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  • Decoding noises in HIV computational genotyping
    Virology (IF 3.353) Pub Date : 2017-09-14
    MingRui Jia, Timothy Shaw, Xing Zhang, Dong Liu, Ye Shen, Amara E. Ezeamama, Chunfu Yang, Ming Zhang

    Lack of a consistent and reliable genotyping system can critically impede HIV genomic research on pathogenesis, fitness, virulence, drug resistance, and genomic-based healthcare and treatment. At present, mis-genotyping, i.e., background noises in molecular genotyping, and its impact on epidemic surveillance is unknown. For the first time, we present a comprehensive assessment of HIV genotyping quality. HIV sequence data were retrieved from worldwide published records, and subjected to a systematic genotyping assessment pipeline. Results showed that mis-genotyped cases occurred at 4.6% globally, with some regional and high-risk population heterogeneities. Results also revealed a consistent mis-genotyping pattern in gp120 in all studied populations except the group of men who have sex with men. Our study also suggests novel virus diversities in the mis-genotyped cases. Finally, this study reemphasizes the importance of implementing a standardized genotyping pipeline to avoid genotyping disparity and to advance our understanding of virus evolution in various epidemiological settings.

    更新日期:2017-09-15
  • Five distinct reassortants of H5N6 highly pathogenic avian influenza A viruses affected Japan during the winter of 2016–2017
    Virology (IF 3.353) Pub Date : 2017-09-09
    Nobuhiro Takemae, Ryota Tsunekuni, Kirill Sharshov, Taichiro Tanikawa, Yuko Uchida, Hiroshi Ito, Kosuke Soda, Tatsufumi Usui, Ivan Sobolev, Alexander Shestopalov, Tsuyoshi Yamaguchi, Junki Mine, Toshihiro Ito, Takehiko Saito

    To elucidate the evolutionary pathway, we sequenced the entire genomes of 89 H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated in Japan during winter 2016–2017 and 117 AIV/HPAIVs isolated in Japan and Russia. Phylogenetic analysis showed that at least 5 distinct genotypes of H5N6 HPAIVs affected poultry and wild birds during that period. Japanese H5N6 isolates shared a common genetic ancestor in 6 of 8 genomic segments, and the PA and NS genes demonstrated 4 and 2 genetic origins, respectively. Six gene segments originated from a putative ancestral clade 2.3.4.4 H5N6 virus that was a possible genetic reassortant among Chinese clade 2.3.4.4 H5N6 HPAIVs. In addition, 2 NS clusters and a PA cluster in Japanese H5N6 HPAIVs originated from Chinese HPAIVs, whereas 3 distinct AIV-derived PA clusters were evident. These results suggest that migratory birds were important in the spread and genetic diversification of clade 2.3.4.4 H5 HPAIVs.

    更新日期:2017-09-13
  • Expression of the chemokine receptors CCR1 and CCR2B is up-regulated in peripheral blood B cells upon EBV infection and in established lymphoblastoid cell lines
    Virology (IF 3.353) Pub Date : 2017-09-09
    Irina Kholodnyuk, Zanna Rudevica, Ainars Leonciks, Barbro Ehlin-Henriksson, Elena Kashuba

    In immunocompetent individuals, EBV establishes in B cells an asymptomatic lifelong latent infection controlled by the immune system. Chemokine receptors regulate immune system function. CCR1 and CCR2 share protein sequence similarity and exert responses to multiple chemokines. The role of these receptors in B cells is largely unknown. We show that the mRNA and functional protein expression of CCR1 and CCR2 is induced in ex vivo B cells upon EBV infection and in established lymphoblastoid cell lines (LCLs). The CCR1 and CCR2B ORF transcripts were determined in LCLs. In contrast, in both the EBV-negative and EBV-positive Burkitt lymphoma cell lines, neither the CCR1, CCR2A, and CCR2B ORF transcripts nor their corresponding proteins were detected. Our data suggest that CCR1/CCR2B could be involved in clearing EBV-infected latency III B cells in immunocompetent individuals via directing the migration of these cells and attracting the chemokines-expressing immune cells.

    更新日期:2017-09-13
  • Hepatic immunopathology during occult hepacivirus re-infection
    Virology (IF 3.353) Pub Date : 2017-09-13
    Cordelia Manickam, Amanda J. Martinot, Rhianna A. Jones, Valerie Varner, R. Keith Reeves

    Despite drug advances for Hepatitis C virus (HCV), re-infections remain prevalent in high-risk populations. Unfortunately, the role of preexisting viral immunity and how it modulates re-infection is unclear. GBV-B infection of common marmosets is a useful model to study tissue immune responses in hepacivirus infections, and in this study we re-challenged 4 animals after clearance of primary viremia. Although only low-to-absent viremia was observed following re-challenge, GBV-B viral RNA was detectable in liver, confirming re-infection. Microscopic hepatic lesions indicated severe-to-mild lymphocyte infiltration and fibrosis in 3 out of 4 animals. Further, GBV-B-specific T cells were elevated in animals with moderate-to-severe hepatopathology, and up to 3-fold increases in myeloid dendritic and activated natural killer cells were observed after infection. Our data indicate that occult hepacivirus re-infections occur and that new liver pathology is possible even in the presence of anti-hepacivirus T cells and in the absence of high viremia.

    更新日期:2017-09-13
  • Autographa californica multiple nucleopolyhedrovirus PK1 is a factor that regulates high-level expression of very late genes in viral infection
    Virology (IF 3.353) Pub Date : 2017-09-13
    Changyong Liang, Xia Su, Guodong Xu, Xuejuan Dai, Shuling Zhao

    The remarkable ability of baculovirus is to hyperexpress very late genes, but the mechanisms remain unclear. Here we report the effect of PK1, a baculovirus-encoded serine/threonine kinase, on very late gene hyperexpression. PK1 knockout does not completely disrupt very late gene expression, but down regulates the hyperexpression. Those truncated PK1s that exhibit kinase activity in vitro rescue the decline of very late hyperexpression, while other truncated PK1s and a point mutant PK1 (D137A) without kinase activity fail to rescue the decline of very late hyperexpression, suggesting that PK1 regulates very late gene expression by its kinase activity. In addition, those PK1 mutants that can rescue the hyperexpression are able to interact with very late promoter containing 5′ UTR. Based on the above data, we hypothesize that PK1 binds to very late promoter containing 5′ UTR to regulate the hyperexpression of very late genes by its kinase activity.

    更新日期:2017-09-13
  • Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia
    Virology (IF 3.353) Pub Date : 2017-09-11
    Wei Wang, Lianwei Yang, Xiumin Huang, Wenkun Fu, Dequan Pan, Linli Cai, Jianghui Ye, Jian Liu, Ningshao Xia, Tong Cheng, Hua Zhu
    更新日期:2017-09-13
  • Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies
    Virology (IF 3.353) Pub Date : 2017-09-13
    Clemens Lier, Stephan Becker, Nadine Biedenkopf

    Zaire Ebolavirus (EBOV) causes a severe feverish disease with high case fatality rates. Transcription of EBOV is dependent on the activity of the nucleocapsid protein VP30 which represents an essential viral transcription factor. Activity of VP30 is regulated via phosphorylation at six N-terminal serine residues. Recent data demonstrated that dynamic phosphorylation and dephosphorylation of serine residue 29 is essential for transcriptional support activity of VP30. To analyze the spatio/temporal dynamics of VP30 phosphorylation, we generated a peptide antibody recognizing specifically VP30 phosphorylated at serine 29. Using this antibody we could demonstrate that (i) the majority of VP30 molecules in EBOV-infected cells is dephosphorylated at the crucial position serine 29, (ii) both, VP30 phosphorylation and dephosphorylation take place in viral inclusion bodies that are induced by the nucleoprotein NP and (iii) NP influences the phosphorylation state of VP30.

