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  • Chalcid wasp paleoviruses bridge the evolutionary gap between bracoviruses and nudiviruses
    Virology (IF 2.657) Pub Date : 2020-01-16
    Yu Zhang; Jianhua Wang; Guan-Zhu Han

    Polydnaviruses are obligate mutualists of parasitoid wasps and are divided into two genera, Bracovirus and Ichnovirus. Bracoviruses are thought to originate from a single integration of an ancestral nudivirus into the ancestor of microgastroid complex ∼100 million years ago. However, all the known nudiviruses are only distantly related to bracoviruses, and much remains obscure about the origin of bracoviruses. Here we employ a paleovirological method to screen endogenous nudivirus-like elements across arthropods. Interestingly, we identify many endogenous nudivirus-like elements within the genome of Eurytoma brunniventris, a species of the Chalcidoidea superfamily. Among them, we find 14 core gene sequences are likely to be derived from a betanudivirus (designated EbrENV-β), suggesting that betanudivirus has been circulating in parasitoid wasps. Phylogenomic analysis suggests that EbrENV-β is the known closest relative of bracoviruses. Synteny analyses show the order of core genes is not well conserved between EbrENV-β and nudiviruses, revealing the dynamic nature of the evolution of nudivirus genome structures. Our findings narrow down the evolutionary gap between bracoviruses and nudiviruses and provide novel insights into the origin and evolution of polydnaviruses.

  • The raspberry bushy dwarf virus 1b gene enables pollen grains to function efficiently in horizontal pollen transmission
    Virology (IF 2.657) Pub Date : 2020-01-14
    Masamichi Isogai; Takanori Matsudaira; Kotaro Miyoshi; Takuya Shimura; Sayaka Torii; Nobuyuki Yoshikawa

    Horizontal pollen transmission by the raspberry bushy dwarf virus 1b deletion mutant (RBΔ1bstop), which is defective in virus virulence, was significantly decreased compared to wild-type raspberry bushy dwarf virus (wtRBDV). We assessed accumulation of viral genomic (g) RNAs in pollen grains from RBΔ1bstop-infected plants and found that the pollen grains had less viral gRNA than those from wtRBDV-infected plants. In addition, pollen grains from 1b-expressing transgenic plants (1b-plants) infected with RBΔ1bstop were more efficient in horizontal virus transmission to healthy plants after pollination than pollen from RBΔ1bstop-infected wild type plants. Moreover, viral gRNA accumulation in pollen grains from RBΔ1bstop-infected 1b-plants was higher than in pollen from RBΔ1bstop-infected wild type plants. We suggest that 1b increases the amount of viral gRNAs released from elongating pollen grains.

  • Molecular characterization of H3 subtype avian influenza viruses based on poultry-related environmental surveillance in China between 2014 and 2017
    Virology (IF 2.657) Pub Date : 2020-01-11
    Shumei Zou; Jing Tang; Ye Zhang; Lijun Liu; Xiyan Li; Yao Meng; Xiang Zhao; Lei Yang; Yuelong Shu; Dayan Wang

    The H3 subtype avian influenza virus (AIV) poses a threat to both animal and human health. In this study, phylogenetic analysis showed that the H3 AIVs had various genomic constellations and extensive reassortments, increasing genetic diversity and the emergence of new pathogenic viruses that might infect human beings. Molecular analysis demonstrated that the major molecular markers linked to drug resistance were identified in M genes of three studied viruses, and there might be wide range of resistant virus infections in poultry in the future. Although all the H3 viruses preferentially bound to the avian-type receptor, the growth kinetics experiments showed that the selected H3 viruses were capable of efficient replication in mammalian cells, suggesting a potential cross-species transmission of H3 viruses. Overall, our results emphasize the need for continued surveillance of H3 outbreaks and may also help us improve knowledge on H3 AIVs prevention and control.

  • TF protein of Sindbis virus antagonizes host type I interferon responses in a palmitoylation-dependent manner
    Virology (IF 2.657) Pub Date : 2020-01-07
    K.J. Rogers; S. Jones-Burrage; W. Maury; S. Mukhopadhyay

    Sindbis virus (SINV) produces the small membrane protein TF from the 6K gene via a (-1) programmed ribosomal frameshifting. While several groups have shown that TF-deficient virus exhibits reduced virulence, the mechanism(s) by which this occurs remain unknown. Here, we demonstrate a role for TF in antagonizing the host interferon response. Using wild-type and type 1 interferon receptor-deficient mice and primary cells derived from these animals, we show that TF controls the induction of the host interferon response at early times during infection. Loss of TF production leads to elevated interferon and a concurrent reduction in viral loads with a loss of pathogenicity. Palmitoylation of TF has been shown to be important for particle assembly and morphology. We find that palmitoylation of TF also contributes to the ability of TF to antagonize host interferon responses as dysregulated palmitoylation of TF reduces virulence in a manner similar to loss of TF.

  • SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses
    Virology (IF 2.657) Pub Date : 2020-01-07
    Marco R. Straus; Jonathan T. Kinder; Michal Segall; Rebecca Ellis Dutch; Gary R. Whittaker

    Viruses possessing class I fusion proteins require proteolytic activation by host cell proteases to mediate fusion with the host cell membrane. The mammalian SPINT2 gene encodes a protease inhibitor that targets trypsin-like serine proteases. Here we show the protease inhibitor, SPINT2, restricts cleavage-activation efficiently for a range of influenza viruses and for human metapneumovirus (HMPV). SPINT2 treatment resulted in the cleavage and fusion inhibition of full-length influenza A/CA/04/09 (H1N1) HA, A/Aichi/68 (H3N2) HA, A/Shanghai/2/2013 (H7N9) HA and HMPV F when activated by trypsin, recombinant matriptase or KLK5. We also demonstrate that SPINT2 was able to reduce viral growth of influenza A/CA/04/09 H1N1 and A/X31 H3N2 in cell culture by inhibiting matriptase or TMPRSS2. Moreover, inhibition efficacy did not differ whether SPINT2 was added at the time of infection or 24 h post-infection. Our data suggest that the SPINT2 inhibitor has a strong potential to serve as a novel broad-spectrum antiviral.

  • Fitness advantage of inter-species TYLCV recombinants induced by beneficial intra-genomic interactions rather than by specific mutations
    Virology (IF 2.657) Pub Date : 2020-01-07
    Cica Urbino; Zohra Fatima Regragui; Martine Granier; Michel Peterschmitt

    Tomato yellow leaf curl virus (TYLCV) and its related viruses are prone to recombination. It was reported that random homologous recombination between 20% diverging TYLCV related species is rarely deleterious and may be associated with a fitness advantage. Indeed, TYLCV-IS76, a recombinant between the 20% divergent TYLCV and tomato yellow leaf curl Sardinia virus, exhibited a higher fitness than that of parental viruses. As this typical fitness advantage was observed with TYLCV-IS76 representatives of different pedigrees, it was thought that it is induced by beneficial intra-genomic interactions rather than by specific mutations. This hypothesis was further supported with TYLCV-IS141, a TYLCV recombinant with a short TYLCSV inherited fragment of around 141 nts, slightly longer than that of TYLCV-IS76. Indeed, the typical fitness advantage was detected irrespective of the position of the recombination breakpoint (loci 76 or 141) and the sequences of the TYLCV and TYLCSV inherited fragments.

  • Bacteriophage P1 does not show spatial preference when infecting Escherichia coli
    Virology (IF 2.657) Pub Date : 2020-01-07
    Kailun Zhang; Ryland F. Young; Lanying Zeng
  • Extracellular vesicles restrict dengue virus fusion in Aedes aegypti cells
    Virology (IF 2.657) Pub Date : 2019-12-28
    Megan N. Freitas; Andrew D. Marten; Gavin A. Moore; Maya O. Tree; Sean P. McBrayer; Michael J. Conway

    Aedes aegypti is the primary vector of dengue virus (DENV), and acquires this virus from a vertebrate host during blood feeding. Previous literature has shown that vertebrate blood factors such as complement protein C5a and low-density lipoprotein (LDL) influence DENV acquisition in the mosquito. Here, we show that extracellular vesicles in cell culture medium inhibit DENV infection in mosquito cells. Specifically, extracellular vesicles enter into mosquito cells and inhibit an early stage of infection. Extracellular vesicles had no effect on virus cell attachment or entry. Instead, extracellular vesicles restricted virus membrane fusion. Extracellular vesicles only inhibited DENV infection in mosquito cells and not vertebrate cells. These data highlight a novel virus-vector-host interaction that limits virus infection in mosquito cells by restricting virus membrane fusion.

