|Journal of General Virology current issue|
The 5' end of eukaryotic mRNA contains the type-1 (m7GpppNm) or type-2 (m7GpppNmNm) cap structure. Many viruses have evolved various mechanisms to develop their own capping enzymes (e.g. flavivirus and coronavirus) or to llsquo;stealrrsquo; caps from host mRNAs (e.g. influenza virus). Other viruses have developed llsquo;cap-mimickingrrsquo; mechanisms by attaching a peptide to the 5' end of viral RNA (e.g. picornavirus and calicivirus) or by having a complex 5' RNA structure (internal ribosome entry site) for translation initiation (e.g. picornavirus, pestivirus and hepacivirus). Here we review the diverse viral RNA capping mechanisms. Using flavivirus as a model, we summarize how a single methyltransferase catalyses two distinct N-7 and 2'-O methylations of viral RNA cap in a sequential manner. For antiviral development, a structural feature unique to the flavivirus methyltransferase was successfully used to design selective inhibitors that block viral methyltransferase without affecting host methyltransferases. Functionally, capping is essential for prevention of triphosphate-triggered innate immune activation; N-7 methylation is critical for enhancement of viral translation; and 2'-O methylation is important for subversion of innate immune response during viral infection. Flaviviruses defective in 2'-O methyltransferase are replicative, but their viral RNAs lack 2'-O methylation and are recognized and eliminated by the host immune response. Such mutant viruses could be rationally designed as live attenuated vaccines. This concept has recently been proved with Japanese encephalitis virus and dengue virus. The findings obtained with flavivirus should be applicable to other RNA viruses.
Human infection by H7N9 influenza virus was first identified in China in March 2013. As of 12 August 2013, a total of 135 documented cases with 44 fatalities had been reported. Genetic and laboratory analyses of the novel H7N9 viruses isolated from patients indicate that these viruses possess several polymerase gene mutations previously associated with human adaptation and potential pandemic capabilities. However, the function of these mutations in the emergence and pathogenicity of the viruses is not well known. In this study, we demonstrate that the PB2 E627K mutation, which occurs in over 70 % of the H7N9 patient isolates, promotes the replication of H7N9 virus by enhancing PB2 polymerase activity and enhances virulence in mice. Our results show the PB2 E627K mutation has played an important role in this H7N9 influenza outbreak and in the pathogenicity of the H7N9 virus.
Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11 482 nt (ABTV) and 11 876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family.
The threat of highly pathogenic avian influenza (HPAI) H5N1 viruses to cause the next pandemic remains a major concern. Here, we evaluated the cross-protection induced by natural infection of human seasonal influenza strains or immunization with trivalent inactivated influenza vaccine (TIV) against HPAI H5N1 (A/Vietnam/1203/2004) virus in ferrets. Groups were treated with PBS (group A), infected with H1N1 (group B) or H3N2 (group C) virus, or immunized with TIV (group D). Twelve weeks after the last treatment, serological assays revealed that groups B and C, but not group D, sustained moderate immunogenicity against homologous viruses; cross-reactivity against the H5N1 virus was not detected in any group. Following challenge with A/Vietnam/1203/2004 (H5N1) virus, only groups B and C exhibited attenuated viral loads leading to 100 % survival. Our data suggest that natural infection with human seasonal strains could potentially provide better heterosubtypic protection against HPAI H5N1 virus infection compared to TIV immunization.
In the present work we investigated the importance of the Raf/MEK/ERK signalling pathway in the multiplication of the arenavirus Juniiacute;n (JUNV) in monkey and human cell cultures. We established that JUNV induces a biphasic activation of ERK and we proved that a specific inhibitor of the ERK pathway, U0126, impairs viral replication. Furthermore, U0126 exerted inhibitory action against the arenaviruses Tacaribe and Pichinde. Moreover, treatment with known ERK activators such as phorbol 12-myristate 13-acetate and serum increased viral yields whereas ERK silencing by small interfering RNAs caused the inhibition of viral multiplication. Therefore, activation of the Raf/MEK/ERK signalling pathway is required to ensure efficient JUNV replication and may constitute a host target for the development of novel effective therapeutic strategies to deal with arenavirus infections.
A full-length genome infectious clone is a powerful tool for functional assays in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank accession no. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered virus from the cDNA clone replicated poorly in baby hamster kidney (BHK-21) cells and in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within the NS2B and NS4A proteins were recovered in the passaged viruses leading to viruses with a large-plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were in the order: NS2B-T102Mllt;NS4A-R79Kllt;NS2B-T102M+NS4A-R79K. An in vivo virulence assay in mice showed that the recombinant virus containing double mutations showed similar virulence to the WT SA14 (GenBank accession no. M55506). This study reports the first chemically synthesized JEV. A reverse genetics assay demonstrated that substitutions of NS2B-T102M and NS4A-R79K altered JEV replication.
The human zinc finger antiviral protein (hZAP) gene is spliced to yield a short (hZAP-S) and a long (hZAP-L) isoform. The long isoform possesses a poly(ADP-ribose) polymerase (PARP)-like domain in its C-terminus predicted to be inactive due to alterations in its triad motif compared with bona fide PARPs. Using Sindbis virus as prototype member of alphaviruses we confirmed that hZAP-L is a more potent inhibitor of alphaviruses than hZAP-S. Specific small interfering RNA knockdown of hZAP-L but not hZAP-S demonstrated a role of endogenous hZAP-L in restriction of alphavirus replication. Whilst single amino-acid substitutions in the triad motif of hZAP-Lrrsquo;s PARP-like domain reduced the antiviral activity, exchange of all three triad motif residues to alanine or to the amino acids of active PARPs virtually abolished the antiviral effect. Contrary to previous assumptions, these results indicate an essential function of the PARP-like domain in hZAP-L's antiviral activity.
Tick-borne encephalitis virus (TBEV) is a major arbovirus that causes thousands of cases of severe neurological illness in humans annually. However, virulence factors and pathological mechanisms of TBEV remain largely unknown. To identify the virulence factors, we constructed chimeric viruses between two TBEV strains of the Far-Eastern subtype, Sofjin-HO (highly pathogenic) and Oshima 5-10 (low pathogenic). The replacement of the coding region for the structural and non-structural proteins from Sofjin into Oshima showed a partial increase of the viral pathogenicity in a mouse model. Oshima-based chimeric viruses with the variable region of the 3' UTR of Sofjin, which had a deletion of 207 nt, killed 100 % of mice and showed almost the same virulence as Sofjin. Replacement of the variable region of the 3' UTR from Sofjin into Oshima did not increase viral multiplication in cultured cells and a mouse model at the early phase of viral entry into the brain. At the terminal phase of viral infection in mice, the virus titre of the Oshima-based chimeric virus with the variable region of the 3' UTR of Sofjin reached a level identical to that of Sofjin and showed a similar histopathological change in the brain tissue. This is the first report to show that the variable region of the 3' UTR is a critical virulence factor in mice. These findings encourage further study to understand the mechanisms of the pathogenicity of TBEV, and to develop preventative and therapeutic strategies for tick-borne encephalitis.
From 1 January 2009 to 31 May 2013, 15 287 respiratory specimens submitted to the Clinical Virology Laboratory at the Childrenrrsquo;s Hospital Colorado were tested for human coronavirus RNA by reverse transcription-PCR. Human coronaviruses HKU1, OC43, 229E and NL63 co-circulated during each of the respiratory seasons but with significant year-to-year variability, and cumulatively accounted for 7.4nndash;15.6 % of all samples tested during the months of peak activity. A total of 79 (0.5 % prevalence) specimens were positive for human betacoronavirus HKU1 RNA. Genotypes HKU1 A and B were both isolated from clinical specimens and propagated on primary human trachealnndash;bronchial epithelial cells cultured at the airnndash;liquid interface and were neutralized in vitro by human intravenous immunoglobulin and by polyclonal rabbit antibodies to the spike glycoprotein of HKU1. Phylogenetic analysis of the deduced amino acid sequences of seven full-length genomes of Colorado HKU1 viruses and the spike glycoproteins from four additional HKU1 viruses from Colorado and three from Brazil demonstrated remarkable conservation of these sequences with genotypes circulating in Hong Kong and France. Within genotype A, all but one of the Colorado HKU1 sequences formed a unique subclade defined by three amino acid substitutions (W197F, F613Y and S752F) in the spike glycoprotein and exhibited a unique signature in the acidic tandem repeat in the N-terminal region of the nsp3 subdomain. Elucidating the function of and mechanisms responsible for the formation of these varying tandem repeats will increase our understanding of the replication process and pathogenicity of HKU1 and potentially of other coronaviruses.
Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, detailed mechanisms of viral replication in the brain and features of viral pathogenesis remain poorly understood. We carried out a comparative analysis of replication of neurotropic flaviviruses: West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus (TBEV), in primary cultures of mouse brain neurons. All the flaviviruses multiplied well in primary neuronal cultures from the hippocampus, cerebral cortex and cerebellum. The distribution of viral-specific antigen in the neurons varied: TBEV infection induced accumulation of viral antigen in the neuronal dendrites to a greater extent than infection with other viruses. Viral structural proteins, non-structural proteins and dsRNA were detected in regions in which viral antigens accumulated in dendrites after TBEV replication. Replication of a TBEV replicon after infection with virus-like particles of TBEV also induced antigen accumulation, indicating that accumulated viral antigen was the result of viral RNA replication. Furthermore, electron microscopy confirmed that TBEV replication induced characteristic ultrastructural membrane alterations in the neurites: newly formed laminal membrane structures containing virion-like structures. This is the first report describing viral replication in and ultrastructural alterations of neuronal dendrites, which may cause neuronal dysfunction. These findings encourage further work aimed at understanding the molecular mechanisms of viral replication in the brain and the pathogenicity of neurotropic flaviviruses.
The neuroinflammatory response to West Nile virus (WNV) infection can be either protective or pathological depending on the context. Although several studies have examined chemokine profiles within brains of WNV-infected mice, little is known about how various cell types within the central nervous system (CNS) contribute to chemokine expression. Here, we assessed chemokine expression in brain microvascular endothelial cells and astrocytes, which comprise the major components of the bloodnndash;brain barrier (BBB), in response to a non-pathogenic (WNV-MAD78) and a highly pathogenic (WNV-NY) strain of WNV. Higher levels of the chemokine CCL5 were detected in WNV-MAD78-infected brain endothelial monolayers compared with WNV-NY-infected cells. However, the opposite profile was observed in WNV-infected astrocytes, indicating that pathogenic and non-pathogenic strains of WNV provoke different CCL5 profiles at the BBB. Thus, cells comprising the BBB may contribute to a dynamic pro-inflammatory response within the CNS that evolves as WNV infection progresses.
We analysed natural recombination in 79 Human enterovirus A strains representing 13 serotypes by sequencing of VP1, 2C and 3D genome regions. The half-life of a non-recombinant tree node in coxsackieviruses 2, 4 and 10 was only 3.5 years, and never more than 9 years. All coxsackieviruses that differed by more than 7 % of the nucleotide sequence in any genome region were recombinants relative to each other. Enterovirus 71 (EV71), on the contrary, displayed remarkable genetic stability. Three major EV71 clades were stable for 19nndash;29 years, with a half-life of non-recombinant viruses between 13 and 18.5 years in different clades. Only five EV71 strains out of over 150 recently acquired non-structural genome regions from coxsackieviruses, while none of 80 contemporary coxsackieviruses had non-structural genes transferred from the three EV71 clades. In contrast to earlier observations, recombination between VP1 and 2C genome regions was not more frequent than between 2C and 3D regions.
The recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV), a betacoronavirus, is associated with severe pneumonia and renal failure. The environmental origin of MERS-CoV is as yet unknown; however, its genome sequence is closely related to those of two bat coronaviruses, named BtCoV-HKU4 and BtCoV-HKU5, which were derived from Chinese bat samples. A hallmark of highly pathogenic respiratory viruses is their ability to evade the innate immune response of the host. CoV accessory proteins, for example those from severe acute respiratory syndrome CoV (SARS-CoV), have been shown to block innate antiviral signalling pathways. MERS-CoV, similar to SARS-CoV, has been shown to inhibit type I IFN induction in a variety of cell types in vitro. We therefore hypothesized that MERS-CoV and the phylogenetically related BtCoV-HKU4 and BtCoV-HKU5 may encode proteins with similar capabilities. In this study, we have demonstrated that the ORF4b-encoded accessory protein (p4b) of MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 may indeed facilitate innate immune evasion by inhibiting the type I IFN and NF-B signalling pathways. We also analysed the subcellular localization of p4b from MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 and demonstrated that all are localized to the nucleus.
Bagaza virus (BAGV) and Israel turkey meningoencephalomyelitis virus (ITV) are classified in the genus Flavivirus of the family Flaviviridae. Serologically, they are closely related, belonging to the Ntaya serocomplex. Nucleotide sequences available to date consist of several complete sequences of BAGV isolates, but only partial sequences of ITV isolates. Sequence comparisons of partial envelope (E) and NS5 regions reveal a close genetic relationship between these viruses. Despite this, BAGV and ITV are considered as separate virus species in the database of the International Committee on Taxonomy of Viruses. In this work, complete nucleotide sequences for five ITV isolates are provided, thereby permitting a phylogenetic comparison with other complete sequences of flaviviruses in the Ntaya serogroup. We conclude that BAGV and ITV are the same virus species and propose that both viruses be designated by a new unified name: Avian meningoencephalomyelitis virus.
Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. The emergence of very virulent IBDV (vvIBDV) has brought more challenges for effective prevention of this disease. The molecular basis for the virulence of vvIBDV is not fully understood. In this study, 20 IBDV strains were analysed phylogenically and clustered in three branches based on their full-length B segments. The amino acid triplet located at positions 145/146/147 of VP1 was found highly conserved in branch I non-vvIBDVs as asparagine/glutamic acid/glycine (NEG), in branch II vvIBDVs as threonine/glutamic acid/glycine (TEG) and in branch III vvIBDVs as threonine/aspartic acid/asparagine (TDN). Further studies showed that the three amino acids play a critical role in the replication and pathogenicity of vvIBDV. Substitution of the TDN triplet with TEG or NEG reduced viral replication and pathogenicity of the vvIBDV HuB-1 strain in chickens. However, the replication of the attenuated IBDV Gt strain was reduced in chicken embryo fibroblast cells, whilst it was enhanced in the bursa by substituting NEG with TEG or TDN. The exchange of the three amino acids was also found to be capable of affecting the polymerase activity of VP1. The important role of segment B in the pathogenicity of IBDV was confirmed in this study. These results also provided new insights into the mechanism of the virulence of vvIBDVs and may offer new targets for their attenuation to develop potential vaccines using reverse genetics.
Orthoreoviruses have been associated with a variety of diseases in domesticated poultry and wild-living birds. In 2002, a reovirus strain named Tvaauml;rminne avian virus (TVAV), was identified in Finland in a crow showing neurological disorders. The objective of this study was the molecular characterization of this novel reovirus strain. Genome sequencing was performed by combining semiconductor sequencing and traditional capillary sequencing. Sequence and phylogenetic analyses showed that TVAV shares low nucleotide sequence identity with other reoviruses (range for each gene, 31nndash;72 %) including strains belonging to the species Avian orthoreovirus. The most closely related reovirus strain was an isolate identified in Steller sea lion. Our data indicate that TVAV is a divergent reovirus of avian origin that may be the first representative of a distinct virus species within the genus Orthoreovirus.
A novel reovirus, designated Cimodo virus (CMDV), was isolated from mosquitoes collected in a rainforest region in Coocirc;te drrsquo;Ivoire. The entire genome comprised 24 835 bp divided into 12 segments ranging from 585 to 4080 bp. The icosahedral non-enveloped virions were 80 nm in diameter. Eight major viral proteins of about 150, 135, 120, 80, 66, 59, 42 and 30 kDa were identified and seven proteins were mapped to the corresponding genome segments by liquid chromatography mass spectrometry. Predicted protein genes diverged by ggt;77 % encoded amino acids from their closest reovirus relatives. The deep phylogenetic branching suggests that CMDV defines an as-yet-unidentified genus within the subfamily Spinareovirinae.
Porcine parvovirus (PPV) is a small DNA virus with restricted coding capacity. The 5 kb genome expresses three major non-structural proteins (NS1, NS2 and SAT), and two structural proteins (VP1 and VP2). These few viral proteins are pleiotropic and interact with cellular components throughout viral replication. In this regard, very few cell lines have been shown to replicate the virus efficiently. Cell lines were established from a primary culture of bovine cells that allowed allotropic variants of PPV to be distinguished. Three cell lines were differentially sensitive to infection by two prototype PPV strains, NADL-2 and Kresse. In the first cell line (D10), infection was restricted early in the infectious cycle and was not productive. Infection of the second cell line (G11) was 1000 times less efficient with the NADL-2 strain compared with porcine cells, while production of infectious virus of the Kresse strain was barely detectable. Restriction points in these cells were the initial generation of DNA replication intermediates and NS1 production. Infection with chimeras between NADL-2 and Kresse showed that residues outside the previously described allotropic determinant were also partially responsible for the restriction to Kresse replication in G11 cells. F4 cells were permissive to both strains, although genome replication and infectious virus production were lower than in the porcine cells used for comparison. These results highlight the dependent nature of parvovirus tropism on host factors and suggest that cells from a non-host origin can fully support a productive infection by both strains.
Some respiratory tract infections remain unexplained despite extensive testing for common pathogens. Nasopharyngeal aspirates (NPAs) from 120 Chilean infants from Santiago with acute lower respiratory tract infections were analysed by viral metagenomics, revealing the presence of nucleic acids from anelloviruses, adenovirus-associated virus and 12 known respiratory viral pathogens. A single sequence read showed translated protein similarity to cycloviruses. We used inverse PCR to amplify the complete circular ssDNA genome of a novel cyclovirus we named CyCV-ChileNPA1. Closely related variants were detected using PCR in the NPAs of three other affected children that also contained anelloviruses. This report increases the current knowledge of the genetic diversity of cycloviruses whose detection in multiple NPAs may reflect a tropism for human respiratory tissues.
Recently, several novel human polyomaviruses (HPyVs) have been detected. HPyV6, 7, 9 and 10 are not associated with any disease so far. Trichodysplasia spinulosa (TS)-associated polyomavirus (TSPyV) can cause the rare skin disease TS. We have evaluated cutaneous DNA prevalence and viral loads of five HPyVs in HIV-infected men compared to healthy male controls. 449 forehead swabs were analysed by HPyV-specific real-time PCR. HPyV6, HPyV7, TSPyV and HPyV10 were found significantly more frequently on the skin of 210 HIV-infected compared to 239 HIV-negative men (HPyV6, 39.0 vs 27.6 %; HPyV7, 21.0 vs 13.4 %; TSPyV, 3.8 vs 0.8 %; HPyV10, 9.3 vs 3.4 %; Pllt;0.05, respectively). HPyV9 was not detected. Multiple infections were more frequent in HIV-positive men, but HPyV-DNA loads did not differ significantly in both groups. In contrast to HPyV6, 7 and 10, TSPyV and HPyV9 do not seem to be a regular part of the human skin microbiome.
Human cytomegalovirus (HCMV) is known to evade extrinsic pro-apoptotic pathways not only by downregulating cell surface expression of the death receptors TNFR1, TRAIL receptor 1 (TNFRSF10A) and TRAIL receptor 2 (TNFRSF10B), but also by impeding downstream signalling events. Fas (CD95/APO-1/TNFRSF6) also plays a prominent role in apoptotic clearance of virus-infected cells, so its fate in HCMV-infected cells needs to be addressed. Here, we show that cell surface expression of Fas was suppressed in HCMV-infected fibroblasts from 24 h onwards through the late phase of productive infection, and was dependent on de novo virus-encoded gene expression but not virus DNA replication. Significant levels of the fully glycosylated (endoglycosidase-H-resistant) Fas were retained within HCMV-infected cells throughout the infection within intracellular membranous structures. HCMV infection provided cells with a high level of protection against Fas-mediated apoptosis. Downregulation of Fas was observed with HCMV strains AD169, FIX, Merlin and TB40.
The catalytic subunit of the herpes simplex virus 1 DNA polymerase (HSV-1 Pol) is essential for viral DNA synthesis and production of infectious virus in cell culture. While mutations that affect 5'nndash;3' polymerase activity have been evaluated in animal models of HSV-1 infection, mutations that affect other functions of HSV-1 Pol have not. In a previous report, we utilized bacterial artificial chromosome technology to generate defined HSV-1 pol mutants with lesions in the previously uncharacterized pre-NH2-terminal domain. We found that the extreme N-terminal 42 residues (deletion mutant polN43) were dispensable for replication in cell culture, while residues 44nndash;49 (alanine-substitution mutant polA6) were required for efficient viral DNA synthesis and production of infectious virus. In this study, we sought to address the importance of these conserved elements in viral replication in a mouse corneal infection model. Mutant virus polN43 exhibited no meaningful defect in acute or latent infection despite strong conservation of residues 1nndash;42 with HSV-2 Pol. The polA6 mutation caused a modest defect in replication at the site of inoculation, and was severely impaired for ganglionic replication, even at high inocula that permitted efficient corneal replication. Additionally, the polA6 mutation resulted in reduced latency establishment and subsequent reactivation. Moreover, we found that the polA6 replication defect in cultured cells was exacerbated in resting cells as compared to dividing cells. These results reveal an important role for the conserved motif at residues 44nndash;49 of HSV-1 Pol for ganglionic viral replication.
Pigs can be severely harmed by influenza, and represent important reservoir hosts, in which new human pathogens such as the recent pandemic swine-origin H1N1 influenza A virus can arise by mutation and reassortment of genome segments. To obtain novel, safe influenza vaccines for pigs, and to investigate the antigen-specific immune response, we modified an established live-virus vaccine against Aujeszkyrrsquo;s disease of swine, pseudorabies virus (PrV) strain Bartha (PrV-Ba), to serve as vector for the expression of haemagglutinin (HA) of swine-origin H1N1 virus. To facilitate transgene insertion, the genome of PrV-Ba was cloned as a bacterial artificial chromosome. HA expression occurred under control of the human or murine cytomegalovirus immediate early promoters (P-HCMV, P-MCMV), but could be substantially enhanced by synthetic introns and adaptation of the codon usage to that of PrV. However, despite abundant expression, the heterologous glycoprotein was not detectably incorporated into mature PrV particles. Replication of HA-expressing PrV in cell culture was only slightly affected compared to that of the parental virus strain. A single immunization of pigs with the PrV vector expressing the codon-optimized HA gene under control of P-MCMV induced high levels of HA-specific antibodies. The vaccinated animals were protected from clinical signs after challenge with a related swine-origin H1N1 influenza A virus, and challenge virus shedding was significantly reduced.
Trim 5aalpha; was the first member of the tripartite motif (TRIM) family of proteins that was identified to potently restrict human immunodeficiency virus type 1 (HIV-1) replication. The breadth of antiretroviral activity of TRIM family members is an active area of investigation. In this study, we demonstrate that human Trim 37 possesses anti-HIV-1 activity. This antiretroviral activity and the manner in which it was displayed were implicated by (1) decreased viral replication upon Trim 37 transient overexpression in virus-producing cells, (2) correlation of the reduction of viral infectivity with Trim 37 virion incorporation, (3) increased HIV-1 replication during siRNA depletion of Trim 37 expression, and (4) reduction in viral DNA synthesis upon Trim 37 transient overexpression. Our findings provide the first demonstration, to our knowledge, of the potent antiviral activity of human Trim 37, and implicate an antiviral mechanism whereby Trim 37 interferes with viral DNA synthesis.