    更新日期:2017-09-13
  • Treatment with PTEN-Long protein inhibits hepatitis C virus replication
    Virology (IF 3.353) Pub Date : 2017-08-04
    Qi Wu, Zhubing Li, Qiang Liu

    Hepatitis C virus (HCV) infection is a confirmed risk factor for hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) possesses tumor suppression function that is frequently defective in HCC tumors. PTEN-Long, a translation isoform of PTEN, functions in a cell non-autonomous manner. In this study, we demonstrated that intracellular overexpression of PTEN-Long inhibits HCV replication. More importantly, we showed that treatment with extracellular PTEN-Long protein inhibits HCV replication in a dose-dependent manner. Furthermore, we showed that PTEN-Long interacts with HCV core protein and this interaction is required for HCV replication inhibition by PTEN-Long. In summary, we demonstrated, for the first time, that PTEN-Long protein, an isoform of the canonical PTEN and in the form of extracellular protein treatment, inhibits HCV replication. Our study offers an opportunity for developing additional anti-HCV agents.

    更新日期:2017-09-13
  • Lipidation increases antiviral activities of coronavirus fusion-inhibiting peptides
    Virology (IF 3.353) Pub Date : 2017-08-10
    Jung-Eun Park, Tom Gallagher
    更新日期:2017-09-13
  • Droplet digital PCR for rapid enumeration of viral genomes and particles from cells and animals infected with orthopoxviruses
    Virology (IF 3.353) Pub Date : 2017-08-10
    Jeffrey L. Americo, Patricia L. Earl, Bernard Moss

    Droplet digital polymerase chain reaction (ddPCR) was adapted for quantifying the number of orthopoxviral genomes in purified virus samples, infected cell lysates and tissues of infected animals. In contrast to the more commonly used qPCR, the newer ddPCR provides absolute numbers of DNA copies in samples without need for standard curves and has the ability to detect rare mutants in a population. The genome/infectious unit ratio for several sucrose gradient-purified orthopoxviruses varied from 5 to 10, which correlated well with values obtained using the Virocyt, a dedicated fluorescence flow cytometer. By employing a nuclease step to digest unencapsulated DNA, the genome/infectious unit ratios of virus in crude cell lysates approached that of purified virus particles. The speed, accuracy, sensitivity, and dynamic range of less than one to millions of infectious units in a sample make this semi-automated method well suited to a variety of laboratory, animal and clinical studies.

    更新日期:2017-09-13
  • The poly-proline tail of SIVmac Vpx provides gain of function for resistance to a cryptic proteasome-dependent degradation pathway
    Virology (IF 3.353) Pub Date : 2017-08-10
    Nannan Zhang, Haoran Guo, Jiaxin Yang, Guanchen Liu, Shuang Li, Siying Li, Dongyin Wang, Rui Li, Chang Shu, Hongmei Xu, Zhentong Wei, Honglan Huang, Songling Zhang, Pujun Gao, Shan Cen, Richard Markham, Yongsheng Wang, Xiao-Fang Yu, Wei Wei

    The lentiviral accessory protein Vpx is critical for viral infection of myeloid cells and acts by hijacking CRL4(DCAF1) E3 ubiquitin ligase to induce the degradation of the host restriction factor SAMHD1. It has been observed that the sequences from HIV-2 and SIVsmm/SIVmac Vpx contain a poly-proline tail which is distinct from other SIV Vpx proteins. However, the role of this region in Vpx function is controversial. Herein, we found proteasome-dependent degradation of a Vpx mutant lacking the poly-proline tail in the nucleus in a CRL4(DCAF1) E3 ligase-independent fashion. Unlike wild-type Vpx, the poly-proline tail mutant Vpx is partly defective in enhancing viral infection in macrophages. Our findings suggest that during Vpx evolution, Vpx of the HIV-2/SIVsm/SIVmac lineage is targeted by a CRL4(DCAF1) E3 ligase-independent ubiquitination pathway, and have gained this interesting region, allowing them to maintain nuclear accumulation as part of their adaptation to host cell regulation.

    更新日期:2017-09-13
  • Identification and characterization of a long non-coding RNA up-regulated during HIV-1 infection
    Virology (IF 3.353) Pub Date : 2017-08-10
    Thomas S. Postler, Shara N. Pantry, Ronald C. Desrosiers, Sankar Ghosh

    Long non-coding RNAs (lncRNAs) are rapidly emerging as important regulators of a diverse array of cellular functions. Here, we describe a meta-analysis of two independent RNA-seq studies to identify lncRNAs that are differentially expressed upon HIV-1 infection. Only three lncRNA genes exhibited altered expression of ≥ 2-fold in HIV-1-infected cells. Of these, the uncharacterized lncRNA LINC00173 was chosen for further study. Both transcript variants of LINC00173 (lnc173 TSV1 and 2) could be detected by qPCR, localized predominantly to the nucleus and were reproducibly up-regulated during infection. Knock-out of the LINC00173 locus did not have detectable effects on HIV-1 replication. Interestingly, however, stimulation of Jurkat T cells with PMA/ionomycin resulted in a decrease of lnc173 expression, and Jurkat cells deficient for lnc173 on average expressed higher levels of specific cytokines than control cells. These data suggest that lnc173 may have a role in the regulation of cytokines in T cells.

    更新日期:2017-09-13
  • Parvovirus B19 integration into human CD36+ erythroid progenitor cells
    Virology (IF 3.353) Pub Date : 2017-08-12
    Tyler Janovitz, Susan Wong, Neal S. Young, Thiago Oliveira, Erik Falck-Pedersen

    The pathogenic autonomous human parvovirus B19 (B19V) productively infects erythroid progenitor cells (EPCs). Functional similarities between B19V nonstructural protein (NS1), a DNA binding endonuclease, and the Rep proteins of Adeno-Associated Virus (AAV) led us to hypothesize that NS1 may facilitate targeted nicking of the human genome and B19 vDNA integration. We adapted an integration capture sequencing protocol (IC-Seq) to screen B19V infected human CD36+ EPCs for viral integrants, and discovered 40,000 unique B19V integration events distributed throughout the human genome. Computational analysis of integration patterns revealed strong correlations with gene intronic regions, H3K9me3 sites, and the identification of 41 base pair consensus sequence with an octanucleotide core motif. The octanucleotide core has homology to a single region of B19V, adjacent to the P6 promoter TATA box. We present the first direct evidence that B19V infection of erythroid progenitor cells disrupts the human genome and facilitates viral DNA integration.

    更新日期:2017-09-13
  • Detection and characterization of an H4N6 avian-lineage influenza A virus in pigs in the Midwestern United States
    Virology (IF 3.353) Pub Date : 2017-08-23
    Eugenio J. Abente, Phillip C. Gauger, Rasna R. Walia, Daniela S. Rajao, Jianqiang Zhang, Karen M. Harmon, Mary Lea Killian, Amy L. Vincent

    H4Nx viruses were reported in swine in Canada and China, but had not been recognized in swine in the USA. In late 2015, an avian-origin H4N6 influenza A virus was isolated from pigs in the United States during a routine diagnostic investigation of clinical respiratory disease in the herd. Serological analysis from additional pigs at the farm and other pigs within the swine production system indicated that the virus did not efficiently transmit from pig-to-pig and the mode of transmission to swine could not be determined. The isolate was characterized at the molecular level and the pathogenesis and transmission was experimentally evaluated in pigs. Although the virus replicated in the lungs of pigs and caused mild pulmonary lesions, there was no evidence of replication in the upper respiratory tract or transmission to indirect contacts, supporting the findings on the farm.