  • Hepatitis E viral infection causes testicular damage in mice
    Virology (IF 2.657) Pub Date : 2019-12-28
    Jianwen Situ; Wenjing Wang; Feiyan Long; Weimin Yang; Chenchen Yang; Daqiao Wei; Wenhai Yu; Fen Huang
  • The use of cells from ANPEP knockout pigs to evaluate the role of aminopeptidase N (APN) as a receptor for porcine deltacoronavirus (PDCoV)
    Virology (IF 2.657) Pub Date : 2019-12-24
    Ana Stoian; Raymond R.R. Rowland; Vlad Petrovan; Maureen Sheahan; Melissa S. Samuel; Kristin M. Whitworth; Kevin D. Wells; Jianqiang Zhang; Benjamin Beaton; Mark Cigan; Randall S. Prather

    The coronaviruses, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) represent important sources of neonatal diarrhea on pig farms. The requirement for aminopeptidase N (APN) as a receptor for TGEV, but not for PEDV, is well established. In this study, the biological relevance of APN as a receptor for PDCoV was tested using CRISPR/Cas9 to knockout the APN gene, ANPEP, in pigs. Porcine alveolar macrophages (PAMs) from ANPEP knockout (KO) pigs showed resistance to PDCoV infection. However, lung fibroblast-like cells, derived from the ANPEP KO PAM cultures, supported PDCoV infection to high levels. The results suggest that APN is a receptor for PDCoV in PAMs but is not necessary for infection of lung-derived fibroblast cells. The infection of the ANPEP KO pigs with PDCoV further confirmed that APN is dispensable as a receptor for PDCoV.

  • Poxvirus encoded eIF2α homolog, K3 family proteins, is a key determinant of poxvirus host species specificity
    Virology (IF 2.657) Pub Date : 2019-12-23
    Jingxin Cao; Jessie Varga; Yvon Deschambault

    Protein kinase R plays a key role in innate antiviral immune responses of vertebrate animals. Most mammalian poxviruses encode two PKR antagonists, E3 (dsRNA binding) and K3 (eIF2α homolog) proteins. In this study, the role of K3 family proteins from poxviruses with distinct host tropisms in determining the virus host range was examined in a vaccinia E3L deletion mutant virus. It was found that K3 orthologs from the species-specific poxviruses (taterapox virus, sheeppox virus, myxoma virus, swinepox virus and yaba monkey tumor virus) restored the virus replication competency in cells derived from their natural host or related animal species. Further, it was found that the residues located in the helix insert region of the protein, K45 of vaccinia K3 and Y47 of the sheep poxvirus ortholog 011, are critical for the virus host species specificity. These observations demonstrate that poxvirus K3 proteins are major determinants of the virus host specificity.

  • CSFV protein NS5A activates the unfolded protein response to promote viral replication
    Virology (IF 2.657) Pub Date : 2019-12-14
    Zhang Chengcheng; Zhao Fuxi; Guo Mengjiao; Ruan Baoyang; Wang Xuefeng; Wu Yantao; Zhang Xiaorong

    Classical swine fever is a world organization for animal health listed disease and is caused by classical swine fever virus (CSFV). CSFV can induced unfolded protein response (UPR) and whether NS5A protein plays a role in this process remains unknown. Here, we demonstrate that CSFV induced all the three signal pathways ATF6, IRE1 and PERK of UPR. Furthermore, this phenomenon may be mediated by the NS5A protein since expression of NS5A alone can achieve the same effect. In the current study, we show that NS5A can interact with GRP78 as measured by using the CO-IP and GST pulldown assays. This interaction plays a positive role in the promotion of CSFV replication. Overexpression or knockdown of GRP78 mediated by lentivirus can enhance or decrease viral replication, respectively. Our findings provide the evidence that CSFV infection can activate the cellular UPRs, in which NS5A and GRP78 play key roles in the process.

  • 更新日期:2019-12-13
  • A novel human monoclonal antibody potently neutralizes human adenovirus serotype 7 By primarily targeting the adenovirus hexon protein
    Virology (IF 2.657) Pub Date : 2019-12-12
    Jiansheng Lu; Rong Wang; Ying Huang; Yunzhou Yu; Xiaowei Zhou; Peitang Huang; Zhixin Yang

    Human adenovirus serotype 7 (HAdV7), belonging to species B, has caused severe lower respiratory tract diseases and even deaths recently. However, no adenovirus vaccine or therapeutic is available thus far. In this study, a HAdV7-specific human monoclonal antibody (HMAb), 3-3E, isolated from single plasma cells obtained from the peripheral blood mononuclear cells of HAdV7-infected patients showed potent HAdV7 neutralization activity. The results showed HMAb 3-3E only binds to the hexon protein of intact HAdV7 or the recombinant hexon protein and it does not bind to other intact virion particles. This could mean the antibody recognizes a conformational epitope of the hexon protein. Further, HMAb 3-3E potently neutralized HAdV7 in vitro at low concentrations. In vivo studies showed HMAb 3-3E protected from HAdV7 infection in a murine model. Therefore, HMAb 3-3E is promising as a safe and effective prophylactic and therapeutic treatment for HAdV7 infection.

  • MiR-BART1-5p targets core 2β-1,6-acetylglucosaminyltransferase GCNT3 to inhibit cell proliferation and migration in EBV-associated gastric cancer
    Virology (IF 2.657) Pub Date : 2019-12-11
    Juanjuan Liu, Yan Zhang, Wen Liu, Qianqian Zhang, Hua Xiao, Hui Song, Bing Luo

    GCNT3 (core 2β-1,6-acetylglucosaminyltransferase) is a novel core mucin synthase. It is known that abnormal expression of GCNT3 promotes the progression of several human cancers. However, its relationship with Epstein-Barr virus (EBV) has not been comprehensively studied. We found GCNT3 expression in EBV-associated gastric cancer cells and tissues to be lower than in EBV-negative gastric cancer cells and tissues, and high expression was significantly associated with advanced tumor-lymph node metastasis. Luciferase reporter assay revealed that miR-BART1-5p directly targeted GCNT3. In addition, miR-BART1-5p mimics transfection was observed to reduce cell proliferation and migration, while miR-BART1-5p inhibitor increased cell proliferation and migration following transfection. In conclusion, both miR-BART1-5p and knockdown of GCNT3 inhibited cell proliferation and migration. In addition, EBV may regulate GCNT3 by affecting the NF-kB signaling pathway. E-cadherin, N-cadherin, vimentin, and p-ERK were found to be downstream molecules of the miR-BART1-5p/GCNT3 pathway.

  • Zika virus NS5 localizes at centrosomes during cell division
    Virology (IF 2.657) Pub Date : 2019-12-06
    Aditi S. Kesari, Veronica J. Heintz, Shishir Poudyal, Andrew S. Miller, Richard J. Kuhn, Douglas J. LaCount

    Zika virus (ZIKV) nonstructural protein 5 (NS5) plays a critical role in viral RNA replication and mediates key virus-host cell interactions. As with other flavivirus NS5 proteins, ZIKV NS5 is primarily found in the nucleus. We previously reported that the NS5 protein of dengue virus, another flavivirus, localized to centrosomes during cell division. Here we show that ZIKV NS5 also relocalizes from the nucleus to centrosomes during mitosis. In infected cells with supernumerary centrosomes, NS5 was present at all centrosomes. Transient expression of NS5 in uninfected cells confirmed that centrosomal localization was independent of other viral proteins. Live-cell imaging demonstrated that NS5-GFP accumulated at centrosomes shortly after break down of nuclear membrane and remained there through mitosis. Cells expressing NS5-GFP took longer to complete mitosis than control cells. Finally, an analysis of ZIKV NS5 binding partners revealed several centrosomal proteins, providing potential direct links between NS5 and centrosomes.

  • The human papillomavirus 16 E5 gene potentiates MmuPV1-Dependent pathogenesis
    Virology (IF 2.657) Pub Date : 2019-12-05
    Alexandra D. Torres, Megan E. Spurgeon, Andrea Bilger, Simon Blaine-Sauer, Aayushi Uberoi, Darya Buehler, Stephanie M. McGregor, Ella Ward-Shaw, Paul F. Lambert

    The papillomavirus E5 gene contributes to transformation and tumorigenesis; however, its exact function in these processes and viral pathogenesis is unclear. While E5 is present in high-risk mucosotropic HPVs that cause anogenital and head and neck cancers, it is absent in cutaneous HPVs and the recently discovered mouse papillomavirus (MmuPV1), which causes papillomas and squamous cell carcinomas of the skin and mucosal epithelia in laboratory mice. We infected K14E5 transgenic mice, which express the high-risk mucosotropic HPV16 E5 gene in stratified epithelia, with MmuPV1 to investigate the effects of E5 on papillomavirus-induced pathogenesis. Skin lesions in MmuPV1-infected K14E5 mice had earlier onset, higher incidence, and reduced frequency of spontaneous regression compared to those in non-transgenic mice. K14E5 mice were also more susceptible to cervicovaginal cancers when infected with MmuPV1 and treated with estrogen compared to non-transgenic mice. Our studies support the hypothesis that E5 contributes to papillomavirus-induced pathogenesis.