HIV-1 transcription depends on cellular transcription factors that bind to sequences in the long-terminal repeat (LTR) promoter. Each HIV-1 subtype has a specific LTR promoter configuration, and minor sequence changes in transcription factor binding sites (TFBSs) or their arrangement can influence transcriptional activity, virus replication and latency properties. Previously, we investigated the proviral latency properties of different HIV-1 subtypes in the SupT1 T cell line. Here, subtype-specific latency and replication properties were studied in primary PHA-activated T lymphocytes. No major differences in latency and replication capacity were measured among the HIV-1 subtypes. Subtype B and AE LTRs were studied in more detail with regard to a putative AP-1 binding site using luciferase reporter constructs. c-Jun, a member of the AP-1 transcription factor family, can activate both subtype B and AE LTRs, but the latter showed a stronger response, reflecting a closer match with the consensus AP-1 binding site. c-Jun overexpression enhanced Tat-mediated transcription of the viral LTR, but in the absence of Tat inhibited basal promoter activity. Thus, c-Jun can exert a positive or negative effect via the AP-1 binding site in the HIV-1 LTR promoter, depending on the presence or absence of Tat.
Baculoviruses infect insects, producing two distinct phenotypes during the viral life cycle: the budded virus (BV) and the occlusion-derived virus (ODV) for intra- and inter-host spread, respectively. Since the 1980s, several countries have been using Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) as a biological control agent against the velvet bean caterpillar, A. gemmatalis. The genome of AgMNPV isolate 2D (AgMNPV-2D) carries at least 152 potential genes, with 24 that possibly code for structural proteins. Proteomic studies have been carried out on a few baculoviruses, with six ODV and two BV proteomes completed so far. Moreover, there are limited data on virion proteins carried by AgMNPV-2D. Therefore, structural proteins of AgMNPV-2D were analysed by MALDI- quadrupole-TOF and liquid chromatography MS/MS. A total of 44 proteins were associated with the ODV and 33 with the BV of AgMNPV-2D. Although 38 structural proteins were already known, we found six new proteins in the ODV and seven new proteins carried by the AgMNPV-2D BV. Eleven cellular proteins that were found on several other enveloped viruses were also identified, which are possibly carried with the virion. These findings may provide novel insights into baculovirus biology and their host interaction. Moreover, our data may be helpful in subsequent applied studies aiming to improve AgMNPV use as a biopesticide and a biotechnology tool for gene expression or delivery.
|Journal of General Virology Publish Ahead of Print|
Anelloviruses are a family of small single stranded circular DNA viruses with a vast genetic diversity. Human infections with the prototype anellovirus, torque teno virus (TTV), are ubiquitous and related viruses have been described in a number of other mammalian hosts. Despite over 15 years of investigation, however, there is still little known about the pathogenesis and possible disease associations of anellovirus infections, arising in part due to the lack of a robust cell culture system for viral replication or tractable small animal model. We report the identification of diverse anelloviruses in several species of wild rodents. The viruses are highly prevalent in wood mice (Apodemus sylvaticus) and field voles (Microtus agrestis), detectable at a low frequency in bank voles (Myodes glareolus) but absent from house mice (Mus musculus). The viruses identified have a genomic organisation consistent with other anelloviruses but form two clear phylogenetic groups that are as distinct from each other as from defined genera.
Feline morbillivirus (FmoPV) is an emerging virus in domestic cats and considered to be associated with tubulointerstitial nephritis. Although FmoPV was first described in China in 2012, there has been no report of the isolation of this virus in other countries. In this study, we describe the isolation and characterization of FmoPV from domestic cats in Japan. By using RT-PCR, we found that 3 of 13 urine samples from cats brought to veterinary hospitals were positive for FmoPV. FmoPV strains SS1 to SS3 were isolated from the RT-PCR-positive urine samples. CRFK cells exposed to FmoPV showed cytopathic effects with syncytia formation, and FmoPV N protein was detected by indirect immunofluorescence assays. In addition, pleomorphic virus particles with apparent glycoprotein envelope spikes were observed by electron microscopy. By sequence analysis of FmoPV H and L genes, we found that FmoPVs showed genetic diversity, however signatures of positive selection were not identified.
The orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) is a transcription factor stimulated by many factors and plays pivotal roles in metabolism, proliferation, and apoptosis. The expression of NR4A1 in Huh7.5.1 cells was significantly upregulated by HCV infection. The silencing of NR4A1 inhibited the entry of HCV and reduced the specific infectivity of secreted HCV particles, but had only minor or no effect on the genome replication and translation, virion assembly, and viral release steps of the virus life cycle. Further experiments demonstrated that the silencing of NR4A1 affected virus entry through pan-downregulation of the expression of HCV receptors SR-BI, Occludin, Claudin-1, and EGFR, but not CD81. The reduced specific infectivity of HCV in the knockdown cells was due to the decreased apolipoprotein E (ApoE) expression. These results explained the delayed spread of HCV in NR4A1 knockdown Huh7.5.1 cells. Thus, NR4A1 plays a role in HCV replication through regulating the expression of HCV receptors and ApoE and facilitates HCV entry and spread.
Although many cells undergo transformation, few of them actually develop into tumors, due to successful mechanisms of immunosurveillance. To investigate whether an infectious agent may play a role in this process, the growth of a plasmacytoma was followed in mice infected by lactate dehydrogenase-elevating virus. Acutely infected animals were significantly protected against tumor growth. The mechanisms responsible for this protection were analyzed in mice deficient for relevant immune cells or molecules, and after in vivo cell depletion. This effect of viral infection correlated with natural killer cell activation and with interferon- production. It was also related to activation of NK/T cells. Therefore, our results indicate that infections with benign microorganisms may protect the host against cancer development, through non-specific stimulation of its innate immune system and especially of natural killer cells.
Replication initiator protein (Rep) is indispensible for rolling-circle replication of geminiviruses, a group of plant infecting circular single stranded DNA (ssDNA) viruses. However, the mechanism of DNA unwinding by circular ssDNA virus encoded helicases is unknown. To understand geminivirus Rep function, we compared the sequence and secondary structure of Rep with those of bovine papillomavirus-E1 and employed charged residue-to-alanine scanning mutagenesis to generate a set of single-substitution mutants in Walker A (K227), in Walker B (D261, 262), and within or adjacent to the B' motif (K272, K286 and K289). All mutants were asymptomatic and viral accumulation could not be detected by Southern blot from both tomato and N. benthamiana plants. Furthermore, the K272 and K289 mutants were deficient in DNA binding and unwinding. Biochemical studies and modelling data based on comparison with the known structures of SF3 helicases suggest that the conserved lysine (K289) located in a predicted bbeta;-hairpin loop may interact with ssDNA, while lysine 272 in the B' motif (K272) located on the outer surface of the protein is presumably involved in coupling ATP-induced conformational changes to DNA binding. To the best of our knowledge, this is the first time that the roles of the B' motif and the adjacent bbeta;-hairpin loop in geminivirus replication have been elucidated.
Despite tremendous efforts to improve the methodology for constructing flavivirus infectious cDNAs, the manipulation of flavivirus cDNAs remains a difficult task in bacteria. Here, we successfully propagated DNA-launched type 2 dengue virus (DENV2) and Japanese encephalitis virus (JEV) infectious cDNAs by introducing seven repeats of the tetracycline-response element (7XTRE) and a minimal cytomegalovirus (CMVmin) promoter upstream of the viral genome. Insertion of the 7XTRE-CMVmin sequence upstream of the DENV2 or JEV genome decreased the cryptic E. coli promoter (ECP) activity of the viral genome in bacteria, as measured using fusion constructs containing DENV2 or JEV segments and the reporter gene Renilla luciferase in an empty vector. The growth kinetics of recombinant viruses derived from DNA-launched DENV2 and JEV infectious cDNAs were similar to those of parental viruses. Similarly, RNA-launched DENV2 infectious cDNAs were generated by inserting 7XTRE-CMVmin, five repeats of the GAL4 upstream activating sequence (5XGAL4), or five repeats of BamHI linkers (5XBamHI) upstream of the DENV2 genome. All three tandem repeat sequences decreased the ECP activity of the DENV2 genome in bacteria. Notably 7XTRE-CMVmin stabilized RNA-launched JEV infectious cDNAs and reduced the ECP activity of the JEV genome in bacteria. The growth kinetics of recombinant viruses derived from RNA-launched DENV2 and JEV infectious cDNAs displayed patterns similar to those of the parental viruses. These results support a novel methodology for constructing flavivirus infectious cDNAs, which will facilitate research in virology, viral pathogenesis, and vaccine development of flaviviruses and other RNA viruses.
Osteosarcoma cells U2OS are partially susceptible to adeno-associated virus 2 infection, allowing efficient synthesis of Rep proteins and, in a low percentage of cells, capsid production. It is not clear if this partial susceptibility to infection is due to the bone cell-like nature of these cells, or is rather a result of their transformed properties. Here we grew osteosarcoma cells in a biomimetic three-dimensional bone-like matrix composed of calcium phosphate and chitosan and tested whether this would increase or reduce their permissiveness to virus. The osteosarcoma cells grew in the matrix and began to express the alkaline phosphatase bone cell differentiation marker. This was accompanied by a block to their infection by adeno-associated virus, as indicated by Rep and capsid production. Infection of cells growing in three-dimensional tissue-like matrices could be, in a wider context, a practical way to mimic in vivo conditions.
Equine herpesvirus type 1 (EHV-1) was shown to use an unusual receptor for cellular entry, major histocompatibility complex class I (MHC-I) molecules. Here we demonstrate that the closely related equine herpesvirus EHV-4 also uses this strategy for cellular invasion, both in equine cells in culture and in the heterologous, non-permissive murine mastocytoma cell line (P815) after stable transfection with horse MHC-I genes. Using a panel of P815 cell lines transfected with individual horse MHC-I genes, we provide support for the hypothesis that EHV-1 and EHV-4 target classical polymorphic MHC-I molecules as viral entry receptors. All known equine MHC-I molecules from the two principal classical polymorphic loci specify alanine at position 173 (A173), while other MHC-I loci encode different amino acids at this position and did not permit viral entry. Site-directed mutagenesis of position 173 diminished or enhanced viral entry, depending upon the initial amino acid. However, there are other, as yet undefined, constraints to this process: MHC-I genes from two non-classical loci carry A173 but did not enable viral entry in P815 transfectants. Our study suggests that the capacity to bind MHC-I molecules arose in the common ancestor of EHV-1 and EHV-4. The widespread occurrence of A173 in classical polymorphic horse MHC-I molecules indicates that horses of most MHC haplotypes should be susceptible to infection via this entry portal.
Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. The invertebrate iridovirus 31 (IIV31) was originally isolated from pill bug adults of Armadillidium vulgare (class Crustacea, order Isopoda, sub-order Oniscidea) found in Southern California on the campus of the University of California, Riverside. IIV31 virions are icosahedral, have a diameter of about 135 nm, and contain a dsDNA genome of 220.22 kbp in length, 35.09 % GC content and 203 open reading frames. Here, we describe the complete genome sequence of this virus and its annotation. This is the eighth genome sequence of an invertebrate iridovirus reported.