    更新日期:2017-09-13
  • Neural precursor cells derived from induced pluripotent stem cells exhibit reduced susceptibility to infection with a neurotropic coronavirus
    Virology (IF 3.353) Pub Date : 2017-08-17
    Vrushali Mangale, Brett S. Marro, Warren C. Plaisted, Craig M. Walsh, Thomas E. Lane

    The present study examines the susceptibility of mouse induced pluripotent stem cell-derived neural precursor cells (iPSC-NPCs) to infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Similar to NPCs derived from striatum of day 1 postnatal GFP-transgenic mice (GFP-NPCs), iPSC-derived NPCs (iPSC-NPCs) are able to differentiate into terminal neural cell types and express MHC class I and II in response to IFN-γ treatment. However, in contrast to postnatally-derived NPCs, iPSC-NPCs express low levels of carcinoembryonic antigen-cell adhesion molecule 1a (CEACAM1a), the surface receptor for JHMV, and are less susceptible to infection and virus-induced cytopathic effects. The relevance of this in terms of therapeutic application of NPCs resistant to viral infection is discussed.

    更新日期:2017-09-13
  • Herpes simplex virus 1 infection of T cells causes VP11/12-dependent phosphorylation and degradation of the cellular protein Dok-2
    Virology (IF 3.353) Pub Date : 2017-08-23
    Soumia Lahmidi, Ulrike Strunk, James R. Smiley, Angela Pearson, Pascale Duplay

    Previous studies have shown that HSV-1 infection of lymphocytes induces the tyrosine phosphorylation of several proteins that might correspond to viral or host proteins. VP11/12, a viral tegument protein, is the major HSV-induced tyrosine phosphorylated protein identified thus far. In this report, we demonstrated that the cellular adaptor proteins Dok-2 and Dok-1 are tyrosine phosphorylated upon HSV-1 infection. In addition, HSV-1 induced the selective degradation of Dok-2. Finally, we provide evidence that Dok-2 interacts with VP11/12, and that HSV-induced tyrosine phosphorylation and degradation of Dok-2 require VP11/12. Inactivation of either the Src Family Kinases binding motifs or the SHC binding motif of VP11/12 eliminated the interaction of Dok-2 with VP11/12. Elimination of the binding of Dok-2 to VP11/12 prevented Dok-2 phosphorylation and degradation. We propose that HSV-induced Dok phosphorylation and Dok-2 degradation is an immune evasion mechanism to inactivate T cells that might play an important role in HSV pathogenesis.

    更新日期:2017-09-13
  • APOBEC3B lysine residues are dispensable for DNA cytosine deamination, HIV-1 restriction, and nuclear localization
    Virology (IF 3.353) Pub Date : 2017-08-23
    Amy M. Molan, Heather M. Hanson, Cynthia M. Chweya, Brett D. Anderson, Gabriel J. Starrett, Christopher M. Richards, Reuben S. Harris

    The APOBEC3 DNA cytosine deaminase family comprises a fundamental arm of the innate immune response and is best known for retrovirus restriction. Several APOBEC3 enzymes restrict HIV-1 and related retroviruses by deaminating viral cDNA cytosines to uracils compromising viral genomes. Human APOBEC3B (A3B) shows strong virus restriction activities in a variety of experimental systems, and is subjected to tight post-translational regulation evidenced by cell-specific HIV-1 restriction activity and active nuclear import. Here we ask whether lysines and/or lysine post-translational modifications are required for these A3B activities. A lysine-free derivative of human A3B was constructed and shown to be indistinguishable from the wild-type enzyme in DNA cytosine deamination, HIV-1 restriction, and nuclear localization activities. However, lysine loss did render the protein resistant to degradation by SIV Vif. Taken together, we conclude that lysine side chains and modifications thereof are unlikely to be central to A3B function or regulation in human cells.

    更新日期:2017-09-13
  • The phenotype of the RABV glycoprotein determines cellular and global virus load in the brain and is decisive for the pace of the disease
    Virology (IF 3.353) Pub Date : 2017-08-23
    M.A.R. Bertoune, B. Nickl, T. Krieger, L. Wohlers, G.A. Bonaterra, B. Dietzschold, E. Weihe, M. Bette

    The Rabies lyssavirus glycoprotein (RABV-G) is largely responsible for the neuroinvasiveness of the virus and the induction of antiviral immune responses.To study the effects of RABV-G we compared the G of the attenuated RABV variant SPBN with that of the pathogenic DOG4 strain. Infection via the olfactory route caused 100% mortality in mice with both virus variants. Of note, with the attenuated SPBN, progression of the disease was accelerated, microglia response less pronounced and IL-6 expression higher than in the presence of RABV-G from the pathogenic DOG4.However, while virus spread was less extensive, viral gene expression in individual neurons was actually higher in SPBN–infected brains without causing apoptosis of infected neurons. These differences between the two variants were not observed in infected neuronal cultures indicating that the effects of RABV-G on virus spread and viral gene expression depend on factors only present in the intact brain.

    更新日期:2017-09-13
  • MERS coronavirus nsp1 participates in an efficient propagation through a specific interaction with viral RNA
    Virology (IF 3.353) Pub Date : 2017-08-23
    Yutaka Terada, Kengo Kawachi, Yoshiharu Matsuura, Wataru Kamitani

    MERS-CoV is the only lethal human CoV still endemic in the Arabian Peninsula and neither vaccine nor therapeutics against MERS-CoV infection is available. The nsp1 of CoV is thought to be a major virulence factor because it suppresses protein synthesis through the degradation of host mRNA. In contrast, viral RNA circumvents the nsp1-mediated translational shutoff for an efficient propagation. In this study, we identified amino acid residue in MERS-CoV nsp1 that differ from those of SARS-CoV nsp1, and that appear to be crucial for circumventing the translational shutoff. In addition, reverse genetics analysis suggested the presence of a cis-acting element at the 5′-terminus of the nsp1-coding region, which contributes to the specific recognition of viral RNA that is required for an efficient viral replication. Our results suggest the CoVs share a common mechanism for circumventing the nsp1-mediated translational shutoff.

    更新日期:2017-09-13
  • Interaction of 2A proteinase of human rhinovirus genetic group A with eIF4E is required for eIF4G cleavage during infection
    Virology (IF 3.353) Pub Date : 2017-08-29
    Martina Aumayr, Anna Schrempf, Öykü Üzülmez, Karin M. Olek, Tim Skern

    In enteroviruses, the inhibition of protein synthesis from capped host cell mRNA is catalyzed by the virally encoded 2A proteinase (2Apro), which cleaves eukaryotic initiation factors (eIF) 4GI and 4GII. Despite much investigation, the exact mechanism of 2Apro cleavage remains however unclear. Here, we identify the domains responsible for the eIF4E/HRV2 2Apro interaction using molecular modelling and describe mutations that impair this interaction and delay in vitro cleavage of eIF4G isoforms. Furthermore, we produced HRV1A viruses bearing the mutation L17R, Y32A or Y86A in the 2Apro sequence. All three viruses showed reduced yield and were appreciably delayed during infection in eIF4GI cleavage. Thus, we propose for genetic group A HRVs that the eIF4E/2Apro interaction is essential for successful viral replication. In contrast, HRV4 2Apro and coxsackievirus B4 2Apro failed to form complexes with eIF4E, suggesting that the mechanism of eIF4G isoform cleavage in these and related viruses is different.