  • Inhibitors of the interferon response increase the replication of gorilla simian foamy viruses
    Virology (IF 2.657) Pub Date : 2019-12-02
    Mathilde Couteaudier, Diego Calzada-Fraile, Thomas Montange, Antoine Gessain, Florence Buseyne

    Simian foamy viruses (SFVs) are complex retroviruses that are widespread throughout nonhuman primates. SFVs can also be transmitted to humans, mostly through bites. We previously observed that primary zoonotic gorilla SFV strains grow much more slowly than laboratory-adapted chimpanzee strains. Here, we tested the hypothesis that the growth of SFV is limited by interferon (IFN) using inhibitors of cellular pathways involved in the induction or action of type I IFN. Inhibitors of JAK1/2 (Ruxolitinib) and TBK-1 (BX795) led to a 2- to 4-fold higher percentage of cells infected with zoonotic gorilla SFVs but did not affect the replication of laboratory-adapted chimpanzee SFVs. IKK2 inhibitors (TPCA-1 and BMS345541) had no effect on any of the SFV strains. In conclusion, the addition of molecules that inhibit the type I IFN response to the culture medium can be used as a simple and efficient method to enhance the replication of zoonotic gorilla SFVs.

  • Drosophila immunity against natural and nonnatural viral pathogens
    Virology (IF 2.657) Pub Date : 2019-12-01
    Ghada Tafesh-Edwards, Ioannis Eleftherianos

    The fruit fly Drosophila melanogaster is extensively used as a model species for molecular biology and genetics. It is also widely studied for its innate immune system to expand our understanding of immune host defenses against numerous pathogens. More precisely, studies using both natural and nonnatural Drosophila pathogens have provided a better perspective of viral infection strategies and immunity processes than any other invertebrate. This has made significant advances in identifying and characterizing the innate immune mechanisms by which hosts can combat viral pathogens. However, in-depth studies on antiviral immunity are still lacking due in part to the narrow research focus on the evolution and conservation of antiviral strategies to combat infections caused by both natural and nonnatural viruses. In this review, we will cover three major areas. First, we will describe the well-characterized antiviral immune mechanisms in Drosophila. Second, we will survey the specific pathways induced by natural viruses that have been studied in Drosophila. Finally, we will discuss the pathways activated by nonnatural viruses, drawing comparisons to natural viruses and giving an unprecedented insight into the virus community of Drosophila that is necessary to understand the evolutionary and immune context needed to develop Drosophila as a model for virus research.

  • STAT3 activates the anti-apoptotic form of caspase 9 in oncovirus-infected B lymphocytes
    Virology (IF 2.657) Pub Date : 2019-11-30
    Siva Koganti, Sandeepta Burgula, Sumita Bhaduri-McIntosh

    The cancer-causing Epstein-Barr virus (EBV) activates the transcription factor STAT3 upon infecting B-lymphocytes. STAT3 then activates caspase 7 to degrade cellular claspin, resulting in impaired Chk1 phosphorylation. This blockade of ATR-Chk1 signaling allows EBV-transformed cells to proliferate despite DNA lesions from virus-induced replication stress. In addressing the mechanism of caspase 7 activation, we now report that in newly-infected B-cells, STAT3 transcriptionally activates the initiator caspase, caspase 9. Caspase 9 then activates caspase 7 to impair phosphorylation of Chk1 at S345. Importantly, although cleaved products of caspase 9 are detectable in infected cells, there is simultaneous increase in the alternatively-spliced dominant-negative form of caspase 9 – and – expression of dominant-negative caspase 9 is abrogated when STAT3 activation is impaired. Thus EBV, via STAT3, activates caspase 9 but also shifts the balance of transcripts towards its dominant-negative form to allow activation of caspase 7 while avoiding death of EBV-infected cells.

  • Novel anti-flavivirus drugs targeting the nucleolar distribution of core protein
    Virology (IF 2.657) Pub Date : 2019-11-29
    Makoto Tokunaga, Yoichi Miyamoto, Tatsuya Suzuki, Mayumi Otani, Shinsuke Inuki, Tsuyoshi Esaki, Chioko Nagao, Kenji Mizuguchi, Hiroaki Ohno, Yoshihiro Yoneda, Toru Okamoto, Masahiro Oka, Yoshiharu Matsuura

    The risk of infectious diseases caused by Flavivirus is increasing globally. Here, we developed a novel high-throughput screening (HTS) system to evaluate the inhibitory effects of compounds targeting the nuclear localization of the flavivirus core protein. We screened 4000 compounds based on their ability to inhibit the nuclear localization of the core protein, and identified over 20 compounds including inhibitors for cyclin dependent kinase and glycogen synthase kinase. The efficacy of the identified compounds to suppress viral growth was validated in a cell-based infection system. Remarkably, the nucleolus morphology was affected by the treatment with the compounds, suggesting that the nucleolus function is critical for viral propagation. The present HTS system provides a useful strategy for the identification of antivirals against flavivirus by targeting the nucleolar localization of the core protein.

  • 更新日期:2019-11-30
  • Cloning and characterization of antiviral cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus
    Virology (IF 2.657) Pub Date : 2019-11-26
    Erin B. Taylor, V. Gregory Chinchar, Sylvie M.A. Quiniou, Melanie Wilson, Eva Bengtén

    To determine the role of piscine anti-viral cytotoxic cells, we analyzed the response of channel catfish to Ictalurid herpesvirus 1, commonly designated channel catfish virus (CCV). Peripheral blood leukocytes (PBL) from catfish immunized with MHC-matched, CCV-infected G14D cells (G14D-CCV) showed marked lysis of G14D-CCV but little to no lysis of uninfected allogenic (3B11) or syngeneic (G14D) cells. Expansion of effectors by in vitro culture in the presence of irradiated G14D-CCV cells generated cultures with enhanced cytotoxicity and often broader target range. Cytotoxic effectors expressed rearranged TCR genes, perforin, granzyme, and IFN-γ. Four clonal cytotoxic lines were developed and unique TCR gene rearrangements including γδ were detected. Furthermore, catfish CTL clones were either CD4+/CD8- or CD4-/CD8-. Two CTL lines showed markedly enhanced killing of G14D-CCV targets, while the other displayed a broader target range. Collectively, catfish virus-specific CTL display unique features that illustrate the diversity of the ectothermic vertebrate immune response.

  • STING is dispensable during KSHV infection of primary endothelial cells
    Virology (IF 2.657) Pub Date : 2019-11-25
    Daniel Vogt, Shivam Zaver, Alice Ranjan, Terri DiMaio, Anshu P. Gounder, Jason G. Smith, Michael Lagunoff

    During DNA virus infections, detection of cytosolic DNA by the cGAS-STING pathway leads to activation of IFN-β. Kaposi's Sarcoma Herpesvirus (KSHV), an oncogenic DNA virus, is the etiological agent of Kaposi's Sarcoma, an endothelial cell (EC)-based tumor. To investigate the role of STING during KSHV infection of primary ECs we identified a primary lymphatic EC sample that is defective for STING activation and we also knocked out STING in blood ECs. Ablation of STING in EC does not increase susceptibility to KSHV latent infection nor does it increase KSHV spread after lytic reactivation indicating STING signaling does not restrict KSHV. In contrast, STING ablation increases Adenovirus spread at low MOI, but STING is dispensable for blocking replication. These experiments reveal that the importance of STING depends on the DNA virus and that STING appears more important for restricting spread to bystander cells than for inhibition of viral replication.

  • Differential transmission of Sri Lankan cassava mosaic virus by three cryptic species of the whitefly Bemisia tabaci complex
    Virology (IF 2.657) Pub Date : 2019-11-24
    Yao Chi, Li-Long Pan, Sophie Bouvaine, Yun-Yun Fan, Yin-Quan Liu, Shu-Sheng Liu, Susan Seal, Xiao-Wei Wang

    In recent years, Sri Lankan cassava mosaic virus (SLCMV), a begomovirus (genus Begmovirus, family Geminiviridae) causing cassava mosaic disease in Asia, poses serious threats to cassava cultivation in Asia. However, the transmission of SLCMV in the areas into which it has recently been introduced remain largely unexplored. Here we have compared the transmission efficiencies of SLCMV by three widely distributed whitefly species in Asia, and found that only Asia II 1 whiteflies were able to transmit this virus efficiently. The transmission efficiencies of SLCMV by different whitefly species were found to correlate positively with quantity of virus in whitefly whole body. Further, the viral transmission efficiency was found to be associated with varied ability of virus movement within different species of whiteflies. These findings provide detailed information regarding whitefly transmission of SLCMV, which will help to understand the spread of SLCMV in the field, and facilitate the prediction of virus epidemics.