During infection by picornaviruses, the cellular environment is modified to favor viral replication. This includes the modification of specific host proteins, including the recently discovered viral proteinase cleavage of mRNA decay factor AUF1. This cellular RNA binding protein was previously shown to act as a restriction factor during poliovirus, rhinovirus, or coxsackievirus infection. During infection by these viruses, AUF1 re-localizes to the cytoplasm and is cleaved by the viral 3C/3CD proteinase. In this study, we demonstrate that replication of encephalomyocarditis virus (EMCV), a picornavirus belonging to the cardiovirus genus, is AUF1-independent. During EMCV infection, AUF1 re-localizes to the cytoplasm; however, unlike what is seen during enterovirus infections, AUF1 is not cleaved to detectable levels even at late times after infection. This suggests that AUF1 does not act broadly as an inhibitor of picornavirus infections but may instead act as a selective restriction factor targeting members of the enterovirus genus.
Potyviruses represent one of the most economically important and widely distributed groups of plant viruses. Despite considerable progress towards understanding the cellular and molecular basis of their pathogenicity, many questions remain about the mechanisms by which potyviruses suppress host defences and create an optimal intracellular environment for viral translation, replication, assembly and spread. This review focuses on the multifunctional roles of potyviral proteins and their interplay with various host factors in different compartments of the infected cell. We place special emphasis on the recently discovered and currently putative mechanisms by which potyviruses subvert the normal functions of different cellular organelles in order to establish an efficient and productive infection.
CClade 2.2 highly pathogenic H5N1 viruses have been in continuous circulation in Egyptian poultry since 2006. Their persistence caused significant genetic drift that led to the re-classification of these viruses into sub-clades 2.2.1 and 220.127.116.11. Here, we conducted full genome sequence and phylogenetic analyses of 45 H5N1 isolated during 2006nndash;2013 through systematic surveillance in Egypt, and 53 viruses that were previously sequenced and available in the public domain. Results indicate that H5N1 viruses in Egypt continue to evolve and a new distinct cluster has emerged. Mutations affecting viral virulence, pathogenicity, transmission, receptor-binding preference, and drug resistance were studied. In light of our findings that H5N1 in Egypt continues to evolve, surveillance and molecular studies need to be sustained.
We characterized the full-length genomes of 16 distinct HCV-1 isolates. Among them, four represent the first full-length genomes for subtypes 1d (QC103), 1i (QC181), 1j (QC329) and 1k (QC82), and another four correspond to subtypes 1c (QC165), 1g (QC78), 1h (QC156) and 1e (QC172). Both QC196 and QC87 are assigned into a new subtype 1m, and QC113 and QC74 into another new subtype 1n. The remaining four (QC60, QC316, QC152, QC180) did not classify among the established subtypes and correspond to four new lineages. Subtypes 1j, 1k, 1m, 1n, and the unclassified isolate QC60 were identified in Haitian immigrants. In the updated HCV nomenclature of 2005, a total of 12 subtypes of HCV-1 were designated. Including the data from the present study, all but subtypes 1f now have their full-length genomes defined. Further analysis of partial NS5B sequences available in GenBank denoted a total of 21 unclassified lineages indicating the taxonomic complexity of HCV-1. Among them, six have had their full-length genomes characterized. Based on the available full-length genome sequences, a timescale phylogenetic tree was reconstructed which estimated important time points in the evolution of HCV genotype 1. It revealed that subtype 1a diverged from its nearest relatives 135 years ago and subtype 1b 112 years ago. When subtypes 1a, 1j, 1k, 1m, 1n, and six close relatives (all but one from Haitian immigrants) were considered as a whole, the divergence time was 176 years ago. This diversification was concurrent in time during which the transatlantic slave trade was actively taking place. When taking all the HCV-1 isolates as a single lineage the divergence time was 326 years ago. This analysis suggests the existence of a recent common ancestor for subtype 1a and the Haitian variants; a co-origin for subtypes 1b, 1i and 1d is also implied.
To investigate the diversity of viral flora, we used metagenomics to study the viral communities in a pooled fecal sample of 27 diarrheic piglets from intensive commercial farms in China. The 15 distinct mammalian viruses identified in the pooled diarrheic sample were, in order of abundance of nucleic acids sequence, porcine epidemic diarrhea virus (PEDV), sapovirus, porcine bocavirus-4 (PBoV-4), sapelovirus, torovirus, coronavirus, PBoV-2, stool-associated single-stranded DNA virus (poSCV), astrovirus (AstV), kobuvirus, posavirus-1, porcine enterovirus-9 (PEV-9), porcine circovirus-like (po-circo-like) virus, picobirnavirus (PBV) and torque teno sus virus 2 (TTSuV-2). The every virus prevalence rate was verified from diarrheic and healthy piglets by PCR assay. An average of 5.5 different viruses was shed in diarrheic piglet, and one piglet was in fact co-infected with 11 different viruses. In contrary, healthy piglets shed an average of 3.2 different viruses. Compared to healthy piglets, the co-infection of PEDV and PBoV had a high prevalence rate in diarrhea samples, suggesting a correlation to the appearance of diarrhea in piglets. Furthermore, several recently described viruses were here reported for the first time in China, and novel genotypes were identified. Therefore, our investigation results provided an unbiased survey of viral communities and prevalence in fecal sample of piglets.
Sunguru virus (SUNV), a novel virus belonging to the highly diverse Rhabdoviridae family, was isolated from a domestic chicken in the district of Arua, Uganda in 2011. This is the first documented isolation of a rhabdovirus from a chicken. SUNV is related to, but distinct from, Boteke virus and other members of the unclassified Sandjimba group. The genome is 11,056 kb in length and contains the five core rhabdovirus genes plus an additional C gene (within the open reading frame of the phosphoprotein gene) and a small hydrophobic protein (between the matrix and glycoprotein genes). Inoculation of vertebrate cells resulted in significant growth, with a peak titer of 7.8 log10 PFU/mL observed in baby hamster kidney cells. Little to no growth was observed in invertebrate cells and in live mosquitoes, with Anopheles gambiae mosquitoes demonstrating a 47.4% infection rate in the body but no dissemination to the salivary glands; this suggests that this novel virus is not arthropod-borne like some other members of the family Rhabdoviridae.
Bovine respiratory syncytial virus (BRSV) causes inflammation and obstruction of the small airways, leading to severe respiratory disease in young calves. The virus is closely related to human (H)RSV, a major cause of bronchiolitis and pneumonia in young children. The ability to manipulate the genome of RSV has provided opportunities for the development of stable, live attenuated RSV vaccines. The role of the SH protein in the pathogenesis of BRSV was evaluated in vitro and in vivo using a recombinant (r)BRSV in which the SH gene had been deleted. Infection of bovine epithelial cells and monocytes with rBRSVSH, in vitro, resulted in an increase in apoptosis, and higher levels of tumor necrosis factor-aalpha; and interleukin-1bbeta; compared with cells infected with parental, wild-type (WT) rBRSV. Although replication of rBRSVSH and WT rBRSV, in vitro, were similar, the replication of rBRSVSH was moderately reduced in the lower, but not the upper, respiratory tract of experimentally infected calves. Despite the greater ability of rBRSVSH to induce pro-inflammatory cytokines, in vitro, the pulmonary inflammatory response in rBRSVSH-infected calves was significantly reduced compared to that in calves inoculated with WT rBRSV, 6 days previously. Virus lacking SH appeared to be as immunogenic and effective in inducing resistance to virulent virus challenge, 6 months later, as the parental rBRSV. These findings suggest that rBRSVSH may be an ideal live attenuated virus vaccine candidate, combining safety with a high level of immunogenicity.
Human norovirus (NoV) research greatly relies on cell culture propagable surrogate caliciviruses, including the murine NoVs and the prototype "Recovirus" (ReCV), the Tulane virus. However, the extreme biological diversity of human NoVs cannot be modeled by uniform group of viruses or a single isolate. Based on a diverse group of recently described ReCVs a more advanced model, capable to reflect human NoV biological diversity, is currently being developed. Here we report the genotypic and serotypic relationships among 10 G1 ReCV isolates, including the Tulane virus and nine other, recently cell culture adapted strains. Based on VP1 amino acid sequences and classification constraints established for NoVs, the G1 ReCVs separated into 3 genotypes, with variable organization of the three open reading frames. Interestingly, cross-neutralization plaque assays revealed the existence of 4 distinct serotypes, with two serotypes detected among the G1.2 strains. The amino acid difference between the two G1.2 ReCV serotypes was 12%. This is less than the minimum 13% difference established between NoV genotypes. Interestingly, one of the G1.3 ReCVs was equally neutralized by antisera raised against the G1.3 (6% aa difference) and the G1.1 (25% aa difference) representative strains. These results imply the existence of a large number of human NoV serotypes, but also shared cross-neutralization epitopes between some strains of different genotypes. In conclusion, the ReCV surrogate model is able to address biologically relevant questions about enteric CV diversity.
Revealing the interactions among cypovirus (CPV) proteins would facilitate understanding of viral replication and assembly. In the present study, interactions among proteins encoded by the 10 segments of Dendrolimus punctatus cypovirus (DpCPV) were identified using yeast two-hybrid (Y2H) and far-western blotting assays. In total, 24 pairs of interactions were detected. Twelve pairs of one-direction interactions, four pairs of binary interactions, and four pairs of self-associations were identified in the Y2H assays. Another four pairs of interactions were identified by far-western blotting. The interactions between the methyltransferase domain of turret protein (VP3) and VP4 as well as between polyhedrin and VP4 were further confirmed by far-western blotting and pull-down assays, respectively. In addition, the immuno-gold labeling showed that the A-spike of DpCPV was formed by VP4. In conclusion, we obtained the protein-protein interaction network of DpCPV and identified that its A-spike was formed by VP4 encoded by genomic segment 6.
Currently circulating H5N1 influenza viruses have undergone a complex evolution since the appearance of their progenitor A/Goose/Guangdong/1/96 in 1996. After the eradication of the H5N1 viruses that emerged in Hong Kong in 1997 (HK/97 viruses), new genotypes of H5N1 viruses emerged in the same region in year 2000, that were more pathogenic for both chickens and mice than HK/97 viruses. These, as well as virtually all highly pathogenic H5N1 viruses since 2000, harbor a deletion of amino-acids 80-84 in the unstructured region of NS1 linking its RNA-binding domain to its effector domain. Segments NS harboring this mutation have since been found in non-H5N1 viruses, and we asked whether this 5-aa deletion could have a general effect not limited to the NS1 of H5N1 viruses. We genetically engineered this deletion in the NS segment of a duck-origin avian H1N1 virus, and compared the in vivo and in vitro properties of the wild-type and NSdel8084 viruses. In experimentally infected chickens, the NSdel8084 virus showed both an increased replication potential and an increased pathogenicity. This in vivo phenotype was correlated with a higher replicative efficiency in vitro, both in embryonated eggs and in a chicken lung epithelial cell line. Our data demonstrate that the increased replicative potential conferred by this small deletion is a general feature not restricted to NS1 from H5N1 viruses, and suggest that viruses acquiring this mutation may be positively selected in the future.
The West Nile flaviviruses are an important cause of viral meningoencephalitis in some areas of the world. They exhibit marked variation in pathogenicity, with some viral lineages (such as those from North America) causing high prevalence of severe neurological disease, while others (such as Australian Kunjin virus) rarely causing disease. The aim of this study was to characterise West Nile virus disease in a mouse model and to elucidate the pathogenetic features that distinguish disease variation. Ten-fold dilutions of five WNV strains (New York 1999, MRM16 and three horse isolates of WNV-Kunjin: Boort and two isolates from the 2011 Australian outbreak) were inoculated into mice by the intraperitoneal route. All isolates induced meningoencephalitis in different proportions of infected mice. WNVNY99 was the most pathogenic, the three horse isolates were of intermediate pathogenicity and WNVKUNV-MRM16 was the least, causing mostly asymptomatic disease with seroconversion. Infectivity, but not pathogenicity, was related to challenge dose. By cluster analysis of the recorded clinical signs, histopathological lesions and antigen distribution scores, the cases could be classified into groups corresponding to disease severity. Metrics that were important in determining pathotype included neurological signs (paralysis and seizures), meningoencephalitis, brain antigen scores and replication in extra-neural tissues. Whereas all mice infected with WNVNY99 had extra-neural antigen, those infected with the WNV-Kunjin viruses only occasionally had antigen outside the nervous system. We conclude that the mouse model could be a useful tool for the assessment of pathotype for West Nile viruses.