    更新日期:2017-09-13
  • Resistance of human plasmacytoid dendritic CAL-1 cells to infection with lymphocytic choriomeningitis virus (LCMV) is caused by restricted virus cell entry, which is overcome by contact of CAL-1 cells with LCMV-infected cells
    Virology (IF 3.353) Pub Date : 2017-08-24
    Masaharu Iwasaki, Siddhartha M. Sharma, Brett S. Marro, Juan C. de la Torre

    Plasmacytoid dendritic cells (pDCs), a main source of type I interferon in response to viral infection, are an early cell target during lymphocytic choriomeningitis virus (LCMV) infection, which has been associated with the LCMV's ability to establish chronic infections. Human blood-derived pDCs have been reported to be refractory to ex vivo LCMV infection. In the present study we show that human pDC CAL-1 cells are refractory to infection with cell-free LCMV, but highly susceptible to infection with recombinant LCMVs carrying the surface glycoprotein of VSV, indicating that LCMV infection of CAL-1 cells is restricted at the cell entry step. Co-culture of uninfected CAL-1 cells with LCMV-infected HEK293 cells enabled LCMV to infect CAL-1 cells. This cell-to-cell spread required direct cell-cell contact and did not involve exosome pathway. Our findings indicate the presence of a novel entry pathway utilized by LCMV to infect pDC.

    更新日期:2017-09-13
  • Pathogenicity testing of influenza candidate vaccine viruses in the ferret model
    Virology (IF 3.353) Pub Date : 2017-08-29
    Jessica A. Belser, Adam Johnson, Joanna A. Pulit-Penaloza, Claudia Pappas, Melissa B. Pearce, Wen-Pin Tzeng, M. Jaber Hossain, Callie Ridenour, Li Wang, Li-Mei Chen, David E. Wentworth, Jacqueline M. Katz, Taronna R. Maines, Terrence M. Tumpey

    The development of influenza candidate vaccine viruses (CVVs) for pre-pandemic vaccine production represents a critical step in pandemic preparedness. The multiple subtypes and clades of avian or swine origin influenza viruses circulating world-wide at any one time necessitates the continuous generation of CVVs to provide an advanced starting point should a novel zoonotic virus cross the species barrier and cause a pandemic. Furthermore, the evolution and diversity of novel influenza viruses that cause zoonotic infections requires ongoing monitoring and surveillance, and, when a lack of antigenic match between circulating viruses and available CVVs is identified, the production of new CVVs. Pandemic guidelines developed by the WHO Global Influenza Program govern the design and preparation of reverse genetics-derived CVVs, which must undergo numerous safety and quality tests prior to human use. Confirmation of reassortant CVV attenuation of virulence in ferrets relative to wild-type virus represents one of these critical steps, yet there is a paucity of information available regarding the relative degree of attenuation achieved by WHO-recommended CVVs developed against novel viruses with pandemic potential. To better understand the degree of CVV attenuation in the ferret model, we examined the relative virulence of six A/Puerto Rico/8/1934-based CVVs encompassing five different influenza A subtypes (H2N3, H5N1, H5N2, H5N8, and H7N9) compared with the respective wild-type virus in ferrets. Despite varied virulence of wild-type viruses in the ferret, all CVVs examined showed reductions in morbidity and viral shedding in upper respiratory tract tissues. Furthermore, unlike the wild-type counterparts, none of the CVVs spread to extrapulmonary tissues during the acute phase of infection. While the magnitude of virus attenuation varied between virus subtypes, collectively we show the reliable and reproducible attenuation of CVVs that have the A/Puerto Rico/9/1934 backbone in a mammalian model.

    更新日期:2017-09-13
  • Virus-like particle vaccine primes immune responses preventing inactivated-virus vaccine-enhanced disease against respiratory syncytial virus
    Virology (IF 3.353) Pub Date : 2017-08-29
    Hye Suk Hwang, Young-Tae Lee, Ki-Hye Kim, Eun-Ju Ko, Youri Lee, Young-Man Kwon, Sang-Moo Kang

    Formalin inactivated respiratory syncytial virus (FI-RSV) vaccination caused vaccine-enhanced respiratory disease (ERD) upon exposure to RSV in children. Virus-like particles presenting RSV F fusion protein (F VLP) are known to increase T helper type-1 (Th1) immune responses and avoid ERD in animal models. We hypothesized that F VLP would prime immune responses preventing ERD upon subsequent exposure to ERD-prone FI-RSV. Here, we demonstrated that heterologous F VLP priming and FI-RSV boosting of mice prevented FI-RSV vaccine-enhanced lung inflammation and eosinophilia upon RSV challenge. F VLP priming redirected pulmonary T cells toward effector CD8 T cells producing Th1 cytokines and significantly suppressed pulmonary Th2 cytokines. This study suggests that RSV F VLP priming would modulate and shift immune responses to subsequent exposure to ERD-prone FI-RSV vaccine and RSV infection, suppressing Th2 immune-mediated pulmonary histopathology and eosinophilia.

    更新日期:2017-09-13
  • ORF73 LANA homologs of RRV and MneRV2 contain an extended RGG/RG-rich nuclear and nucleolar localization signal that interacts directly with importin β1 for non-classical nuclear import
    Virology (IF 3.353) Pub Date : 2017-08-29
    Kellie Howard, Lidia Cherezova, Laura K. DeMaster, Timothy M. Rose

    The latency-associated nuclear antigens (LANA) of KSHV and macaque RFHVMn, members of the RV1 rhadinovirus lineage, are closely related with conservation of complex nuclear localization signals (NLS) containing bipartite KR-rich motifs and RG-rich domains, which interact distinctly with importins α and ß1 for nuclear import via classical and non-classical pathways, respectively. RV1 LANAs are expressed in the nucleus of latently-infected cells where they inhibit replication and establish a dominant RV1 latency. Here we show that LANA homologs of macaque RRV and MneRV2 from the more distantly-related RV2 lineage, lack the KR-rich NLS, and instead have a large RG-rich NLS with multiple RG dipeptides and a conserved RGG motif. The RG-NLS interacts uniquely with importin β1, which mediates nuclear import and accumulation of RV2 LANA in the nucleolus. The alternative nuclear import and localization of RV2 LANA homologs may contribute to the dominant RV2 lytic replication phenotype.

    更新日期:2017-09-13
  • Insights into genetic diversity and biological propensities of potentially zoonotic avian influenza H9N2 viruses circulating in Egypt
    Virology (IF 3.353) Pub Date : 2017-08-29
    Mahmoud M. Naguib, Abdel-Satar Arafa, Rokshana Parvin, Martin Beer, Thomas Vahlenkamp, Timm C. Harder

    Low pathogenic avian influenza (LPAI) H9N2 viruses have established endemic status in Egyptian poultry populations since 2012. Recently, four cases of human H9N2 virus infections in Egypt demonstrated the zoonotic potential of these viruses. Egyptian H9N2 viruses obtained from 2011 to 2014 phylogenetically grouped into three clusters (1−3) within subclade B of the G1 lineage. Antigenically, a close clustering of the Egyptian H9N2 viruses with other recent G1-B like H9N2 strains and a significant antigenic distance from viruses outside the G1-B lineage was evident. Recent Egyptian LPAIV H9N2 showed a tendency to increased binding with erythrocytes expressing α 2,6-linked sialic acid which correlated with the Q226L amino acid substitution at the receptor binding unit of the hemagglutinin (Q234L, H9 numbering). Sequence analyses of the N2 neuraminidase (NA) revealed substitutions in the NA hemadsorption site similar to the N2 of prepandemic H3N2/1968, but no distinct antigenic or functional characteristics of the H9N2 NA associated with increased zoonotic potential could be identified.