  • Field porcine reproductive and respiratory syndrome viruses (PRRSV) attenuated by codon pair deoptimization (CPD) in NSP1 protected pigs from heterologous challenge
    Virology (IF 2.657) Pub Date : 2019-11-15
    Changhoon Park, Jong Hyuk Baek, Sun Hee Cho, Jiwoon Jeong, Chanhee Chae, Su-Hwa You, Sang-Ho Cha

    Two type 2 field porcine reproductive and respiratory syndrome viruses (PRRSV) isolated from PRRS-affected swine farms were attenuated by de-optimization of codon pair bias in NSP1. In 3-week-old pigs infection, the attenuated viruses showed significantly lower replication ability than the original viruses without distinct clinical sign and pathological lesions, which were observed in pig infected with the original viruses. Regarding induction of PRRSV specific immunity, the level of the neutralizing antibodies as well as secretion of IFN-γ-SCs in PBMCs was not different between the attenuated viruses and the original viruses. More importantly, pigs infected with the attenuated viruses exhibited significant reduction in respiratory scores, viremia, macroscopic and microscopic lung lesion scores, and PRRSV-antigen with interstitial pneumonia against a heterologous challenge with a type 2 virulent strain. Conclusively, the viruses attenuated by CPD in this study demonstrated potential usefulness as vaccine strains to provide protective immunity against diverse virulent PRRSVs.

  • GADD34 attenuates HIV-1 replication by viral 5′-UTR TAR RNA-mediated translational inhibition
    Virology (IF 2.657) Pub Date : 2019-11-15
    Mohammad Ishaq, Heather Marshall, Ven Natarajan

    Role of GADD34, a protein that is induced following cellular stress, in HIV-1 replication was investigated. GADD34 was induced during the late phase of HIV-1 infection. siRNA-knockdown of GADD34 stimulated whereas overexpression of GADD34 inhibited HIV-1 replication. GADD34 N-terminal ER-binding-helix amino acid region 1–192 alone was found to be sufficient for the inhibition of HIV-1 replication whereas protein-phosphatase -1-binding domain and eIF-2α-phosphatase activity of GADD34 were not crucial for anti-HIV-1 activity. GADD34 did not alter the HIV-1 RNA levels but reduced the viral protein expression suggesting that GADD34 interferes in HIV protein synthesis. Studies on the effect of HIV-1-5′-UTR and its mutants on a human promoter-driven luciferase expression indicated that GADD34-inhibition was mediated by 5′-UTR/TAR RNA, probably by modulating TAR RNA structure. In summary, our data support a novel function of GADD34 as a putative anti-HIV-1 restriction factor.

  • Protein inhibitor of activated STAT1 (PIAS1) inhibits IRF8 activation of Epstein-Barr virus lytic gene expression
    Virology (IF 2.657) Pub Date : 2019-11-11
    Kun Zhang, Dong-Wen Lv, Renfeng Li

    Epstein-Barr virus (EBV), a major human oncogenic pathogen, establishes life-long persistent infections. In latently infected B lymphocytes, the virus persists as an episome in the nucleus. Periodic reactivation of latent virus is controlled by both viral and cellular factors. Our recent studies showed that interferon regulatory factor 8 (IRF8) is required for EBV lytic reactivation while protein inhibitor of activated STAT1 (PIAS1) functions as an EBV restriction factor to block viral reactivation. Here, we show that IRF8 directly binds to the EBV genome and regulates EBV lytic gene expression together with PU.1 and EBV transactivator RTA. Furthermore, our study reveals that PIAS1 antagonizes IRF8/PU.1-mediated lytic gene activation through binding to and inhibiting IRF8. Together, our study establishes IRF8 as a transcriptional activator in promoting EBV reactivation and defines PIAS1 as an inhibitor of IRF8 to limit lytic gene expression.

  • HIV-1 genetic diversity and divergence and its correlation with disease progression among antiretroviral naïve recently infected individuals
    Virology (IF 2.657) Pub Date : 2019-11-08
    Ana Rachel Leda, James Hunter, Ursula Castro de Oliveira, Inacio Junqueira de Azevedo, Esper G. Kallas, Maria Cecilia Araripe Sucupira, Ricardo Sobhie Diaz

    HIV-1 genetic diversity evolution was deeply characterized during the first year of infection among recently-infected patients using deep sequencing technology and correlated with disease progression surrogate markers. RNA and DNA samples from twenty-five individuals (13 female) encoding the protease and reverse transcriptase regions of the pol gene, and the V3 region of the env gene were evaluated at recent infection and during established infection. Infection by a unique HIV-1 strain was inferred in 70.1% of the individuals, with no differences between genders. Infections by multiple strains were associated with higher viral loads and faster CD4+ T cell declines. Either low or high levels of viral loads accompanied low levels of genetic diversity and lower selective pressure. With massive sequence data from 3 distinct genomic HIV-1 regions from plasma and PBMCs over time, we propose a model for HIV-1 genetic diversity, which correlates to basal viral loads of patients.

  • Early antibody responses map to non-protective, PCV2 capsid protein epitopes
    Virology (IF 2.657) Pub Date : 2019-11-08
    M. Ilha, P. Nara, S. Ramamoorthy

    Porcine circovirus type 2 (PCV2) is an economically important cause of post-weaning multisystemic wasting syndrome (PMWS) in weanling piglets. Current commercial vaccines against PCV2 are highly effective. Yet, a recurring emergence of new genotypes in vaccinated herds necessitates a better understanding of protective immunity. The study objectives were to identify previously unrecognized decoy epitopes in the PCV2 capsid and test the hypothesis that early antibody responses would map to decoy epitopes and vice versa. Using a peptide library spanning the PCV2a capsid and weekly sera collections from PCV2a infected animals, three major immunodominant regions mapping the early responses to decoy epitopes were identified. Regions with potential decoy activity were mapped using peptide blocking fluorescent focus inhibition assays to residues 55 YTVKATTVRTPSWAVDMM 72, 106 WPCSPITQGDRGVGSTAV 123 and 124 ILDDNFVTKATALTYDPY 141. Post-vaccination responses largely recognized these same three identified regions and dominated the antibody responses to PCV2 in both infection and vaccination.

  • Metagenomic analysis of virome cross-talk between cultivated Solanum lycopersicum and wild Solanum nigrum
    Virology (IF 2.657) Pub Date : 2019-11-08
    Yuxin Ma, Armelle Marais, Marie Lefebvre, Chantal Faure, Thierry Candresse

    Wild plants and weeds growing close to crops constitute a potential reservoir for future epidemies or for the emergence of novel viruses but the frequency and directionality of viral flow between cultivated and wild plants remains poorly documented in many cases. Here, we studied the diversity of viral populations between tomato (Solanum lycopersicum) and neighboring european black nightshade (Solanum nigrum) using high throughput sequencing (HTS) based metagenomics. A large variability in virome richness with only 17.9% shared Operational Taxonomy Units between tomato and nightshade, but this richness could not be linked to a particular host or to local conditions. A detailed population analysis based on assembled contigs for potato virus Y (PVY), broad wilt bean virus 1 and a new ilarvirus tentatively named Solanum nigrum ilarvirus 1 provides information on the circulation of these viruses between these two Solanum species and enriches our knowledge of the tomato virome.

  • Molecular characterisation of a novel pathogenic avipoxvirus from the Australian magpie (Gymnorhina tibicen)
    Virology (IF 2.657) Pub Date : 2019-11-07
    Subir Sarker, Steven Batinovic, Saranika Talukder, Shubhagata Das, Fiona Park, Steve Petrovski, Jade K. Forwood, Karla J. Helbig, Shane R. Raidal
  • Analysis of the in vitro replication phenotype of African hepatitis B virus (HBV) genotypes and subgenotypes present in Australia identifies marked differences in DNA and protein expression
    Virology (IF 2.657) Pub Date : 2019-11-07
    E. Bannister, V. Sozzi, H. Mason, S. Locarnini, W. Hardikar, P.A. Revill

    Hepatitis B virus infection in Africa is characterised by distinct genotypes with observed differences in natural history and clinical outcomes. Replication-competent infectious cDNA clones of African genotypes were generated from patient-derived sequences identified in African children with chronic hepatitis B infection living in Australia: A1 (wild-type and basal core promotor (BCP) mutant), D2, D6, and E, comparing the replication phenotype to an established D3 cDNA clone in a transient transfection cell culture model. All clones replicated efficiently although less than the European D3 reference clone, and demonstrated marked differences in replication capacity, highest for subgenotypes A1 and D2. The BCP mutation increased the replication levels of the A1 subgenotype compared to wild-type. Intracellular and secreted surface antigen and HBeAg protein expression also varied across genotypes. We observed differences in functional activity in the upstream regulatory region across the genotypes that may contribute to the replication and protein differences observed.