Papillomaviruses are epitheliotropic viruses that have circular double stranded DNA genomes encapsidated in non-enveloped virions. They have been found to infect a variety of mammals, reptiles and birds but they have so far not been found in amphibians. Using a Next-Gen de novo assembly contig-informed recovery, we cloned and Sanger-sequenced the complete genome of a novel papillomavirus from the faecal matter of Adeeacute;lie penguins (Pygoscelis adeliae) nesting on Ross Island, Antarctica. The genome has all the usual features of a papillomavirus and an E9 open reading frame encoding a protein of unknown function that is found in all the avian papillomaviruses. This novel papillomavirus genome shares ~60% pairwise identity with the genomes of the other three known avian papillomaviruses: Fringilla coelebs papillomavirus 1 (FcPV1), Francolinus leucoscepus papillomavirus 1 (FlPV1) and Psittacus erithacus papillomavirus 1 (PePV1). Both pairwise identity analysis and phylogenetic analysis of the major capsid protein gene clearly indicate that it is a new species (Pygoscelis adeliae papillomavirus 1, PaCV1). No evidence of recombination was detected in the genome of PaCV1 but we did detect a recombinant region (119nts) in the E6 gene of FlPV1 with the recombinant region being derived from ancestral FcPV1-like sequences. Previously only paramyxoviruses, orthomyxoviruses, and avian pox viruses have been genetically identified in penguins, however, the majority of the penguin viral identifications have been based on serology or histology. This is the first report of a papillomavirus associated with a penguin species.
The TATA box binding protein (TBP) plays a key role in initiating eukaryotic transcription and is used by many viruses for viral transcription. We previously reported increased TBP levels during the infection with the baculovirus Autographa californica multicapsid nuclear polyhedrovirus (AcMNPV). The TBP antiserum used in that study, however, cross-reacted with a baculoviral protein. Here, we report that with a TBP-specific antiserum increased amounts of nuclear TBP were detected upon infection of S. frugipeda and TN-368 cells. TBP levels increased until 72 h p.i. while tbp transcripts decreased by 16 h p.i. which suggests a virus-induced influence on the TBP protein levels. To address a potential modification of the TBP degradation pathway during infection, we investigated the possible role of viral ubiquitin. Infection studies with AcMNPV recombinants carrying a mutated viral ubiquitin gene revealed that the TBP increase during infection was not altered. In addition, pulse-chase experiments indicated a high TBP half-life of about 60 h in uninfected cells, suggesting that a virus-induced increase of TBP stability was unlikely. This increase in TBP correlated with a redistribution to nuclear domains resembling sites of viral DNA synthesis. Furthermore, we observed colocalization of TBP with host RNA polymerase II (RNAPII), but only until 8 h p.i. while TBP, but not RNAPII, was present in the enlarged replication domains late during infection. Thus, we suggest that AcMNPV adapted a mechanism to accumulate the higly stable cellular TBP at sites of viral DNA replication and transcription.
Mass bird mortality has been observed in North America after the introduction of West Nile virus (WNV), most notably massive die-offs of American crows (Corvus brachyrhynchos). In contrast, WNV epidemic activity in Europe has been characterized by very low incidences of bird mortality. As the general susceptibility of European corvids to strains of WNV remains in question, European jackdaws (Corvus monedula) were inoculated with WNV strains currently circulating in Greece (Gr-10), Italy (FIN and Ita09) and Hungary (578/10), as well as a North American (NY99) genotype with a demonstrated corvid virulence phenotype. Infection with all strains except WNV-FIN resulted in mortality. Viremia was observed for birds inoculated with all strains and virus was detected in a series of organs upon necropsy. These results suggest that jackdaws could potentially function as a sentinel for following WNV transmission in Europe; however, elicited viremia levels might be too low to allow for efficient transmission of virus to mosquitoes.
All influenza virusesrrsquo; bud and egress from lipid rafts within the apical plasma membrane of infected epithelial cells. As a result all components of progeny virions must be transported to these lipids rafts for assembly and budding. Though the mechanism of transport for other influenza proteins has been elucidated, influenza B glycoprotein NB subcellular localization and transport are not completely understood. To address the aforementioned properties of NB protein, a series of trafficking experiments were conducted. Here we showed that NB co-localized with markers specific for the endoplasmic reticulum and Golgi region. The data from chemical treatment of NB-expressing cells by Brefeldin A, a fungal antibiotic and a known chemical inhibitor of the protein secretory pathway, further confirmed that NB is transported through the ER golgi pathway as it restricted NB localization to the perinuclear region. Using NB deletion mutants, the hydrophobic transmembrane domain was identified as being required for NB transport to the plasma membrane. Furthermore, palmitoylation was also required for transport of NB protein to the plasma membrane. Systematic mutation of NBrrsquo;s cysteines to serines demonstrated that cysteine 49, likely in a palmitoylated form, is also required for transport to the plasma membrane. Surprisingly, further analysis demonstrated that in vitro replication of NBC49S mutant virus was delayed relative to the parental influenza B virus. The results of this study demonstrate NB is the third influenza virus protein to have been shown to be palmitoylated and together these findings may aid in future studies aimed at elucidating NBrrsquo;s function.
Water bodies are important media for the spread and transmission of avian influenza viruses (AIV). The determining factor for an AIV to transmit through diffusion in waters is the term of viability of the virus in water body. To better understand the perpetuation of AIV in nature waters, and thus risks of AIV spread and transmission via it, we systematically studied the inactivation dynamics of two strains (H5N1 and H9N2), at different temperatures in waters of important migratory birds habitats within China: Dongting Lake, Poyang Lake, Qinghai Lake and Hubei segment of the Yangtze River, and we also studied the impact of water-borne microorganisms on perpetuation of AIVs. Our findings indicate that water is very likely an important route for the epidemic spread of the AIVs, especially during the fall and winter seasons. In addition, water-borne microorganisms significantly antagonize the persistence of AIVs.
Human Pegivirus (HPgV; previously called GB virus C/hepatitis G virus) has limited pathogenicity despite causing persistent infection, and is associated with prolonged survival in HIV-infected individuals. Although HPgV RNA is found in and produced by T and B lymphocytes, the primary permissive cell type(s) are unknown. We quantified HPgV RNA in highly purified CD4+ and CD8+ T cells, including naiiuml;ve, central memory, and effector memory populations, and in B cells (CD19+), NK cells (CD56+) cells and monocytes (CD14+) using real time RT-PCR. Single genome sequencing was performed on virus within individual cell types to estimate genetic diversity among cell populations. HPgV RNA was present in CD4+ and CD8+ T lymphocytes (9 of 9 subjects), B lymphocytes (7 of 10), NK cells and monocytes (both 4 of 5). HPgV RNA levels were higher in naiiuml;ve (CD45RA+) CD4+ cells than in central memory and effector memory cells (pllt;0.01). HPgV sequences were highly conserved between patients (0.117 pplusmn; 0.02 substitutions per site; range 0.58nndash;0.14) and within subjects (0.006 pplusmn; 0.003 substitutions per site; range 0.006nndash;0.010). The non-synonymous/synonymous substitution ratio was 0.07 suggesting low selective pressure. CFSE-labeled HPgV RNA-containing particles precipitated by a commercial exosome isolation reagent delivered CSFE to uninfected monocytes, NK cells, T and B lymphocytes, and HPgV RNA was transferred to peripheral blood mononuclear cells with evidence of subsequent viral replication. Thus, HPgV RNA-containing serum particles including microvesicles may contribute to delivery of HPgV to PBMCs in vivo, explaining the apparent broad tropism of this persistent human RNA virus.
The GP129, GP131 and GP133 genes of guinea pig cytomegalovirus (GPCMV) are homologs of human CMV UL128, UL130 and UL131A, respectively, which are essential for infection of endothelial and epithelial cells and for viral transmission to leukocytes. Our previous study demonstrated that a GPCMV strain lacking the 1.6-kb locus that contains the GP129, GP131, and GP133 genes had a growth defect in animals. Here we demonstrated that the wild-type strain but not the 1.6-kb deleted strain formed capsids in macrophages prepared from the peritoneal fluid. To understand the mechanism, we prepared GPCMV strains defective in each of GP129, GP131 and GP133 and found that they were all essential for the infection of peritoneal, splenic and PBMC-derived macrophages/monocytes and expression of immediate-early antigens in the macrophages/monocytes, although they were dispensable for infection of fibroblasts. Monocyte/macrophage-tropism could be one of the important determinants for viral dissemination in vivo.
We identified breast cancer-associated protein (BCA3) as a novel binding partner of Mason-Pfizer monkey virus protease (M-PMV PR). The interaction was confirmed by co-immunoprecipitation and immunocolocalization of M-PMV PR and BCA3. Full-length but not C-terminally truncated BCA3 was incorporated into M-PMV virions. We ruled out the potential role of the G-patch domain, a glycine-rich domain located at the C-terminus of M-PMV PR, in BCA3 interaction and virion incorporation. Expression of BCA3 does not affect M-PMV particle release and proteolytic processing; however, it slightly increases M-PMV infectivity.
A novel virus was detected from diseased ducks and completely determined. The virus was shown to have a picornavirus-like genome layout. Interestingly, the genome contained a total of up to six 2As, including four 2As (2A1 to 2A4) each having an NPGP motif, an AIG1-like 2A5, and a parechovirus-like 2A6. The 5'UTR was predicted to possess a hepacivirus/pestivirus (HP)-like internal ribosome entry site (IRES). However, the subdomain IIIe consisted of a 3-nt stem and five unpaired bases, distinct from those found in all other HP-like IRESs. The virus was most closely related to duck hepatitis A virus (DHAV), with amino acid identities of 37.7%, 39%, and 43.7% in the P1, P2, and P3 regions, respectively. Based on these investigations, together with phylogenetic analyses, the virus could be considered as the founding member of a novel picornavirus genus that we tentatively named llsquo;Aalivirusrrsquo;, with llsquo;Aalivirus Arrsquo; as the type species.
We isolated a new species of virus from mosquitoes in the Philippines in 2005. The virion was elliptical in shape and had a short single projection. The virus was named Tanay virus (TANAV) after the locality. Genomic RNA of TANAV was a positive strand, 9,562nt+poly-A, and poly-cistronic. The longest open reading frame (ORF) contained putative RNA-dependent RNA polymerase (RdRP), however, conserved short motifs in the RdRP were permuted. TANAV was phylogenetically close to Negevirus, a recently proposed taxon of viruses isolated from hemophagic insects, and to some plant viruses, such as Citrus leprosis virus C, Hibiscus green spot virus and Blueberry necrotic ring blotch virus. In this article, we described the TANAV and the permuted structure of its RdRP, and discussed its phylogeny together with plant viruses and the Negevirus.
A new type III interferon, interferon lambda 4 (IFNL4), and its single nucleotide polymorphism (SNP) ss469415590 causing a frame-shift have been recently reported to strongly affect anti-viral therapy for chronic hepatitis C virus (HCV) infection in African and Caucasian populations compared to previously reported interleukin-28B (IL-28B) SNPs, rs12979860 and rs8099917. To compare the predictability for treatment outcome among those polymorphisms, we estimated haplotype structure of IFNL4/IL-28B consisting of the three SNPs in 4,630 Japanese chronic hepatitis C patients and 1,122 healthy controls and then compared their impact on response to pegylated-interferon (PEG-IFN) plus ribavirin (RBV) combined therapy in 903 HCV-1b infected patients. A total of five haplotypes were identified, although two major haplotypes accounted for ggt; 99% of the variation. The SNPs were tightly linked but not in absolute linkage disequilibrium. We could not find any difference in the predictive impact of any of these 3 SNPs with regard to susceptibility to HCV and treatment response. However, patients with favorable rs8099917 TT, linked to unfavorable genotype of ss469415590 and rs12979860, showed poor initial viral response compared to those with all favorable genotypes (p = 0.0022). These findings suggest that, in part, ss469415590 and rs12979860 may have better predictive impact on response to PEG-IFN plus RBV therapy in the Japanese population, especially in patients having any of minor haplotypes consisting of these SNPs.