    更新日期:2017-09-13
  • Heartland virus infection in hamsters deficient in type I interferon signaling: Protracted disease course ameliorated by favipiravir
    Virology (IF 3.353) Pub Date : 2017-08-31
    Jonna B. Westover, Johanna D. Rigas, Arnaud J. Van Wettere, Rong Li, Brady T. Hickerson, Kie-Hoon Jung, Jinxin Miao, Erin S. Reynolds, Bettina L. Conrad, Skot Nielson, Yousuke Furuta, Saravanan Thangamani, Zhongde Wang, Brian B. Gowen

    Heartland virus (HRTV) is an emerging tick-borne virus (Bunyaviridae, Phlebovirus) that has caused sporadic cases of human disease in several central and mid-eastern states of America. Animal models of HRTV disease are needed to gain insights into viral pathogenesis and advancing antiviral drug development. Presence of clinical disease following HRTV challenge in hamsters deficient in STAT2 function underscores the important role played by type I interferon-induced antiviral responses. However, the recovery of most of the infected animals suggests that other mechanisms to control infection and limit disease offer substantial protection. The most prominent disease sign with HRTV infection in STAT2 knockout hamsters was dramatic weight loss with clinical laboratory and histopathology demonstrating acute inflammation in the spleen, lymph node, liver and lung. Finally, we show that HRTV disease in hamsters can be prevented by the use of favipiravir, a promising broad-spectrum antiviral in clinical development for the treatment of influenza.

    更新日期:2017-09-13
  • Genetic variation and co-evolutionary relationship of RNA polymerase complex segments in influenza A viruses
    Virology (IF 3.353) Pub Date : 2017-09-01
    Wentian Chen, Qi Xu, Yaogang Zhong, Hanjie Yu, Jian Shu, Tianran Ma, Zheng Li

    The RNA polymerase complex (RNApc) in influenza A viruses (IVs) is composed of the PB2, PB1 and PA subunits, which are encoded by the three longest genome segments (Seg1-3) and are responsible for the replication of vRNAs and transcription of viral mRNAs. However, the co-evolutionary relationships of the three segments from the known 126 subtypes IVs are unclear. In this study, we performed a detailed analysis based on a total number of 121,191 nucleotide sequences. Three segment sequences were aligned before the repeated, incomplete and mixed sequences were removed for homologous and phylogenetic analyses. Subsequently, the estimated substitution rates and TMRCAs (Times for Most Recent Common Ancestor) were calculated by 175 representative IVs. Tracing the cladistic distribution of three segments from these IVs, co-evolutionary patterns and trajectories could be inferred. The further correlation analysis of six internal protein coding segments reflect the RNApc segments have the closer correlation than others during continuous reassortments. This global approach facilitates the establishment of a fast antiviral strategy and monitoring of viral variation.

    更新日期:2017-09-13
  • Association of toll-like receptor polymorphisms with susceptibility to chikungunya virus infection
    Virology (IF 3.353) Pub Date : 2017-09-06
    Sudip Kumar Dutta, Anusri Tripathi

    Chikungunya virus (CHIKV) infection leads to activation of innate immune response by triggering Toll-like receptor (TLR) pathways resulting in elevated cytokines and type-I interferon levels. Genetic variations of these genes may influence human CHIKV-susceptibility and disease progression. Present study aimed to identify role of TLR polymorphisms in CHIKV-susceptibility and their association with cytokines and clinical symptoms. This is the first study illustrating certain genotypes of TLR-7 and TLR-8 SNPs viz. CT(p = 0.002)]; rs3853839[GC(p<0.001), CC(p = 0.039)] and rs3764879[GC(p<0.001)] were considerably associated with CHIKV susceptibility. Increased risk of CHIKV infection among male patients with CC-genotype (rs179010) (p = 0.028) and female patients with GT-genotype (rs5741880) (p = 0.019) was observed. Significant higher IFN-α (P = 0.002) levels among chikungunya TNF-α (P = 0.034) patients was reported. Chikungunya patients with rs179010-CC genotype showed significantly high IFN-α level(p = 0.003). Thus, these TLR variants might act as potential prognostic biomarkers for predicting CHIKV susceptibility among uninfected individuals.

    更新日期:2017-09-13
  • An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody protects mice from morbidity without interfering with the development of protective immunity to subsequent homologous challenge
    Virology (IF 3.353) Pub Date : 2017-09-06
    Jason R. Wilson, Jessica A. Belser, Juliana DaSilva, Zhu Guo, Xiangjie Sun, Shane Gansebom, Yaohui Bai, Thomas J. Stark, Jessie Chang, Paul Carney, Min Z. Levine, John Barnes, James Stevens, Taronna R. Maines, Terrence M. Tumpey, Ian A. York

    The emergence of A(H7N9) virus strains with resistance to neuraminidase (NA) inhibitors highlights a critical need to discover new countermeasures for treatment of A(H7N9) virus-infected patients. We previously described an anti-NA mAb (3c10-3) that has prophylactic and therapeutic efficacy in mice lethally challenged with A(H7N9) virus when delivered intraperitoneally (i.p.). Here we show that intrananasal (i.n.) administration of 3c10-3 protects 100% of mice from mortality when treated 24 h post-challenge and further characterize the protective efficacy of 3c10-3 using a nonlethal A(H7N9) challenge model. Administration of 3c10-3 i.p. 24 h prior to challenge resulted in a significant decrease in viral lung titers and deep sequencing analysis indicated that treatment did not consistently select for viral variants in NA. Furthermore, prophylactic administration of 3c10-3 did not inhibit the development of protective immunity to subsequent homologous virus re-challenge. Taken together, 3c10-3 highlights the potential use of anti-NA mAb to mitigate influenza virus infection.

    更新日期:2017-09-13
  • Isolate fitness and tissue-tropism determine superinfection success
    Virology (IF 3.353) Pub Date : 2017-09-06
    S.J. Harper, S.J. Cowell, W.O. Dawson

    The mechanism of cross-protection, the deliberate infection of plants with a “mild” virus isolate to protect against “severe” isolates, has long been a topic of debate. In our model system, Citrus tristeza virus (CTV), this appears to be genotype-specific superinfection-exclusion, suggesting a simple recipe for cross-protection. However, this concept failed in field trials, which led us to examine the process of superinfection-exclusion more closely. We found that exclusion relies on the relative fitness of the primary versus the challenge isolates, and the host infected, and that significant differences in superinfection success could occur between isolates that differ by as few as 3 nucleotides. Furthermore, we found that exclusion was not uniform throughout the plant, but was tissue-specific. These data suggest that cross-protection is not a simple like-for-like process but a complex interaction between the primary and challenge isolates and the host.

    更新日期:2017-09-13
  • Immunogenicity of ORFV-based vectors expressing the rabies virus glycoprotein in livestock species
    Virology (IF 3.353) Pub Date : 2017-09-09
    Mathias Martins, Lok R. Joshi, Fernando S. Rodrigues, Deniz Anziliero, Rafael Frandoloso, Gerald F. Kutish, Daniel L. Rock, Rudi Weiblen, Eduardo F. Flores, Diego G. Diel

    The parapoxvirus Orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host-innate and pro-inflammatory responses and has been proposed as a vaccine delivery vector for use in animal species. Here we describe the construction and characterization of two recombinant ORFV vectors expressing the rabies virus (RABV) glycoprotein (G). The RABV-G gene was inserted in the ORFV024 or ORFV121 gene loci, which encode for IMPs that are unique to parapoxviruses and inhibit activation of the NF-κB signaling pathway. The immunogenicity of the resultant recombinant viruses (ORFV∆024RABV-G or ORFV∆121RABV-G, respectively) was evaluated in pigs and cattle. Immunization of the target species with ORFV∆024RABV-G and ORFV∆121RABV-G elicited robust neutralizing antibody responses against RABV. Notably, neutralizing antibody titers induced in ORFV∆121RABV-G-immunized pigs and cattle were significantly higher than those detected in ORFV∆024RABV-G-immunized animals, indicating a higher immunogenicity of ORFVΔ121-based vectors in these animal species.