  • Nucleocapsid proteins from other swine enteric coronaviruses differentially modulate PEDV replication
    Virology (IF 2.657) Pub Date : 2019-11-07
    Suttipun Sungsuwan, Anan Jongkaewwattana, Peera Jaru-Ampornpan

    Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) share tropism for swine intestinal epithelial cells. Whether mixing of viral components during co-infection alters pathogenicity outcomes or viral replication is not known. In this study, we investigated how different coronavirus nucleocapsid (CoV N) proteins interact and affect PEDV replication. We found that PDCoV N and TGEV N can competitively interact with PEDV N. However, the presence of PDCoV or TGEV N led to very different outcomes on PEDV replication. While PDCoV N significantly suppresses PEDV replication, overexpression of TGEV N, like that of PEDV N, increases production of PEDV RNA and virions. Despite partial interchangeability in nucleocapsid oligomerization and viral RNA synthesis, endogenous PEDV N cannot be replaced in the production of infectious PEDV particles. Results from this study give insights into functional compatibilities and evolutionary relationship between CoV viral proteins during viral co-infection and co-evolution.

  • The respiratory syncytial virus polymerase can perform RNA synthesis with modified primers and nucleotide analogs
    Virology (IF 2.657) Pub Date : 2019-11-06
    Barbara Ludeke, Rachel Fearns

    Respiratory syncytial virus (RSV) is significant for public health, capable of causing respiratory tract disease in infants, the elderly and the immunocompromised. The RSV polymerase is an attractive target for antiviral drug development, but as yet, there is no high throughput assay for analyzing RSV polymerase activity, specifically. In this study, using a primer elongation assay as a basis, we analyzed the tolerance of the RSV polymerase for modifications at the 5′ end of the primer, and nucleotide analogs. The RSV polymerase was found to accept primers containing 5′ biotin or digoxygenin modifications, and nucleotide analogs that are reactive or fluorescent, including 5-ethynyl UTP, 8-azido ATP, 2-aminopurine, and thieno-GTP. These findings provide a menu of options for developing non-isotopic high throughput assays for RSV polymerase RNA synthesis activity, and yield insight regarding the molecular biology of the polymerase complex.

  • In experimental challenge with infectious clones of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), MrNV alone can cause mortality in freshwater prawn (Macrobrachium rosenbergii)
    Virology (IF 2.657) Pub Date : 2019-11-05
    Warachin Gangnonngiw, Malinee Bunnontae, Kornsunee Phiwsaiya, Saengchan Senapin, Arun K. Dhar

    To overcome the lack of immortal shrimp cell lines for shrimp viral research, we constructed and tested DNA infectious clones of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) often found together in freshwater prawn (M. rosenbergii) exhibiting white tail disease (WTD). Full-length cDNAs of MrNV and XSV genomic RNA were individually inserted into the baculovirus pFastBacDUAL shuttle vector. Individual Sf9 (insect cell line) transfection resulted in production of RNA (RT-PCR) and capsid proteins (immunofluorescence) for both viruses. Presence of respective virions was confirmed by density gradient purification followed by RT-PCR and transmission electron microscopy. Infectivity was by tested in immersion-challenge tests with M. rosenbergii post-larvae (PL) using both semi-purified viruses, individually or combined, and confirmed by histological analysis (morphology and immunofluorescence) and quantitative RT-PCR. Mortality accompanied by WTD lesions occurred with MrNV alone or in combination with XSV but not with XSV alone, despite its replication.

  • IFITM3 and type I interferons are important for the control of influenza a virus replication in murine macrophages
    Virology (IF 2.657) Pub Date : 2019-11-05
    Sarah L. Londrigan, Linda M. Wakim, Jeffrey Smith, Anne J. Haverkate, Andrew G. Brooks, Patrick C. Reading

    Abortive infection of macrophages serves as a “dead end” for most seasonal influenza A virus (IAV) strains, and it is likely to contribute to effective host defence. Interferon (IFN)-induced transmembrane protein 3 (IFITM3) restricts the early stages of IAV replication in epithelial cells, but IFITM3 restriction of IAV replication in macrophages has not been previously investigated. Herein, macrophages isolated from IFITM3-deficient mice were more susceptible to initial IAV infection, but late-stage viral replication was still controlled through abortive infection. Strikingly, IFNα/β receptor (IFNAR)-deficient macrophages infected with IAVwere not only more susceptible to initial infection, but these cells also supported productive viral replication. Significantly, we have established that abortive IAV infection in macrophages is controlled through a type I IFN-dependent mechanism, where late-stage IAV replication can proceed in the absence of type I IFN responses. These findings provide novel mechanistic insight into macrophage-specific processes that potently shut down IAV replication.

  • Characterization of pUL5, an HCMV protein interacting with the cellular protein IQGAP1
    Virology (IF 2.657) Pub Date : 2019-11-03
    Giulia Anselmi, Maria Giuliani, Giacomo Vezzani, Rossella Ferranti, Michela Gentile, Mirko Cortese, Diego Amendola, Nicola Pacchiani, Romina D'Aurizio, Luca Bruno, Yasushi Uematsu, Marcello Merola, Domenico Maione

    Among the Herpesviridae, human cytomegalovirus (HCMV) owns the largest genome and displays a huge coding potential. Here, we characterized the UL5 gene product (pUL5) of the clinical isolate TR strain. The protein was predicted as a 166-amino-acid membrane protein with a theoretical mass of 19 kDa. Recombinant virus expressing pUL5 with a tag allowed the identification of two pUL5 non-glycosylated species of approximately 19 and 9 kDa, expressed with early and late kinetic respectively. Experiments in infection confirmed that the lower molecular weight species was translated from an internal ATG in the UL5 open reading frame. Confocal microscopy analysis showed that pUL5 localized within the assembly compartment, but is not incorporated in the virion, as shown by Western blot on purified viral particles. Finally, pull-down experiments coupled with mass spectrometry analysis identified IQGAP1 as a pUL5 interactor, giving new hints on possible roles of pUL5 during HCMV infection.

  • Mutations of the segment-specific nucleotides at the 3’ end of influenza virus NS segment control viral replication
    Virology (IF 2.657) Pub Date : 2019-11-01
    Paloma Rodriguez, Laura Marcos-Villar, Noelia Zamarreño, Emilio Yángüez, Amelia Nieto

    The vRNAs of influenza A viruses contain 12 and 13 nucleotide-long sequences at their 3′ and 5′ termini respectively that are highly conserved and constitute the vRNA promoter. These sequences and the next three segment-specific nucleotides show inverted partial complementarity and are followed by several unpaired nucleotides of poorly characterized function at the 3′ end. We have performed systematic point-mutations at the segment-specific nucleotides 15–18 of the 3′-end of a NS-like vRNA segment. All NS-like vRNAs containing mutations at position 15, and some at positions 16–18 showed reduced transcription/replication efficiency in a transfection/infection system. In addition, the replication of recombinant viruses containing mutations at position 15 was impaired both in single and multi-cycle experiments. This reduction was the consequence of a decreased expression of the NS segment. The data indicate that NS1 plays a role in the transcription/replication of its own segment, which elicits a global defect on virus replication.

  • A convenient in vivo cap donor delivery system to investigate the cap snatching of plant bunyaviruses
    Virology (IF 2.657) Pub Date : 2019-11-01
    Wenzhong Lin, Ran Wu, Ping Qiu, Jing jin, Yunyue Yang, Jinglin Wang, Zhonglong Lin, Jie Zhang, Zujian Wu, Zhenguo Du

    Like their animal-infecting counterparts, plant bunyaviruses use capped RNA leaders cleaved from host cellular mRNAs to prime viral genome transcription in a process called cap-snatching, but in vivo systems to investigate the details of this process are lacking for them. Here, we report that Rice stripe tenuivirus (RSV) and Tomato spotted wilt tospovirus (TSWV) cleave capped RNA leaders from mRNAs transiently expressed by agroinfiltration, which makes it possible to artificially deliver defined cap donors to the two plant bunyaviruses with unprecedented convenience. With this system, some ideas regarding how plant bunyaviruses select and use capped RNA leaders can be tested easily. We were also able to obtain clear evidence that the capped RNA leaders selected by TSWV are generally longer than those by RSV. TSWV frequently uses the prime-and-realign mechanism in transcription primed by capped RNA leaders shorter than a certain length, like that has been demonstrated recently for RSV.