We have examined the role of HMG CoA reductase (HMGCR) during PCV2 (porcine circovirus 2) infection. The results demonstrated that levels of endogenous HMGCR were not significantly different in PCV2 infected cells and mock infected cells. However, the level of phosphorylated HMGCR, as an inactivated form of HMGCR, was increased in PCV2-infected cells. Furthermore, HMGCR was upregulated by overexpression, silenced by siRNA, or inactivated using its dominant-negative form in PK-15 cells. The results showed that PCV2 infection was inhibited by HMGCR overexpression, whereas it was significantly increased in HMGCR-silenced cells and HMGCR inhibitor-treated cells. Moreover, there was a robust apoptotic response at 48 hours post infection (hpi) in the HMGCR-inactivated cells, and this response was significantly greater than that observed in the PK-15 cells. A modest apoptotic response was also observed in the HMGCR-silenced cells. Caspase-3 activity was also analyzed in the PCV2-infected cells at 48 hpi. As expected, caspase-3 activity was significantly increased in the HMGCR-inactivated cells and the HMGCR-silenced cells compared with PK-15 cells. PCV2 replication was dose-dependently increased in the HMGCR-inactivated cells when treated with increasing amounts of HMGCR inhibitor. Altogether, HMGCR was negatively associated with PCV2 infection and PCV2-induced apoptotic cell death. These data demonstrated that HMGCR can be used as a candidate target for disease control and antivirus research related to PCV2. Furthermore, the cells generated in this study can be used to evaluate the potential effects of HMGCR on PCV2 replication.
Understanding the potential for host range shifts and expansions of RNA viruses is critical to predicting the evolutionary and epidemiological paths of these pathogens. Because arthropod-borne viruses (arboviruses) experience frequent spillover from their amplification cycles and are generalists by nature, they are likely to experience a relatively high frequency of success in a range of host environments. Despite this, the potential for host expansion, the genetic correlates of adaptation to novel environments and the costs of such adaptations in originally competent hosts are not well characterized for arboviruses. In the studies presented here, we utilized experimental evolution of St. Louis encephalitis virus (SLEV; Flaviviridae, flavivirus) in vitro in the Dermacentor andersoni line of tick cells to model adaptation to a novel invertebrate host. Our results demonstrate that levels of adaptation and costs in alternate hosts are highly variable among lineages, but also that significant fitness increases in tick cells are achievable with only modest change in consensus genetic sequence. In addition, although accumulation of diversity may at times buffer against phenotypic costs within the SLEV swarm, an increased proportion of variants with an impaired capacity to infect and spread on vertebrate cell culture accumulated with tick cell passage. Isolation and characterization of a subset of these variants suggests that the NS3 gene is an important host range determinant for SLEV.
Mature dendritic cells (mDCs) are the most potent antigen presenting cells (APCs) known today since they are the only APCs able to induce naiiuml;ve T cells. Therefore they play a crucial role during the induction of effective antiviral immune responses. Interestingly, the surface molecule CD83 expressed on mDCs, is targeted by several viruses. As CD83 was shown to exert costimulatory functions on mDCs its downmodulation represents a viral immune escape mechanism. Mechanistically, it has been shown that HSV-1 infection leads to proteasomal degradation of CD83 resulting in a strongly diminished T cell stimulatory capacity of the infected mDC. Earlier data from our laboratory suggest that the viral immediate early protein ICP0 (infected cell protein 0) plays an important role in this process. In the present study we show that ICP0 is sufficient to induce CD83-degradation in the absence of any other viral factor. However, the mechanism of ICP0 mediated CD83-degradation is not yet understood. Here we provide evidence that ubiquitination of lysine residues is, despite of the published E3 ubiquitin ligase activity of ICP0, not necessary for CD83-degradation. This finding is underlined by the observation that expression of an ICP0 mutant lacking the E3 ubiquitin ligase domain in mDCs still induces CD83-degradation. Finally, inhibition of E1 activating enzyme using the specific inhibitor PYR-41 did not prevent CD83-degradation. Taken together, our data provide strong evidence that ICP0 alone induces CD83-degradation independently of its E3 ubiquitin ligase function and of the ubiquitin machinery.
Heterodera glycines, the soybean cyst nematode (SCN), is a subterranean root pathogen that causes the most damaging disease of soybean in the United States. A novel nematode virus genome, soybean cyst nematode virus 5 (SbCNV-5), was identified in RNASeq data from SCN eggs and second-stage juveniles. The SbCNV-5 RNA dependent RNA polymerase and RNA helicase domains have homology to pestiviruses in the Flaviviridae, suggesting SbCNV-5 is a positive-polarity single-stranded RNA virus. SbCNV-5 RNA was present in all nematode developmental stages indicating a transovarial mode of transmission, but it is also potentially sexually transmitted via the male. SbCNV-5 was common in SCN laboratory cultures and in nematode populations isolated from the field. Transmission electron microscopy of sections from a female SCN showed virus particles budding from the endoplasmic reticulum and in endosomes. The size of the viral genome is 19,191 nucleotides, which makes it much larger than known pestiviruses. Additionally, the presence of a methyltransferase in the SbCNV-5 genome is atypical for a pestivirus. When cDNA sequences were mapped to the genome of SbCNV-5, a disproportionate number aligned to the 3rrsquo; non-translated region, suggesting SbCNV-5 produces a subgenomic RNA, which was confirmed by RNA blot analysis. Since subgenomic RNAs and methyltransferases do not occur in pestiviruses, we conclude that SbCNV-5 is a new flavivirus infecting the soybean cyst nematode.
Pigeon circovirus (PiCV) has a ~2kb genome circular single stranded DNA genome. All but one of the known PiCV isolates have been found infecting pigeons in various parts of the world. In this study we screened 324 swab and tissue samples from Polish pigeons and recovered 30 complete genomes, 16 of which came from birds displaying no obvious pathology. Together with 17 other publically available PiCV complete genomes sampled throughout the Northern Hemisphere and Australia, we find that PiCV displays a similar degree of genetic diversity to that of the related Psittacine infecting circovirus species, Beak and feather disease virus (BFDV). We show that, as is the case with its pathology and epidemiology, PiCV also displays patterns of recombination, genomic secondary structure and natural selection that are generally very similar to those of BFDV. It is likely that breeding facilities play a significant role in the emergence of new recombinant PiCV variants and given that ~50% of the domestic pigeon population is infected subclinically, all pigeon breeding stocks should be routinely screened for this virus.
The cucumber mosaic virus (CMV) 2b silencing suppressor protein allows the virus to overcome resistance to replication and local movement in inoculated leaves of plants treated with salicylic acid (SA), a resistance-inducing plant hormone. In Arabidopsis thaliana plants systemically infected with CMV, the 2b protein also primes the induction of SA biosynthesis during this compatible interaction. We found that CMV infection of susceptible tobacco (Nicotiana tabacum) also induced SA accumulation. Utilization of mutant 2b proteins expressed during infection of tobacco showed that the N and C terminal domains, which had previously been implicated in regulation of symptom induction, were both required for subversion of SA-induced resistance, while all mutants tested except those affecting the putative phosphorylation domain had lost the ability to prime SA accumulation and expression of the SA-induced marker gene PR-1.
The incidence and severity of hepatitis E virus (HEV) infection in pregnant women is high in developing countries. Transplacental transmission of HEV in the third trimester of pregnancy has been found to be associated with a high fetal mortality. Based on this evidence and in absence of report on HEV replication in extra hepatic sites, this study was carried out to investigate if HEV replication occurs in the placenta of the infected mothers. The study included 68 acute viral hepatitis (AVH) and 22 acute liver failure (ALF) pregnant patients. Viral RNA was extracted from blood and placenta. HEV replication in placenta was confirmed by a replicative negative strand-specific rt-PCR. Viral load was estimated by real time PCR. Immunohistochemical studies were also carried out for in situ detection of HEV in placental tissue sections. Replicative HEV RNA was detectable only in the placenta in ALF and AVH cases and not in the blood samples.Also, positive staining of placental tissue sections stained with Hep E virus antibody against the viral structural protein ORF3 was identified. HEV replication in placenta also correlated with fetal and maternal mortality in ALF. It is demonstrated for the first time that HEV replication does occur in human placenta and placenta may be a site of extrahepatic replication of Hepatitis E virus in humans.
The Newcastle disease virus (NDV) matrix (M) protein is a highly basic and nucleocytoplasmic shuttling viral protein. Previous study has demonstrated that the N-terminal 100 amino acids of NDV M protein are somewhat acidic overall, but the remainder of the polypeptide is strongly basic. In this study, we investigated the role of the N-terminal basic residues in the subcellular localization of M protein and in the replication and pathogenicity of NDV. We found that mutation of the basic residue arginine (R) to alanine (A) at position 42 disrupted Mrrsquo;s nuclear localization. Moreover, a recombinant virus with R42A mutation in the M protein reduced viral replication in DF-1 cells and attenuated the virulence and pathogenicity of the virus in chickens. This is the first report to show that a basic residue mutation in the NDV M protein abrogates its nuclear localization and attenuates viral replication and pathogenicity.
During three seasons of mumps outbreaks in the Netherlands (September 2009 nndash; August 2012), 822 mumps cases were laboratory confirmed at the national laboratory (RIVM). Most patients were vaccinated young adults. Given the protracted endemic circulation, we studied the genetic diversity and changes of mumps virus over a period of three years. Phylogenetic analysis of the SH gene (316 bp) was performed on a representative set of 808 PCR positive specimens. Additionally, the HN gene (1749 bp) and F gene (1617 bp) were sequenced for a subset of samples (n=17). Correlations between different sequence types and epidemiological and clinical data were investigated. The outbreaks in the Netherlands were dominated by two SH gene sequence types within genotype G, termed MuVs/Delft.NLD/03.10 (variant 1) and MuVs/Scheemda.NLD/12.10 (variant 2). Sequence analysis of the HN and F genes indicated that the outbreaks were initiated by separately introduced genetic lineages. The predominance of variant 2 by the end of the first outbreak season could not be explained by any of the epidemiological factors investigated. Orchitis was more frequently reported in males infected with variant 2, irrespective of age and vaccination status. These findings illustrate genetic heterogeneity of an emerging mumps genotype and raise questions about the mechanisms driving mumps epidemiology and immunity, in relation to vaccination.
Typical avian influenza A viruses are restricted from replicating efficiently and causing disease in humans. However, an avian virus can become adapted to humans by mutating or recombining with currently circulating human viruses. These viruses have the potential to cause pandemics in an immunologically naiiuml;ve human population. It is critical that we understand the molecular basis of host range restriction and how this can be overcome. Here we review our current understanding of the mechanisms by which influenza viruses adapt to replicate efficiently in a new host. We predominantly focus on the influenza polymerase which remains one of the least understood host range barriers.