    更新日期:2017-09-13
  • Temporal characterization of the non-structural Adenovirus type 2 proteome and phosphoproteome using high-resolving mass spectrometry
    Virology (IF 3.353) Pub Date : 2017-09-12
    Malin Källsten, Arina Gromova, Hongxing Zhao, Alberto Valdés, Anne Konzer, Ulf Pettersson, Sara Bergström Lind
    更新日期:2017-09-13
  • Tobacco mosaic virus infection disproportionately impacts phloem associated translatomes in Arabidopsis thaliana and Nicotiana benthamiana
    Virology (IF 3.353) Pub Date : 2017-07-12
    Tamara D. Collum, James N. Culver

    In this study we use vascular specific promoters and a translating ribosome affinity purification strategy to identify phloem associated translatome responses to infection by tobacco mosaic virus (TMV) in systemic hosts Arabidopsis thaliana ecotype Shahdara and Nicotiana benthamiana. Results demonstrate that in both hosts the number of translatome gene alterations that occurred in response to infection is at least four fold higher in phloem specific translatomes than in non-phloem translatomes. This finding indicates that phloem functions as a key responsive tissue to TMV infection. In addition, host comparisons of translatome alterations reveal both similarities and differences in phloem responses to infection, representing both conserved virus induced phloem alterations involved in promoting infection and virus spread as well as host specific alterations that reflect differences in symptom responses. Combined these results suggest phloem tissues play a disproportion role in the mediation and control of host responses to virus infection.

    更新日期:2017-09-13
  • Modeling the evolution of SIV sooty mangabey progenitor virus towards HIV-2 using humanized mice
    Virology (IF 3.353) Pub Date : 2017-07-24
    Kimberly Schmitt, Dipu Mohan Kumar, James Curlin, Leila Remling-Mulder, Mark Stenglein, Shelby O’Connor, Preston Marx, Ramesh Akkina

    HIV-2 is thought to have originated from an SIV progenitor native to sooty mangabeys. To model the initial human transmission and understand the sequential viral evolution, humanized mice were infected with SIVsm and serially passaged for five generations. Productive infection was seen by week 3 during the initial challenge followed by chronic viremia and gradual CD4+ T cell decline. Viral loads increased by the 5th generation resulting in more rapid CD4+ T cell decline. Genetic analysis revealed several amino acid substitutions that were nonsynonymous and fixed in multiple hu-mice across each of the 5 generations in the nef, env and rev regions. The highest rate of substitution occurred in the nef and env regions and most were observed within the first two generations. These data demonstrated the utility of hu-mice in modeling the SIVsm transmission to the human and to evaluate its potential sequential evolution into a human pathogen of HIV-2 lineage.

    更新日期:2017-09-13
  • Deep sequencing of RSV from an adult challenge study and from naturally infected infants reveals heterogeneous diversification dynamics
    Virology (IF 3.353) Pub Date : 2017-08-03
    Jessica W. Lau, Young-In Kim, Ryan Murphy, Ruchi Newman, Xiao Yang, Michael Zody, John DeVincenzo, Yonatan H. Grad

    As RNA virus mutation occurs during replication within host cells, we hypothesized that viral evolution during acute infections in healthy hosts reflects host immune pressure. We therefore investigated the within-host diversification of human respiratory syncytial virus (RSV), a highly prevalent cause of acute respiratory infections. We evaluated healthy adults experimentally infected with an identical inoculum and infants hospitalized with naturally acquired infections. In aggregate, viral diversification in adults peaked at day 3, with overrepresentation of diversity in the matrix protein 2 (M2) and non-structural protein 2 (NS2) genes. In one subject, delayed viral clearance was accompanied by a late peak of diversity at day 10 in known and predicted B and T cell epitopes. In contrast, infant infections showed much less viral diversity. Our findings suggest multiple overlapping mechanisms for early control of acute viral infections, which may differ between age groups and host immune responses.

    更新日期:2017-09-13
  • Polioviruses that bind a chimeric Pvr–nectin-2 protein identify capsid residues involved in receptor interaction
    Virology (IF 3.353) Pub Date : 2017-08-08
    Yi Lin, Vincent R. Racaniello

    Amino acid changes in the C’C”D region in poliovirus receptor domain 1 disrupt poliovirus binding. To examine further the role of the C’C”D region in poliovirus infection, we substituted this region of Pvr into the corresponding region of a murine homolog, nectin-2. The chimeric receptor, nectin-2Pvr(c'c"d), rendered transformed L cells susceptible to infection with poliovirus P1/Mahoney, but not with polioviruses P2/Lansing and P3/Leon, due to lack of binding. Twenty-four variants of P2/Lansing were selected that replicate in nectin-2Pvr(c'c"d) producing cell lines. Sequence analysis revealed 30 amino acid changes at 28 capsid residues. One change, K1103R, is found in nearly all isolates and is located at one end of the VP1 BC loop. Other alterations are located on the canyon surface, at the protomer interface, and along the perimeter of the canyon south wall. Unlike poliovirus-Pvr binding, the VP1 BC loop is required for infection of cells producing nectin-2Pvr(c'c"d).

    更新日期:2017-09-13
  • The histone deacetylase inhibitor SAHA simultaneously reactivates HIV-1 from latency and up-regulates NKG2D ligands sensitizing for natural killer cell cytotoxicity
    Virology (IF 3.353) Pub Date : 2017-07-06
    Maria Giovanna Desimio, Erica Giuliani, Margherita Doria

    In pilot HIV-1 eradication studies, patients’ immune responses were ineffective at killing viral reservoirs reactivated through latency reversing agents (LRAs) like suberoylanilide hydroxamic acid (SAHA). We hypothesized that T cells harboring reactivated HIV-1 express MIC and ULBP ligands for the activating NKG2D receptor of natural killer (NK) cells. Here, we demonstrated that MICA/B and ULBP2 are induced by SAHA on primary T cells harboring reactivated virus. Using latently HIV-1-infected J-Lat 6.3/8.4/9.2 and J1.1 cell lines, we showed that SAHA reverts latency and, simultaneously, up-regulates MICA/B and ULBP2 acting at the transcriptional level and through ATR activation, thus sensitizing T cells with reactivated virus to NKG2D-mediated killing by NK cells. Moreover, IL-2 and IL-15 potently boosted NKG2D expression and cytotoxicity of NK cells against SAHA-reactivated p24+ target cells. Therefore, immunotherapy with cytokines enhancing NKG2D-mediated NK-cell cytotoxicity combined with administration of LRAs up-modulating NKG2D ligands, represents a promising approach towards HIV-1 eradication.

    更新日期:2017-09-13
  • Validation of reference genes for quantifying changes in gene expression in virus-infected tobacco
    Virology (IF 3.353) Pub Date : 2017-07-07
    Eseul Baek, Ju-Yeon Yoon, Peter Palukaitis

    To facilitate quantification of gene expression changes in virus-infected tobacco plants, eight housekeeping genes were evaluated for their stability of expression during infection by one of three systemically-infecting viruses (cucumber mosaic virus, potato virus X, potato virus Y) or a hypersensitive-response-inducing virus (tobacco mosaic virus; TMV) limited to the inoculated leaf. Five reference-gene validation programs were used to establish the order of the most stable genes for the systemically-infecting viruses as ribosomal protein L25 > β-Tubulin > Actin, and the least stable genes Ubiquitin-conjugating enzyme (UCE) < PP2A < GAPDH. For local infection by TMV, the most stable genes were EF1α > Cysteine protease > Actin, and the least stable genes were GAPDH < PP2A < UCE. Using two of the most stable and the two least stable validated reference genes, three defense responsive genes were examined to compare their relative changes in gene expression caused by each virus.