  • Genomic and transcriptional analyses of novel parvoviruses identified from dead peafowl
    Virology (IF 2.657) Pub Date : 2019-10-31
    Xiaoping Liu, Hanzhong Wang, Xiaoqian Liu, Yong Li, Jing Chen, Jun Zhang, Xi Wang, Shu Shen, Hualin Wang, Fei Deng, Manli Wang, Wuxiang Guan, Zhihong Hu

    To identify potential pathogens responsible for a disease outbreak of cultured peafowls in China in 2013, metagenomic sequencing was conducted. The genomes of two closely related parvoviruses, namely peafowl parvovirus 1 (PePV1) and PePV2, were identified with size of 4438 bp and 4348 bp, respectively. Phylogenetic analysis revealed that both viruses are novel parvoviruses, belonging to the proposed genus Chapparvovirus of Parvoviridae. The transcriptional profile of PePV1 was analyzed by transfecting a nearly complete PePV1 genome into HEK-293T cells. Results revealed that PePV1 employs one promoter and two polyadenylation sites to start and terminate its transcriptions, with one donor site and two acceptor sites for pre-mRNA splicing. PePV1 DNA and structural protein were detected in several tissues of a dead peafowl, which appeared to have suffered enteritis, pneumonia and viremia. These results provide novel information of chapparvoviruses, and call for attention to the potential pathogens.

  • Transmission dynamics between infected waterfowl and terrestrial poultry: Differences between the transmission and tropism of H5N8 highly pathogenic avian influenza virus (clade among ducks, chickens and turkeys
    Virology (IF 2.657) Pub Date : 2019-10-31
    Anita Puranik, Marek J. Slomka, Caroline J. Warren, Saumya S. Thomas, Sahar Mahmood, Alexander M.P. Byrne, Andrew M. Ramsay, Paul Skinner, Samantha Watson, Helen E. Everett, Alejandro Núñez, Ian H. Brown, Sharon M. Brookes

    H5N8 highly-pathogenic avian influenza viruses (HPAIVs, clade have spread globally via migratory waterfowl. Pekin ducks infected with a UK virus (H5N8-2014) served as the donors of infection in three separate cohousing experiments to attempt onward transmission chains to sequentially introduced groups of contact ducks, chickens and turkeys. Efficient transmission occurred among ducks and turkeys up to the third contact stage, with all (100%) birds becoming infected. Introduction of an additional fourth contact group of ducks to the turkey transmission chain demonstrated retention of H5N8-2014's waterfowl-competent adaptation. However, onward transmission ceased in chickens at the second contact stage where only 13% became infected. Analysis of viral progeny at this contact stage revealed no emergent polymorphisms in the intra-species (duck) transmission chain, but both terrestrial species included changes in the polymerase and accessory genes. Typical HPAIV pathogenesis and mortality occurred in infected chickens and turkeys, contrasting with 5% mortality among ducks.

  • EGR1 upregulation following Venezuelan equine encephalitis virus infection is regulated by ERK and PERK pathways contributing to cell death
    Virology (IF 2.657) Pub Date : 2019-10-31
    Bibha Dahal, Shih-Chao Lin, Brian D. Carey, Jonathan L. Jacobs, Jonathan D. Dinman, Monique L. van Hoek, Andre A. Adams, Kylene Kehn-Hall

    Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18 h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.

  • Human pegivirus 2 exhibits minimal geographic and temporal genetic diversity
    Virology (IF 2.657) Pub Date : 2019-10-31
    Kenn Forberg, Mary A. Rodgers, George J. Dawson, Silvia Sauleda, Ana Olivo, Ana Vallari, Marta Bes, Maria Piron, Gavin A. Cloherty, Michael G. Berg

    We applied an NGS based target capture approach to amplify HPgV-2 sequences from metagenomic libraries and enable full genome characterization. Despite expanded geographical sampling, sequence variability remains low, with diversity concentrated in approximately 3.3% of all amino acids. Serial samples from one HPgV-2 positive individual co-infected with comparable titers of HIV, HCV, and GBV-C showed that HPgV-2 remains highly stable over several weeks compared to other RNA viruses, despite a similarly error-prone polymerase. The consistent epidemiological association with and structural similarities to HCV, and the weak positive correlation of HCV and HPgV-2 titers shown here, suggests it may benefit from co-infection. While minimal selective pressure on HPgV-2 to evolve could suggest fitness, the rarity of HPgV-2 and the tight phylogenetic clustering of global strains likely indicates origination from a common source and a virus that is ill-suited to its host. Sporadic infections may explain the limited genetic diversity observed worldwide.

  • Detection of hypermutated human papillomavirus type 16 genome by Next-Generation Sequencing.
    Virology (IF 2.657) Pub Date : 2015-09-12
    Kousho Wakae,Satoru Aoyama,Zhe Wang,Kouichi Kitamura,Guangyan Liu,Ahasan Md Monjurul,Miki Koura,Mieko Imayasu,Naoya Sakamoto,Mitsuhiro Nakamura,Satoru Kyo,Satoru Kondo,Hiroshi Fujiwara,Tomokazu Yoshizaki,Iwao Kukimoto,Katsushi Yamaguchi,Shuji Shigenobu,Tomoaki Nishiyama,Masamichi Muramatsu

    Human papillomavirus type 16 (HPV16) is a major cause of cervical cancer. We previously demonstrated that C-to-T and G-to-A hypermutations accumulated in the HPV16 genome by APOBEC3 expression in vitro. To investigate in vivo characteristics of hypermutation, differential DNA denaturation-PCR (3D-PCR) was performed using three clinical specimens obtained from HPV16-positive cervical dysplasia, and detected hypermutation from two out of three specimens. One sample accumulating hypermutations in both E2 and the long control region (LCR) was further subjected to Next-Generation Sequencing, revealing that hypermutations spread across the LCR and all early genes. Notably, hypermutation was more frequently observed in the LCR, which contains a viral replication origin and the early promoter. APOBEC3 expressed abundantly in an HPV16-positive cervix, suggesting that single-stranded DNA exposed during viral replication and transcription may be efficient targets for deamination. The results further strengthen a role of APOBEC3 in introducing HPV16 hypermutation in vivo.

  • A computational analysis of the structural determinants of APOBEC3's catalytic activity and vulnerability to HIV-1 Vif.
    Virology (IF 2.657) Pub Date : 2014-12-03
    Shivender M D Shandilya,Markus-Frederik Bohn,Celia A Schiffer

    APOBEC3s (A3) are Zn(2+) dependent cytidine deaminases with diverse biological functions and implications for cancer and immunity. Four of the seven human A3s restrict HIV by 'hypermutating' the reverse-transcribed viral genomic DNA. HIV Virion Infectivity Factor (Vif) counters this restriction by targeting A3s to proteasomal degradation. However, there is no apparent correlation between catalytic activity, Vif binding, and sequence similarity between A3 domains. Our comparative structural analysis reveals features required for binding Vif and features influencing polynucleotide deaminase activity in A3 proteins. All Vif-binding A3s share a negatively charged surface region that includes residues previously implicated in binding the highly-positively charged Vif. Additionally, catalytically active A3s share a positively charged groove near the Zn(2+) coordinating active site, which may accommodate the negatively charged polynucleotide substrate. Our findings suggest surface electrostatics, as well as the spatial extent of substrate accommodating region, are critical determinants of substrate and Vif binding across A3 proteins with implications for anti-retroviral and anti-cancer therapeutic design.

  • Host restriction of murine gammaherpesvirus 68 replication by human APOBEC3 cytidine deaminases but not murine APOBEC3.
    Virology (IF 2.657) Pub Date : 2014-04-15
    Nana Minkah,Kevin Chavez,Parth Shah,Thomas Maccarthy,Hui Chen,Nathaniel Landau,Laurie T Krug

    Humans encode seven APOBEC3 (A3A-A3H) cytidine deaminase proteins that differ in their expression profiles, preferred nucleotide recognition sequence and capacity for restriction of RNA and DNA viruses. We identified APOBEC3 hotspots in numerous herpesvirus genomes. To determine the impact of host APOBEC3 on herpesvirus biology in vivo, we examined whether murine APOBEC3 (mA3) restricts murine gammaherpesvirus 68 (MHV68). Viral replication was impaired by several human APOBEC3 proteins, but not mA3, upon transfection of the viral genome. The restriction was abrogated upon mutation of the A3A and A3B active sites. Interestingly, virus restriction by A3A, A3B, A3C, and A3DE was lost if the infectious DNA was delivered by the virion. MHV68 pathogenesis, including lung replication and splenic latency, was not altered in mice lacking mA3. We infer that mA3 does not restrict wild type MHV68 and restriction by human A3s may be limited in the herpesvirus replication process.