Five neutralizing antigenic sites have been described for serotype O foot-and-mouth disease viruses (FMDV) based on monoclonal antibody (mAb) escape mutant studies. However, a mutant virus selected to escape neutralization of mAb binding at all five sites was previously shown to confer complete cross-protection with the parental virus in guinea pig challenge studies, suggesting that amino acid residues outside of the mAb binding sites contribute to antibody-mediated in vivo neutralization of FMDV. Comparison of the ability of bovine antisera to neutralize a panel of serotype O FMDV identified three novel putative sites at VP2-74, VP2-191 and VP3-85, where amino acid substitutions correlated with changes in sero-reactivity. The impact of these positions was tested using site-directed mutagenesis to effect substitutions at critical amino acid residues within an infectious copy of FMDV O1 Kaufbeuren (O1K). Recovered viruses containing additional mutations at VP2-74 and VP2-191 exhibited greater resistance to neutralization with both O1K guinea pig and O BFS bovine antisera than a virus that was engineered to include only mutations at the five known antigenic sites. The changes at VP2-74 and VP3-85 are adjacent to critical amino acids that define antigenic sites 2 and 4, respectively. However VP2-191 (17AAring; away from VP2-72), located at the 3-fold axis and more distant from previously identified antigenic sites exhibited the most profound effect. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and will improve our strategies for vaccine strain selection and rational vaccine design.
The full-length genome sequence of a porcine picobirnavirus detected in Italy in 2004 was determined. The S segment was 1730 nucleotide (nt) in length, coding for a putative RNA-dependent RNA polymerase. Two distinct sub-populations of L segment (LA and LB) were identified in the sample, with the sizes ranging from 2351 to 2666 nt. The ORF1 coding for a protein of unknown function, contained repetitions of the ExxRxNxxxE motif in variable number. The capsid protein coding ORF2 spanned nt 810-2447 in the LB variants and started at nt 734 in the LA variants. However, a termination codon was present only in one of all LA segment variants. Three dimensional modelling of the porcine PBV capsids suggested structural differences in the protruding domain, tentatively involved as antigens in humoral immune response. Altogether, these findings suggest simultaneous presence of two different picobirnavirus strains sharing the same S segment but displaying genetically diverse L segments. In addition, the sample probably contained a mixture of PBVs with aberrant RNA replication products. Altered structure in the L segments could be tolerated and retained in the presence of functionally integer cognate genes and represent a mechanism of virus diversification.
Respiratory syncytial virus (RSV) is one of the primary causative agents of upper and lower respiratory tract infections in young children, in particular infants. Recently, we reported the protective efficacy of a RSV vaccine formulation consisting of a truncated version of the fusion (F) protein formulated with a TLR agonist and an immunostimulatory peptide in a carrier system (F/TriAdj). To evaluate the duration of immunity induced by this vaccine candidate, we carried out long-term trials. The F was formulated with TriAdj containing either poly(I:C) or CpG ODN and administered intranasally (IN) to mice. One year after the second vaccination all mice were challenged with RSV. Both F/TriAdj formulations mediated the induction of high levels of IgG1, IgG2a and virus neutralizing antibodies, and IgA in the lungs. Based on the numbers of IFN- and IL-5 secreting cells in the spleens, the immune response was slightly Th1-biased. This was confirmed by the presence of F85nndash;93-specific CD8+ effector T cells in the lungs of both F/TriAdj(polyI:C)- and F/TriAdj(CpG)-immunized mice. Both F/TriAdj formulations induced RSV-specific CD8 T cells. However, F/TriAdj(polyI:C) generated significantly higher IgG affinity maturation and higher numbers of RSV-specific CD8+ effector memory T cells in lungs and CD8+ central memory T cells in spleens and lymph nodes than F/TriAdj(CpG). After RSV challenge, no virus replication and no evidence of vaccine-induced pathology were detected in mice immunized with either of the F/TriAdj formulations, which demonstrates that the duration of immunity induced with these vaccines is at least one year.
Significant loss of RNA followed by severely reduced cellular protein pool, a phenomenon termed host shutoff, is associated with a number of lytic virus infections and is a critical player in virus pathogenesis. Until recently, viral DNA exonucleases were associated only with processing of viral genomic DNA and encapsidation. However, recent observations have identified a combined host shutoff and exonuclease function for the highly conserved viral exonucleases in - herpesviruses which include Kaposirrsquo;s sarcoma-associated herpesvirus, Epstein-Barr virus and the murine model murine gammaherpesvirus-68. In this study, we show that although ablation of the MHV-68 exonuclease ORF37 caused a restrictive phenotype in interferon-aalpha;/bbeta; receptor-positive cells, lack of ORF37 was tolerated in cells lacking the interferon- aalpha; /bbeta; receptor: the ORF37 deletant virus was capable of forming infectious particles and caused loss of RNA in interferon-aalpha;/bbeta; receptor knockout cells. Moreover, ORF37 deletant virus was able to establish lytic infection in lungs of mice lacking the interferon-aalpha;/bbeta; receptor. These observations provide further evidence that this family of viral nucleases play a key role in providing a window of opportunity for virus growth by overcoming type I IFN-dependent responses by host shutoff of cellular transcripts.
Morphogenesis of member viruses of genus Enterovirus of Picornaviridae is still poorly understood despite decades-long investigations. However, we recently provided evidence that 2CATPase gives specificity to poliovirus encapsidation through an interaction with capsid protein VP3. Polypeptide 2CATPase is a highly conserved nonstructural protein of enteroviruses with important roles in RNA replication, encapsidation and uncoating. We have identified a site (K279/R280) near the C-terminus of the polypeptide that is required for morphogenesis. The aim of our current project was to search for additional functional sites near the C-terminus of the 2CATPase polypeptide, with particular interest in those that are required for encapsidation. We selected for analysis a cysteine-rich site of the polypeptide and constructed four mutants in which cysteines or a histidine was changed to an alanine. The RNA transcripts were transfected into HeLa cells yielding 2 lethal, one temperature-sensitive and one quasi-infectious mutant. All four mutants exhibited normal protein translation in vitro and 3 of them possessed severe RNA replication defects. The quasi-infectious mutant (C286A) yielded variants with a pseudoreversion at the original site (A286D) but some also contained one additional mutation: A138Vor M293V. The temperature-sensitive mutant (C272A/H273A) exhibited an encapsidation, and possibly also an uncoating defect, at 37ddeg;C. Variants of this mutant revealed suppressor mutations at 3 different sites in the 2CATPase polypeptide: A138V, M293V, or K295R. We conclude that the cysteine-rich site near the C-terminus of 2CATPase is involved in encapsidation, possibly through an interaction with an upstream segment located between boxes A and B of the nucleotide-binding domain.
A thorough characterization of the genetic diversity of viruses present in vector and vertebrate host populations is essential for the early detection of and response to emerging pathogenic viruses, yet genetic characterization of many important viral groups remains incomplete. The Simbu serogroup of the genus Orthobunyavirus, family Bunyaviridae is an example. The Simbu serogroup currently consists of a highly diverse group of related arboviruses that infect both humans and economically important livestock species. Here, we report complete genome sequences for 11 viruses within this group, with a focus on the large and poorly characterized Manzanilla and Oropouche species complexes. Phylogenetic and pairwise divergence analyses indicate the presence of high-levels of genetic diversity within these two species complexes, on par with that seen among the five other species complexes in the Simbu serogroup. Based on previously reported divergence thresholds between species, the data suggest that these two complexes should actually be divided into at least five species. Together these five species form a distinct phylogenetic clade apart from the rest of the Simbu serogroup. Pairwise sequence divergences among viruses of this clade and viruses in other Simbu serogroup species complexes are similar to levels of divergence among the other orthobunyavirus serogroups. The genetic data also suggest relatively high levels of natural reassortment, with three potential reassortment events present, including two well-supported events involving viruses known to infect humans.
In Europe and Asia, Ixodid ticks transmit tick-borne encephalitis virus (TBEV), a flavivirus that causes severe encephalitis in humans but appears to show no virulence for livestock and wildlife. In the British Isles, where TBEV is absent, a closely related tick-borne flavivirus, named Louping ill virus (LIV), is present. However, unlike TBEV, LIV causes a febrile illness in sheep, cattle, grouse and some other species, that can progress to fatal encephalitis. The disease is detected predominantly in animals from upland areas of the United Kingdom and Ireland. This distribution is closely associated with the presence of its arthropod vector, the hard tick Ixodes ricinus. The virus is a positive-strand RNA virus belonging to the Flavivirus genus, exhibiting a high degree of genetic homology to TBEV and other mammalian tick-borne viruses. In addition to causing acute encephalomyelitis in sheep, other mammals and some avian species, the virus is recognised as a zoonotic agent with occasional reports of seropositive individuals, particularly those whose occupation involves contact with sheep. Preventative vaccination in sheep is effective although there is no treatment for disease. Surveillance for LIV in Great Britain (GB) is limited despite an increased awareness of emerging arthropod-borne diseases and potential changes in distribution and epidemiology. This review provides an overview of LIV and highlights areas where further effort is needed to control this disease.
Summary Semaphorins are a large, phylogenetically conserved family of proteins that are involved in a wide range of biological processes including axonal steering, organogenesis, neoplastic transformation, as well as immune responses. In this study, a novel semaphorin homolog gene belonging to the Singapore grouper iridovirus (SGIV), ORF155R (termed as SGIV-sema), was cloned and characterized. The coding region of SGIV-sema is 1728 bp in length, encoding a predicted protein with 575 amino acids (aa). SGIV-sema contains a ~370-aa N-terminal Sema domain, a conserved PSI domain, and an Ig-like domain near the C terminus. SGIV-sema is an early gene product during viral infection and predominantly distributed in the cytoplasm with a speckled and clubbed pattern of appearance. Functionally, SGIV-sema could promote viral replication during SGIV infection in vitro, with no effect on the proliferation of host cells. Intriguingly, ectopically expressed SGIV-sema could alter the cytoskeletal structure of fish cells, characterized by a circumferential ring of microtubules near the nucleus and a disrupted microfilament organization. Furthermore, SGIV-sema was able to attenuate the cellular immune response, as demonstrated by decreased expression of inflammation/immune-related genes such as interleukin-8 (IL-8), IL-15, tumor necrosis factor alpha (TNF-aalpha;), and mediator of IRF3 activation (MITA), in SGIV-sema-expressing cells before and after SGIV infection. Ultimately, our study identified a novel, functional SGIV gene that could regulate cytoskeletal structure, immune responses and facilitate viral replication. Keywords: semaphorin, Singapore grouper iridovirus, viral replication, cytoskeletal structure, immune response
Murine hybridomas producing neutralizing monoclonal antibodies (MAbs) specific of the pandemic influenza virus A/Cal/07/09 hemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extr/6530/10) lacked reactivity with the monoclonals and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to ablate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus hemagglutinin during virus evolution (Shih et al., Proc. Nal. Acad. Sci. USA, 104:6283-6288, 2007). The changes found in the A/Extr/6530/10 HA were not found in escape mutants selected in vitro with one of the monoclonals, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may alter similarly epitopes of the new antigenic site identified in this work on the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of MAbs and human postinfection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.
The multifunctional potyviral Helper component Protease (HcPro) contains variable regions with some functionally conserved domains, such as the FRNK box. Natural variants occur at FRNK box, a conserved central domain, known for its role in RNA binding and RNAi suppression activities, although no dominant natural variant for N182 residue are known to occur. Here, a mutant at HcProN182L was developed to ascertain its indispensible nature in natural populations. Using in vitro studies, we found an increase in the small RNA (sRNA) binding potentiality of HcProN182Lwithout affecting the protein-protein interaction property, suggesting that the presence of N182 is critical to maintain threshold levels of sRNAs, but does not interfere in self-interaction of HcPro. Furthermore, we found that expression of HcProN182L in Nicotiana benthamiana affected plant growth. Transient expression of HcProN182L supported reporter gene expression in 16c GFP transgenic plants more than HcPro, suggesting that replacement of asparagine in FRNK box favors RNA silencing suppression. HcPro was found to be distributed in the nucleus and cytoplasm, whereas HcProN182L was observed only in cytoplasmic inclusion bodies in N. benthamiana leaves, when fused to GFP tag and expressed by agro-infiltration, suggesting mutation favours oligomerisation of HcPro. These findings suggest that amino acid N182 of the conserved FRNK box may impose stringent regulation on the RNA silencing mechanisms and is required to maintain subcellular localisation of the protein for its multi-functionality. Hence, the N182 residue of the FRNK box seems to be indispensable for potyvirus infection during evolution.