    更新日期:2017-09-13
  • The function of DNA binding protein nucleophosmin in AAV replication
    Virology (IF 3.353) Pub Date : 2017-07-10
    Stifani Satkunanathan, Robin Thorpe, Yuan Zhao

    Adeno-associated viruses (AAV) contain minimal viral proteins necessary for their replication. During virus assembly, AAV acquire, inherently and submissively, various cellular proteins. Our previous studies identified the association of AAV vectors with the DNA binding protein nucleophosmin (NPM1). Nucleophosmin has been reported to enhance AAV infection by mobilizing AAV capsids into and out of the nucleolus, indicating the importance of NPM1 in the AAV life cycle; however the role of NPM1 in AAV production remains unknown. In this study, we systematically investigated NPM1 function on AAV production using NPM1 knockdown cells and revealing for the first time the presence of G-quadruplex DNA sequences (GQRS) in the AAV genome, the synergistic NPM1-GQRS function in AAV production and the significant enhancement of NPM1 gene knockdown on AAV vector production. Understanding the role of cellular proteins in the AAV life cycle will greatly facilitate high titre production of AAV vectors for clinical use.

    更新日期:2017-09-13
  • Infection and Colonization of Nicotiana benthamiana by Grapevine leafroll-associated virus 3
    Virology (IF 3.353) Pub Date : 2017-07-12
    Cecilia A. Prator, Chloe M. Kashiwagi, Darko Vončina, Rodrigo P.P. Almeida

    Grapevine leafroll disease is an increasing problem in all grape-growing regions of the world. The most widespread agent of the disease, Grapevine leafroll-associated virus 3 (GLRaV-3), has never been shown to infect species outside of the genus Vitis. Virus transmission to several plant species used as model systems was tested using the vine mealybug, Planococcus ficus. We show that GLRaV-3 is able to infect Nicotiana benthamiana. Working with GLRaV-3 infected N. benthamiana revealed distinct advantages in comparison with its natural host Vitis vinifera, yielding both higher viral protein and virion concentrations in western blot and transmission electron microscopy observations, respectively. Immunogold labelling of thin sections through N. benthamiana petioles revealed filamentous particles in the phloem cells of GLRaV-3 positive plants. Comparison of assembled whole genomes from GLRaV-3 infected V. vinifera vs. N. benthamiana revealed substitutions in the 5′ UTR. These results open new avenues and opportunities for GLRaV-3 research.

    更新日期:2017-09-13
  • Japanese encephalitis virus counteracts BST2 restriction via its envelope protein E
    Virology (IF 3.353) Pub Date : 2017-07-12
    Mei Li, Ping Wang, Zifeng Zheng, Kai Hu, Mudan Zhang, Xinmeng Guan, Ming Fu, Di Zhang, Wei Wang, Gengfu Xiao, Qinxue Hu, Yalan Liu

    It has been well documented that BST2 restricts the release of enveloped viruses by cross-linking newly produced virions to the cell membrane. However, it is less clear whether and how BST2 inhibits the release of enveloped viruses which bud via the secretory pathway. Here, we demonstrated that BST2 restricts the release of Japanese encephalitis virus (JEV) whose budding occurs at the ER-Golgi intermediate compartment, and in turn, JEV infection downregulates BST2 expression. We further found that the JEV envelope protein E, but not other viral components, significantly downregulates BST2 with the viral protein M playing an auxiliary role in the process. Envelope protein E-mediated BST2 downregulation appears to undergo lysosomal degradation pathway. Additional study revealed that the transmembrane domain and the coiled-coil domain (CC) of BST2 are the target domains of viral protein E and that the N- and C-terminal membrane anchors and the CC domain of BST2 are essential for blocking JEV release. Our results together indicate that the release of enveloped viruses whose budding take place in an intracellular compartment can be restricted by BST2.

    更新日期:2017-09-13
  • Highly conserved intragenic HSV-2 sequences: Results from next-generation sequencing of HSV-2 UL and US regions from genital swabs collected from 3 continents
    Virology (IF 3.353) Pub Date : 2017-07-13
    Christine Johnston, Amalia Magaret, Pavitra Roychoudhury, Alexander L. Greninger, Anqi Cheng, Kurt Diem, Matthew P. Fitzgibbon, Meei-li Huang, Stacy Selke, Jairam R. Lingappa, Connie Celum, Keith R. Jerome, Anna Wald, David M. Koelle

    IntroductionUnderstanding the variability in circulating herpes simplex virus type 2 (HSV-2) genomic sequences is critical to the development of HSV-2 vaccines.MethodsGenital lesion swabs containing ≥ 107 log10 copies HSV DNA collected from Africa, the USA, and South America underwent next-generation sequencing, followed by K-mer based filtering and de novo genomic assembly. Sites of heterogeneity within coding regions in unique long and unique short (UL_US) regions were identified. Phylogenetic trees were created using maximum likelihood reconstruction.ResultsAmong 46 samples from 38 persons, 1468 intragenic base-pair substitutions were identified. The maximum nucleotide distance between strains for concatenated UL_US segments was 0.4%. Phylogeny did not reveal geographic clustering. The most variable proteins had non-synonymous mutations in < 3% of amino acids.ConclusionsUnenriched HSV-2 DNA can undergo next-generation sequencing to identify intragenic variability. The use of clinical swabs for sequencing expands the information that can be gathered directly from these specimens.

    更新日期:2017-09-13
  • Inhibition of NF-κB activity by the porcine epidemic diarrhea virus nonstructural protein 1 for innate immune evasion
    Virology (IF 3.353) Pub Date : 2017-07-15
    Qingzhan Zhang, Jinyou Ma, Dongwan Yoo

    Porcine epidemic diarrhea virus emerged in the US is known to suppress the type I interferons response during infection. In the present study using porcine epithelial cells, we showed that PEDV inhibited both NF-κB and proinflammatory cytokines. PEDV blocked the p65 activation in infected cells and suppressed the PRD II-mediated NF-κB activity. Of the total of 22 viral proteins, nine proteins were identified as NF-κB antagonists, and nsp1 was the most potent suppressor of proinflammatory cytokines. Nsp1 interfered the phosphorylation and degradation of IκBα, and thus blocked the p65 activation. Mutational studies demonstrated the essential requirements of the conserved residues of nsp1 for NF-κB suppression. Our study showed that PEDV inhibited NF-κB activity and nsp1 was a potent NF-κB antagonist for suppression of both IFN and early production of pro-inflammatory cytokines.

    更新日期:2017-09-13
  • Silencing the tobacco gene for RNA-dependent RNA polymerase 1 and infection by potato virus Y cause remodeling of cellular organelles
    Virology (IF 3.353) Pub Date : 2017-07-15
    Farshad Rakhshandehroo, Saeed Rezaee, Peter Palukaitis

    RNA-dependent RNA polymerase 1 (RDR1) has been shown to be involved in DNA methylation, RNA silencing and regulating expression of other genes. RDR1 gene expression is stimulated by infection with potato virus Y° (PVY). Transgenic Nicotiana tabacum plants silenced for RDR1 gene expression showed morphological changes in mesophyll cells, associated with remodeling of the nuclei, chloroplasts and mitochondria. RDR1 silencing led to decreased nuclear size, increased heterochromatin content and aggregation, decreased numbers of chloroplasts, plus changes in shape, internal structures and integrity of chloroplasts and mitochondria. RDR1-silenced transgenic plants showed increased PVY accumulation and ultrastructural remodeling was intensified in both chloroplasts and mitochondria of PVY-infected, RDR1-silenced plants. By contrast, heterochromatin condensation was reduced by PVY infection, and in non-transgenic plants the nuclei were translucent and lacked morphology after PVY infection. Thus, RDR1 regulates gene expression leading to remodeling of chromosomes, and PVY infection counteracts these effects on chromosomal remodeling.