  • Deamination intensity profiling of human APOBEC3 protein activity along the near full-length genomes of HIV-1 and MoMLV by HyperHRM analysis.
    Virology (IF 2.657) Pub Date : 2013-12-10
    Kasandra Bélanger,Mathieu Savoie,Halil Aydin,Tyler Milston Renner,Zahra Montazeri,Marc-André Langlois

    Enzymatic deamination of cytidines in DNA is an intrinsic component of antibody maturation and retroviral resistance, but can also be a source of HIV drug resistance and cancer-causing mutations. Here, we developed a high-throughput method based on high resolution melt (HRM) analysis called HyperHRM that can screen genomic DNA for rare hypermutated proviral sequences and accurately quantify the number of C-to-T or G-to-A mutations in each sequence. We demonstrate the effectiveness of the approach by profiling in parallel the intensity of the DNA mutator activity of all seven human APOBEC3 proteins on the near full-length sequence of HIV-1 and the Moloney murine leukemia virus. Additionally, HRM was successfully used to identify hypermutated proviral sequences in peripheral blood mononuclear cells from an HIV-1 patient. These results exemplify the effectiveness of HRM-based approaches for hypermutation quantification and for the detection of hypermutated DNA sequences potentially associated with disease or retroviral drug resistance.

  • Identification and antiviral activity of common polymorphisms in the APOBEC3 locus in human populations.
    Virology (IF 2.657) Pub Date : 2013-06-13
    Nisha K Duggal,Wenqing Fu,Joshua M Akey,Michael Emerman

    There are seven members of the APOBEC3 family in humans (APOBEC3A through APOBEC3H) that have antiviral activity against retroviruses and/or retroelements. To determine whether variants in APOBEC3 genes in human populations have altered antiviral activity, we identified and functionally tested novel single nucleotide variants (SNVs) in APOBEC3 genes present in the 1000 Genome Project dataset. We found that common variants minor allele frequency (> 1%) of APOBEC3A, C, F, and G do not affect protein function. However, we found that two common novel polymorphisms in APOBEC3D decrease antiviral activity against HIV-1, and one polymorphism decreases activity against Alu retrotransposons. We characterized the diversity of APOBEC3 genes in three human populations and find significant evidence that APOBEC3D has evolved under purifying selection in recent human history. These data suggest that the activity of APOBEC3D has been maintained in human populations for a cellular function in host defense.

  • Human APOBEC3 proteins can inhibit xenotropic murine leukemia virus-related virus infectivity.
    Virology (IF 2.657) Pub Date : 2010-12-07
    Hal P Bogerd,Fengwen Zhang,Paul D Bieniasz,Bryan R Cullen

    Xenotropic murine leukemia virus-related virus (XMRV) is a novel retrovirus, related to murine leukemia virus (MLV), that has been implicated in human disease. If XMRV is indeed able to replicate in humans, then one might predict that XMRV would have developed resistance to human innate antiviral resistance factors such as APOBEC3G (hA3G). In fact, we observed that XMRV and MLV are both highly sensitive to inhibition by hA3G and equally resistant to inhibition by murine APOBEC3. While several human prostate cancer cell lines were found to lack hA3G, stable expression of physiological levels of hA3G rendered these cells refractory to XMRV replication. Few human tissues fail to express hA3G, and we therefore hypothesize that XMRV replicates in one or more hA3G-negative reservoir tissues and/or that human XMRV infections are likely to be rare and potentially of zoonotic origin.

  • A novel human DnaJ protein, hTid-1, a homolog of the Drosophila tumor suppressor protein Tid56, can interact with the human papillomavirus type 16 E7 oncoprotein.
    Virology (IF 2.657) Pub Date : 1998-07-31
    B Schilling,T De-Medina,J Syken,M Vidal,K Münger

    We have cloned hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, by virtue of its ability to form complexes with the human papillomavirus E7 oncoprotein. The carboxyl terminal cysteine-rich metal binding domain of E7 is the major determinant for interaction with hTid-1. The carboxyl terminus of E7 is essential for the functional and structural integrity of E7 and has previously been shown to function as a multimerization domain. The hTid-1 protein is a member of the DnaJ-family of chaperones. Its mRNA is widely expressed in human tissues, including the HPV-18-positive cervical carcinoma cell line HeLa and human genital keratinocytes, the normal host cells of the HPVs. The hTid-1 gene has been mapped to the short arm of chromosome 16. The large tumor antigens of polyomaviruses encode functional J-domains that are important for viral replication as well as cellular transformation. The ability of HPV E7 to interact with a cellular DnaJ protein suggests that these two viral oncoproteins may target common regulatory pathways through J-domains.

  • Coronaviruses and arteriviruses display striking differences in their cyclophilin A-dependence during replication in cell culture.
    Virology (IF 2.657) Pub Date : 2017-12-19
    Adriaan H de Wilde,Jessika C Zevenhoven-Dobbe,Corrine Beugeling,Udayan Chatterji,Danielle de Jong,Philippe Gallay,Karoly Szuhai,Clara C Posthuma,Eric J Snijder

    Cyclophilin A (CypA) is an important host factor in the replication of a variety of RNA viruses. Also the replication of several nidoviruses was reported to depend on CypA, although possibly not to the same extent. These prior studies are difficult to compare, since different nidoviruses, cell lines and experimental set-ups were used. Here, we investigated the CypA dependence of three distantly related nidoviruses that can all replicate in Huh7 cells: the arterivirus equine arteritis virus (EAV), the alphacoronavirus human coronavirus 229E (HCoV-229E), and the betacoronavirus Middle East respiratory syndrome coronavirus (MERS-CoV). The replication of these viruses was compared in the same parental Huh7 cells and in CypA-knockout Huh7 cells generated using CRISPR/Cas9-technology. CypA depletion reduced EAV yields by ~ 3-log, whereas MERS-CoV progeny titers were modestly reduced (3-fold) and HCoV-229E replication was unchanged. This study reveals that the replication of nidoviruses can differ strikingly in its dependence on cellular CypA.

  • Stem cell-based anti-HIV gene therapy.
    Virology (IF 2.657) Pub Date : 2011-01-21
    Scott G Kitchen,Saki Shimizu,Dong Sung An

    Human stem cell-based therapeutic intervention strategies for treating HIV infection have recently undergone a renaissance as a major focus of investigation. Unlike most conventional antiviral therapies, genetically engineered hematopoietic stem cells possess the capacity for prolonged self-renewal that would continuously produce protected immune cells to fight against HIV. A successful strategy therefore has the potential to stably control and ultimately eradicate HIV from patients by a single or minimal treatment. Recent progress in the development of new technologies and clinical trials sets the stage for the current generation of gene therapy approaches to combat HIV infection. In this review, we will discuss two major approaches that are currently underway in the development of stem cell-based gene therapy to target HIV: one that focuses on the protection of cells from productive infection with HIV, and the other that focuses on targeting immune cells to directly combat HIV infection.

  • Human bocavirus 1 infection of CACO-2 cell line cultures.
    Virology (IF 2.657) Pub Date : 2017-08-05
    Lucía María Ghietto,Ana Paola Toigo D'Angelo,Franco Agustin Viale,María Pilar Adamo

    Human bocavirus 1 (HBoV1) is a parvovirus associated with pneumonia in infants. It has been detected in different tissues, including colorectal tumors. In this study, we investigated whether Caco-2 cell line, derived from human colon cancer, can be utilized as a model for HBoV1 replication. We demonstrate HBoV1 replication in Caco-2 cultures supplemented with DEAE-dextran after inoculation with respiratory material from infected patients presenting with acute respiratory infection. A viral cycle of rapid development is displayed. However, in spite of HBoV1 DNA 4-fold increment in the supernatants and monolayers by day 1, evidencing that the system allows the virus genome replication after the entry occurred, infectious progeny particles were not produced. These results are consistent with an infection that is limited to a single growth cycle, which can be associated to mutations in the NS1 and VP1/VP2 regions of HBoV1 genome. Further research will contribute to fully elucidate these observations.