The recent identification of a new nanovirus, pea necrotic yellow dwarf virus (PNYDV), from pea in Germany prompted us to survey wild and cultivated legumes for nanovirus infections in several European countries. This led to the identification of two new nanoviruses: black medic leaf roll virus (BMLRV) and pea yellow stunt virus (PYSV), each considered a putative new species. The complete genomes of a PYSV isolate from Austria and three BMLRV isolates from Austria, Azerbaijan and Sweden were sequenced. In addition, the genomes of five isolates of faba bean necrotic yellows virus (FBNYV) from Azerbaijan and Spain and those of four faba bean necrotic stunt virus (FBNSV) isolates from Azerbaijan were completely sequenced, leading to the first identification of FBNSV occurring in Europe. Sequence analyses uncovered evolutionary relationships, extensive reassortment, potential remnants of mixed nanovirus infections as well as intra- and intercomponent recombination events within the nanovirus genomes. In some virus isolates, diverse types of the same genome component (paralogs) were observed, a type of genome complexity not previously described for any member of the Nanoviridae family. Moreover, infectious and aphid-transmissible nanoviruses from cloned genomic DNAs of FBNYV and BMLRV were reconstituted that, for the first time, allow experimental reassortments for studying the genome functions and evolution of these nanoviruses.
The present study was aimed at evaluating an underlying mechanism of the antiviral activity of the sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri (G. fisheri) against white spot syndrome virus (WSSV) infection in haemocytes of the black tiger shrimp Penaeus monodon. Primary culture of haemocytes from P. monodon was performed and inoculated with WSSV, after which the cytopathic effect (CPE), cell viability, and viral load were determined. Haemocytes treated with WSSV-SG premix showed decreased CPE, viral load, and cell mortality from the viral infection. Solid-phase virus-binding assay revealed that SG bound to WSSV in a dose-related manner. Far Western blotting analysis indicated that SG bound to VP 26, and VP 28 proteins of WSSV. In contrast to the native SG, desulfated SG did not reduce CPE and cell mortality, and showed low binding activity with WSSV. The current study suggests that SG from G. fisheri elicits its anti-WSSV activity by binding to viral proteins which are important for the process of viral attachment to the host cells. It is anticipated that the sulfate groups of SG are important for the viral binding.
We previously reported that Pin1 facilitates the human immunodeficiency virus type 1 (HIV-1) uncoating by interacting with the capsid (CA) core through the phosphorylated Ser16-Pro17 motif. However, the specific kinase responsible for Ser16 phosphorylation has remained unknown. Here, we show that virion-associated extracellular signal-regulated kinase 2 (ERK2) phosphorylates Ser16. The characterization of immature virions produced by exposing of CEM/LAV-1 cells to 10 mmicro;M saquinavir indicated that Ser16 is phosphorylated after the initiation of Pr55gag processing. Furthermore, a mass spectrometry-based in vitro kinase assay demonstrated that ERK2 specifically phosphorylated the Ser16 residue in the Ser16-Pro17-motif-containing substrate. The treatment of CEM/LAV-1 cells with the ERK2 inhibitor sc-222229 decreased the Ser16 phosphorylation level inside virions, and the virus partially defective in Ser16 phosphorylation showed an impaired reverse transcription and an attenuated replication owing to an attenuated Pin1-dependent uncoating. Furthermore, the suppression of ERK2 expression by RNA interference in CEM/LAV-1 cells resulted in suppressed ERK2 packaging inside virions and decreased the Ser16 phosphorylation level inside virions. Interestingly, the ERK2-packaging-defective virus showed an impaired reverse transcription and an attenuated HIV-1 replication. Taken together, these findings provide insights into the as yet obscure processes in Pin1-dependent HIV-1 uncoating.
Circulating coxsackievirus A16 (CA16) is a major cause of hand, foot, and mouth disease (HFMD) in Southeast Asia. At present, there is no vaccine against CA16 viruses. Pathogenic animal models that are sensitive to diverse circulating CA16 viruses would be desirable for vaccine development and evaluation. In this study, we isolated and characterized several circulating CA16 viruses from recent HFMD patients. These CA16 viruses currently circulating in humans were highly pathogenic in a newly developed neonatal mouse model; we also observed and analyzed the pathogenesis of representative circulating recombinant form (CRF) CA16 viruses. An inactivated CA16 vaccine candidate, formulated with alum adjuvant and containing sub-microgram quantities of viral proteins, protected neonatal mice born to immunized female mice from lethal-dose challenge with a series of CA16 viruses. Further analysis of humoral immunity showed that antibody elicited from both the immunized dams and their pups can neutralize various lethal viruses by cytopathic effect (CPE) in vitro. Moreover, viral titers and loads in the tissues of challenged pups in the vaccine group were far lower than those in the control group, and some were undetectable. This lethal challenge model using pathogenic CA16 viruses and the vaccine candidates that mediated protection in this model could be useful tools for the future development and evaluation of CA16 vaccine.
In 2009, a novel lyssavirus (subsequently named Ikoma lyssavirus, IKOV) was detected in the brain of an African civet (Civettictis civetta) with clinical rabies in the Serengeti National Park of Tanzania. The degree of nucleotide divergence between genomes of IKOV and other lyssaviruses predicted antigenic distinction from, and lack of protection provided by, available rabies vaccines. In addition, the index case was considered likely to be an incidental spill over event, and therefore the true reservoir of IKOV remained to be identified. The advent of sensitive molecular techniques has led to a rapid increase in the discovery of novel viruses. Detecting viral sequence alone however, only allows for prediction of phenotypic characteristics and not their measurement. In the present study we describe the in-vitro and in-vivo characterisation of IKOV, demonstrating that it is (1) pathogenic by peripheral inoculation in an animal model, (2) antigenically distinct from current rabies vaccine strains, and (3) is poorly neutralised by sera from humans and animals immunised against rabies. In a laboratory mouse model, no protection was elicited by a licensed rabies vaccine. We also investigated the role of bats as reservoirs of IKOV. We found no evidence for infection among 483 individuals of at least 13 bat species sampled across sites in the Serengeti and Southern Kenya.
A novel picornavirus from commercial broiler chickens (Gallus gallus domesticus) has been identified and genetically characterised. The viral genome consists of a single-stranded, positive-sense RNA genome of ggt;9243 nucleotides (nt) excluding the poly (A) tail and as such represents one of the largest picornavirus genomes reported to date. The virus genome is GC-rich with a G+C content of 54.5 %. The genomic organisation is similar to other picornaviruses: 5rrsquo; UTR-L-VP0-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3rrsquo; UTR. The partially characterised 5rrsquo; UTR of ggt;373 nt appears to possess a type II internal ribosomal entry site (IRES), found in Aphthovirus and Cardiovirus. This IRES exhibits significant sequence homology to turkey "Gallivirus A". The 3rrsquo; UTR of 278 nt contains the conserved 48 nt llsquo;barbell-likerrsquo; structure identified in "Passerivirus", "Gallivirus", Avihepatovirus and some Kobuvirus genus members. A predicted large open reading frame (ORF) of 8592 nt encodes a potential polyprotein precursor of 2864 amino acids. In addition, the virus contains a predicted large L protein of 462 amino acids. Pairwise sequence comparisons, along with phylogenetic analysis revealed the highest percentage identity to "passerivirus A" (formerly called turdivirus 1), forming a monophyletic group across the P1, P2 and P3 regions, llt;40, llt;40 and llt;50 % amino acid identity respectively. Reduced identity was observed against "Gallivirus A" and members of the Kobuvirus genus. Quantitative PCR analysis estimated a range of 4 x 105 to 5 x 108 viral genome copies/gram in 22 (73 %) of 30 PCR-positive faeces. Based on sequence and phylogenetic analysis, we propose that this virus is the first member of a potential novel genus within the family Picornaviridae. Further studies are required to investigate the pathogenic potential of this virus within the avian host.
Chronic infections by the hepatotropic viruses hepatitis B virus (HBV) and hepatitis C virus (HCV) are major risk factors for the development of hepatocellular carcinoma (HCC). It is estimated that more than 700,000 individuals per year die from hepatocellular carcinoma, and around 80% of HCC is attributable to HBV or HCV infection. Despite the clear clinical importance of virus-associated HCC, the underlying molecular mechanisms remain largely elusive. Oxidative stress, in particular DNA lesions associated with oxidative damage, play a major contributory role in carcinogenesis, and are strongly linked to the development of many cancers, including HCC. A large body of evidence demonstrates that both HBV and HCV induce hepatic oxidative stress, with increased oxidative DNA damage being observed both in infected individuals and in murine models of infection. Here, we review the impact of HBV and HCV on the incidence and repair of oxidative DNA damage. We begin by giving a brief overview of oxidative stress and the repair of DNA lesions induced by oxidative stress. We then review in detail the evidence surrounding the mechanisms by which both viruses stimulate oxidative stress, before focusing on how the viral proteins themselves may perturb the cellular response to oxidative DNA damage, impacting upon genome stability and thus hepatocarcinogenesis.
An epidemic of diarrhea in adult cows occurred at a total of 105 dairy farms in Yamagata Prefecture, Japan between 2003 and 2010. Reverse transcription-polymerase chain reaction (RT-PCR) diagnostic tests revealed the presence of bovine rotavirus species C (RVCs) in samples from each of 6 farms (5.7%). In this study, we determined the full-length nucleotide sequences of 11 RNA segments from six bovine RVC strains and investigated genetic diversity among them, including two bovine RVC strains identified in our previous study. Comparisons of all segmental nucleotide and the deduced amino acid sequences among bovine RVCs indicated high identities across all genes except for the VP4 gene. Phylogenetic analysis of each gene revealed that the 6 bovine RVCs belonged to a bovine cluster distinct from human and porcine RVCs. Bovine RVC strains could be clearly divided into two lineages of the VP4 genes. The nucleotide sequence identity for VP4 genes between lineage I and II was 83.7-84.8 %. Moreover, bovine RVC strains belonging to lineage I exhibited one amino acid deletion and three amino acid insertions, which differed for those strains belonging to lineage II. Our data suggest that multiple bovine RVCs originated from a common ancestor, but had different genetic backgrounds not only in Yamagata Prefecture but also in the rest of Japan.
Since the discovery of a non-virulent infectious salmon anaemia virus (ISAV) HPR0 variant, many studies have speculated on the functional role of deletions within the highly polymorphic region (HPR) of genomic segment 6, which codes for the haemagglutinin-esterase (HE) protein. To address this issue, mutant HE proteins with deletions in their HPR were generated from the Scottish HPR0 template (NWM10) and fusion inducing activity measured using lipid (octadecyl Rhodamine B) and content mixing assays (Firefly luciferase). Segment 6 HPR was found to have a strong influence on ISA viral fusion, and deletions in this near membrane region predominantly increased the fusion inducing ability of the resulting HE proteins. The position and length of the HPR deletions were not significant factors, suggesting that they may affect fusion non-specifically. In comparison, the amino acid composition of the associated fusion (F) protein was a more crucial criterion. Antibody co-patching and confocal fluorescence demonstrated that HE and F protein were highly co-localised forming defined clusters on the cell surface, post-transfection. The binding of erythrocyte ghosts on the attachment protein caused a reduction in the percentage of co-localisation suggesting that ISAV fusion might be triggered through physical separation of the F and HE protein. In this process HPR deletion appeared to modulate and reduce the strength of interaction between the two glycoproteins, causing more F protein to be released and activated. This work provides a first insight into the mechanism of virulence acquisition through HPR deletion, with fusion enhancement acting as a major contributing factor.