressed patients. The majority of EBV-positive Burkitt lymphoma (BL) tumors are characterized by latency I, but the BL cell lines drift towards latency III during in vitro culture. In this study, the CCR2A and CCR2B expression was assessed in the isogenic EBV-positive BL cell lines with latency I and III using RT-PCR, immunoblotting, and immunostaining analyses. We found that CCR2B is upregulated in the EBV-positive BL cells with latency III. Consequently, we detected the migration of latency III cells toward CCL2. Notably, the G190A mutation, corresponding to SNP CCR2-V64I, was found in one latency III cell line with a reduced migratory response to CCL2. The upregulation of CCR2B may contribute to the enhanced migration of malignant B cells into CCL2-rich compartments. Full article
(This article belongs to the Section Animal Viruses)
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Open AccessConference Report Bioinformatics Meets Virology: The European Virus Bioinformatics Center’s Second Annual Meeting
Viruses 2018, 10(5), 256; https://doi.org/10.3390/v10050256
Received: 9 May 2018 / Revised: 11 May 2018 / Accepted: 11 May 2018 / Published: 14 May 2018
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Abstract
The Second Annual Meeting of the European Virus Bioinformatics Center (EVBC), held in Utrecht, Netherlands, focused on computational approaches in virology, with topics including (but not limited to) virus discovery, diagnostics, (meta-)genomics, modeling, epidemiology, molecular structure, evolution, and viral ecology. The goals of
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The Second Annual Meeting of the European Virus Bioinformatics Center (EVBC), held in Utrecht, Netherlands, focused on computational approaches in virology, with topics including (but not limited to) virus discovery, diagnostics, (meta-)genomics, modeling, epidemiology, molecular structure, evolution, and viral ecology. The goals of the Second Annual Meeting were threefold: (i) to bring together virologists and bioinformaticians from across the academic, industrial, professional, and training sectors to share best practice; (ii) to provide a meaningful and interactive scientific environment to promote discussion and collaboration between students, postdoctoral fellows, and both new and established investigators; (iii) to inspire and suggest new research directions and questions. Approximately 120 researchers from around the world attended the Second Annual Meeting of the EVBC this year, including 15 renowned international speakers. This report presents an overview of new developments and novel research findings that emerged during the meeting. Full article
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