    更新日期:2017-09-13
  • ATM supports gammaherpesvirus replication by attenuating type I interferon pathway
    Virology (IF 3.353) Pub Date : 2017-07-18
    Eric J. Darrah, Kyle P. Stoltz, Mitchell Ledwith, Vera L. Tarakanova

    Ataxia-Telangiectasia mutated (ATM) kinase participates in multiple networks, including DNA damage response, oxidative stress, and mitophagy. ATM also supports replication of diverse DNA and RNA viruses. Gammaherpesviruses are prevalent cancer-associated viruses that benefit from ATM expression during replication. This proviral role of ATM had been ascribed to its signaling within the DNA damage response network; other functions of ATM have not been considered. In this study increased type I interferon (IFN) responses were observed in ATM deficient gammaherpesvirus-infected macrophages. Using a mouse model that combines ATM and type I IFN receptor deficiencies we show that increased type I IFN response in the absence of ATM fully accounts for the proviral role of ATM during gammaherpesvirus replication. Further, increased type I IFN response rendered ATM deficient macrophages more susceptible to antiviral effects of type II IFN. This study identifies attenuation of type I IFN responses as the primary mechanism underlying proviral function of ATM during gammaherpesvirus infection.

    更新日期:2017-09-13
  • The C-terminal region of the Turnip mosaic virus P3 protein is essential for viral infection via targeting P3 to the viral replication complex
    Virology (IF 3.353) Pub Date : 2017-07-20
    Xiaoyan Cui, Hoda Yaghmaiean, Guanwei Wu, Xiaoyun Wu, Xin Chen, Greg Thorn, Aiming Wang

    Like other positive-strand RNA viruses, plant potyviruses assemble viral replication complexes (VRCs) on modified cellular membranes. Potyviruses encode two membrane proteins, 6K2 and P3. The former is known to play pivotal roles in the formation of membrane-associated VRCs. However, P3 remains to be one of the least characterized potyviral proteins. The P3 cistron codes for P3 as well as P3N-PIPO which results from RNA polymerase slippage. In this study, we show that the P3N-PIPO of Turnip mosaic virus (TuMV) is required for viral cell-to-cell movement but not for viral replication. We demonstrate that the C-terminal region of P3 (P3C) is indispensable for P3 to form cytoplasmic punctate inclusions and target VRCs. We reveal that TuMV mutants that lack P3C are replication-defective. Taken together, these data suggest that the P3 cistron has two distinct functions: P3N-PIPO as a dedicated movement protein and P3 as an essential component of the VRC.

    更新日期:2017-09-13
  • Evolutionary dynamics of recent peste des petits ruminants virus epidemic in China during 2013–2014
    Virology (IF 3.353) Pub Date : 2017-07-19
    Jingyue Bao, Qinghua Wang, Lin Li, Chunju Liu, Zhicheng Zhang, Jinming Li, Shujuan Wang, Xiaodong Wu, Zhiliang Wang

    Peste des petits ruminants virus (PPRV) causes a highly contagious disease, peste des petits ruminants (PPR), in sheep and goats which has been considered as a serious threat to the local economy in Africa and Asia. However, the in-depth evolutionary dynamics of PPRV during an epidemic is not well understood. We conducted phylogenetic analysis on genomic sequences of 25 PPRV strains from China 2013–2014 outbreaks. All these strains clustered into a novel clade in lineage 4. An evolutionary rate of 2.61 × 10−6 nucleotide substitutions per site per day was estimated, dating the most recent common ancestor of PPRV China 2013–2014 strains to early August 2013. Transmission network analysis revealed that all the virus sequences could be grouped into five clusters of infection, suggesting long-distance animal transmission play an important role in the spread of PPRV in China. These results expanded our knowledge for PPRV evolution to achieve effective control measures.

    更新日期:2017-09-13
  • Two-amino acids change in the nsp4 of SARS coronavirus abolishes viral replication
    Virology (IF 3.353) Pub Date : 2017-07-21
    Yusuke Sakai, Kengo Kawachi, Yutaka Terada, Hiroko Omori, Yoshiharu Matsuura, Wataru Kamitani

    Infection with coronavirus rearranges the host cell membrane to assemble a replication/transcription complex in which replication of the viral genome and transcription of viral mRNA occur. Although coexistence of nsp3 and nsp4 is known to cause membrane rearrangement, the mechanisms underlying the interaction of these two proteins remain unclear. We demonstrated that binding of nsp4 with nsp3 is essential for membrane rearrangement and identified amino acid residues in nsp4 responsible for the interaction with nsp3. In addition, we revealed that the nsp3-nsp4 interaction is not sufficient to induce membrane rearrangement, suggesting the participation of other factors such as host proteins. Finally, we showed that loss of the nsp3-nsp4 interaction eliminated viral replication by using an infectious cDNA clone and replicon system of SARS-CoV. These findings provide clues to the mechanism of the replication/transcription complex assembly of SARS-CoV and could reveal an antiviral target for the treatment of betacoronavirus infection.

    更新日期:2017-09-13
  • Longitudinal sequencing of HIV-1 infected patients with low-level viremia for years while on ART shows no indications for genetic evolution of the virus
    Virology (IF 3.353) Pub Date : 2017-07-24
    Leen Vancoillie, Laura Hebberecht, Kenny Dauwe, Els Demecheleer, Sylvie Dinakis, Dries Vaneechoutte, Virginie Mortier, Chris Verhofstede

    HIV-infected patients on antiretroviral therapy (ART) may present low-level viremia (LLV) above the detection level of current viral load assays. In many cases LLV is persistent but does not result in overt treatment failure or selection of drug resistant viral variants. To elucidate whether LLV reflects active virus replication, we extensively sequenced pol and env genes of the viral populations present before and during LLV in 18 patients and searched for indications of genetic evolution. Maximum likelihood phylogenetic trees were inspected for temporal structure both visually and by linear regression analysis of root-to-tip and pairwise distances. Viral coreceptor tropism was assessed at different time points before and during LLV. In none of the patients consistent indications for genetic evolution were found over a median period of 4.8 years of LLV. As such these findings could not provide evidence that active virus replication is the main driver of LLV.

    更新日期:2017-09-13
  • Differential use of 3’CITEs by the subgenomic RNA of Pea enation mosaic virus 2
    Virology (IF 3.353) Pub Date : 2017-07-24
    Feng Gao, Anne E. Simon

    The genomic RNA (gRNA) of Pea enation mosaic virus 2 (PEMV2) is the template for p33 and −1 frameshift product p94. The PEMV2 subgenomic RNA (sgRNA) encodes two overlapping ORFs, p26 and p27, which are required for movement and stability of the gRNA. Efficient translation of p33 requires two of three 3’ proximal cap-independent translation enhancers (3’CITEs): the kl-TSS, which binds ribosomes and engages in a long-distance interaction with the 5’end; and the adjacent eIF4E-binding PTE. Unlike the gRNA, all three 3’CITEs were required for efficient translation of the sgRNA, which included the ribosome-binding 3’TSS. A hairpin in the 5’ proximal coding region of p26/p27 supported translation by the 3’CITEs by engaging in a long-distance RNA:RNA interaction with the kl-TSS. These results strongly suggest that the 5’ ends of PEMV2 gRNA and sgRNA connect with the 3’UTR through similar long-distance interactions while having different requirements for 3’CITEs.

    更新日期:2017-09-13
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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