  • Stable complex formation between HIV Rev and the nucleosome assembly protein, NAP1, affects Rev function.
    Virology (IF 2.657) Pub Date : 2009-04-03
    Alan Cochrane,Laura Lea Murley,Mian Gao,Raymond Wong,Kiera Clayton,Nicole Brufatto,Veronica Canadien,Daniel Mamelak,Tricia Chen,Dawn Richards,Mahel Zeghouf,Jack Greenblatt,Christian Burks,Lori Frappier

    The Rev protein of HIV-1 is essential for HIV-1 proliferation due to its role in exporting viral RNA from the nucleus. We used a modified version of tandem affinity purification (TAP) tagging to identify proteins interacting with HIV-1 Rev in human cells and discovered a prominent interaction between Rev and nucleosome assembly protein 1 (Nap1). This interaction was also observed by specific retention of Nap1 from human cell lysates on a Rev affinity column. Nap1 was found to bind Rev through the Rev arginine-rich domain and altered the oligomerization state of Rev in vitro. Overexpression of Nap1 stimulated the ability of Rev to export RNA, reduced the nucleolar localization of Rev, and affected Rev nuclear import rates. The results suggest that Nap-1 may influence Rev function by increasing the availability of Rev.

  • A simple one-step method for the preparation of HIV-1 envelope glycoprotein immunogens based on a CD4 mimic peptide.
    Virology (IF 2.657) Pub Date : 2008-10-07
    Grégoire Martin,Yide Sun,Bernadette Heyd,Olivier Combes,Jeffrey B Ulmer,Anne Descours,Susan W Barnett,Indresh K Srivastava,Loïc Martin

    To counteract the problems associated with the purification of HIV envelope, we developed a new purification method exploiting the high affinity of a peptide mimicking CD4 towards the viral glycoprotein. This miniCD4 was used as a ligand in affinity chromatography and allowed the separation in one step of HIV envelope monomer from cell supernatant and the capture of pre-purified trimer. This simple and robust method of purification yielded to active and intact HIV envelopes as proved by the binding of CCR5 HIV co-receptor, CD4 and a panel of well-characterized monoclonal antibodies. The immunogenicity of miniCD4-purified HIV envelope was further assessed in rats. The analysis of the humoral response indicated that elicited antibodies were able to recognize a broad range of HIV envelopes. Finally, this method based on a chemically synthesized peptide may represent a convenient and versatile tool for protein purification compatible far scale-up in both academic and pharmaceutical researches.

  • Long-term infection with retroviral structural gene vector provides protection against bovine leukemia virus disease in rabbits.
    Virology (IF 2.657) Pub Date : 2004-11-03
    Veronika Altanerova,Dana Holicova,Lucia Kucerova,Cestmir Altaner,Michael D Lairmore,Kathleen Boris-Lawrie

    Bovine leukemia virus (BLV) infection of rabbits is a tractable model system to evaluate vaccination strategies against lymphotropic retroviruses, which represent a global human health problem. We have previously developed genetically simplified BLV structural gene vector (SGV) that replicates BLV structural and enzymatic genes independently of BLV regulatory and accessory genes. Results of a 20-month study in a rabbit model demonstrated that BLV SGV induces an antiviral immunological response and lacks pathogenicity. Here, these chronically infected-BLV SGV rabbits are assessed in a proof-of-principle study of preventative vaccination against challenge with pathogenic BLV. This study commences 24 months after BLV SGV inoculation and proceeds for an additional 20 months. The previously characterized BLV SGV rabbits and age-matched control rabbits were challenged with 1 x 10(8) fetal lamb kidney/BLV producer cells. BLV SGV rabbits seroconverted upon BLV challenge, but did not progress to BLV infection nor clinical disease. By contrast, naive rabbits became infected and succumbed to lymphotropic disease. Our findings provide proof-of-principle that chronic infection with BLV SGV induces protection against BLV infection. The data indicate that SGV based on HTLV or HIV is a promising approach against lymphotropic disease by human retroviruses.

  • Pre-vaccine circulating group a rotavirus strains in under 5 years children with acute diarrhea during 1999-2013 in Cameroon.
    Virology (IF 2.657) Pub Date : 2017-10-21
    Paul Koki Ndombo,Valantine N Ndze,Charles Fokunang,Taku Nadesh Ashukem,Angeline Boula,Mina N Kinkela,Corlins E Ndode,Mapaseka L Seheri,Michael D Bowen,Diane Waku-Kouomou,Mathew D Esona

    The aim of this review was to assess all the studies on rotavirus G and P characterization during the pre-vaccine period (1999-2013) in Cameroon to have a better basis for post-vaccine introduction evaluations. A retrospective study was done through a comprehensive review of published (PubMed, Google Scholar) and accessible unpublished data on rotavirus G and P genotypes circulating in five regions of Cameroon. Descriptive data were expressed as frequencies tables and proportions. A total of 1844 rotavirus positive cases were analyzed. In all, 1534 strains were characterized for the P (VP4) specificity. Six different VP4 genotypes were observed, including P [4], P [6], P [8], P [9], P [10] and P [14]. The most predominant P genotypes were P [8] at 42.6%, and P [6] at 37.9%. Mixed infections were observed at 5.3%, whereas 4.1% of the strains were P non-typeable. A total of 1518 rotavirus strains were characterized for the G (VP7) specificity. VP7 genotypes G1, G2, G3, G4, G5, G6, G8, G9, G10 and G12 were observed. G1 (35.3%), G3 (19.5%), G2 (14.9%) and G12 (10.1%) were the predominant G genotypes while G5 and G10 were least prevalent at 0.06% each. Approximately 5.1% of all strains were G non-typeable whereas 5.3% were mixed G genotypes. A total of 1472 strains were characterized for both G and P genes, from which 38 different G-P combinations were observed. Overall, G1P [8] (22%) was identified as the predominant rotavirus strain circulating in Cameroon followed by G3P [6] (15%). In conclusion, we observed that the genotypes identified in Cameroon during 1999-2013 were partially covered by the two WHO recommended rotavirus vaccines. This review provides comprehensive up-to-date information on rotavirus strain surveillance in Cameroon during the pre-vaccination era.

  • Sensitivity to BST-2 restriction correlates with Orthobunyavirus host range.
    Virology (IF 2.657) Pub Date : 2017-06-20
    Mariana Varela,Ilaria M Piras,Catrina Mullan,Xiaohong Shi,Natasha L Tilston-Lunel,Rute Maria Pinto,Aislynn Taggart,Stephen R Welch,Stuart J D Neil,Felix Kreher,Richard M Elliott,Massimo Palmarini

    Orthobunyaviruses include several recently emerging viruses of significant medical and veterinary importance. There is currently very limited understanding on what determines the host species range of these pathogens. In this study we discovered that BST-2/tetherin restricts orthobunyavirus replication in a host-specific manner. We show that viruses with human tropism (Oropouche virus and La Crosse virus) are restricted by sheep BST-2 but not by the human orthologue, while viruses with ruminant tropism (Schmallenberg virus and others) are restricted by human BST-2 but not by the sheep orthologue. We also show that BST-2 blocks orthobunyaviruses replication by reducing the amount of envelope glycoprotein into viral particles egressing from infected cells. This is the first study identifying a restriction factor that correlates with species susceptibility to orthobunyavirus infection. This work provides insight to help us dissect the adaptive changes that bunyaviruses require to cross the species barrier and emerge into new species.

  • Contributions of CD8 T cells to the pathogenesis of mouse adenovirus type 1 respiratory infection.
    Virology (IF 2.657) Pub Date : 2017-04-15
    Caitlyn T Molloy,Jennifer S Andonian,Harrison M Seltzer,Megan C Procario,Michael E Watson,Jason B Weinberg

    CD8 T cells are key components of the immune response to viruses, but their roles in the pathogenesis of adenovirus respiratory infection have not been characterized. We used mouse adenovirus type 1 (MAV-1) to define CD8 T cell contributions to the pathogenesis of adenovirus respiratory infection. CD8 T cell deficiency in β2m-/- mice had no effect on peak viral replication in lungs, but clearance of virus was delayed in β2m-/- mice. Virus-induced weight loss and increases in bronchoalveolar lavage fluid total protein, IFN-γ, TNF-α, IL-10, CCL2, and CCL5 concentrations were less in β2m-/- mice than in controls. CD8 T cell depletion had similar effects on virus clearance, weight loss, and inflammation. Deficiency of IFN-γ or perforin had no effect on viral replication or inflammation, but perforin-deficient mice were partially protected from weight loss. CD8 T cells promote MAV-1-induced pulmonary inflammation via a mechanism that is independent of direct antiviral effects.

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