|Journal of General Virology current issue|
On 23 March 2014, the World Health Organization issued its first communiqueeacute; on a new outbreak of Ebola virus disease (EVD), which began in December 2013 in Guineeacute;e Forestieegrave;re (Forested Guinea), the eastern sector of the Republic of Guinea. Located on the Atlantic coast of West Africa, Guinea is the first country in this geographical region in which an outbreak of EVD has occurred, leaving aside the single case reported in Ivory Coast in 1994. Cases have now also been confirmed across Guinea as well as in the neighbouring Republic of Liberia. The appearance of cases in the Guinean capital, Conakry, and the transit of another case through the Liberian capital, Monrovia, presents the first large urban setting for EVD transmission. By 20 April 2014, 242 suspected cases had resulted in a total of 147 deaths in Guinea and Liberia. The causative agent has now been identified as an outlier strain of Zaire Ebola virus. The full geographical extent and degree of severity of the outbreak, its zoonotic origins and its possible spread to other continents are sure to be subjects of intensive discussion over the next months.
Type I IFN production is one of the hallmarks of host innate immune responses upon virus infection. Whilst most respiratory viruses carry IFN antagonists, reports on human metapneumovirus (HMPV) have been conflicting. Using deep sequencing, we have demonstrated that HMPV particles accumulate excessive amounts of defective interfering RNA (DIs) rapidly upon in vitro passage, and that these are associated with IFN induction. Importantly, the DIs were edited extensively; up to 70 % of the original A and T residues had mutated to G or C, respectively. Such high editing rates of viral RNA have not, to our knowledge, been reported before. Bioinformatics and PCR assays indicated that
Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment.
Bunyaviruses have evolved a variety of strategies to counteract the antiviral defence systems of mammalian cells. Here we show that the NSs protein of Schmallenberg virus (SBV) induces the degradation of the RPB1 subunit of RNA polymerase II and consequently inhibits global cellular protein synthesis and the antiviral response. In addition, we show that the SBV NSs protein enhances apoptosis in vitro and possibly in vivo, suggesting that this protein could be involved in SBV pathogenesis in different ways.
Bokeloh bat lyssavirus (BBLV), a novel lyssavirus, was isolated from a Nattererrrsquo;s bat (Myotis nattererii), a chiropteran species with a widespread and abundant distribution across Europe. As a novel lyssavirus, the risks of BBLV to animal and human health are unknown and as such characterization both in vitro and in vivo was required to assess pathogenicity and vaccine protection. Full genome sequence analysis and antigenic cartography demonstrated that the German BBLV isolates are most closely related to European bat lyssavirus type 2 (EBLV-2) and Khujand virus and can be characterized within phylogroup I. In vivo characterization demonstrated that BBLV was pathogenic in mice when inoculated peripherally causing clinical signs typical for rabies encephalitis, with higher pathogenicity observed in juvenile mice. A limited vaccination-challenge experiment in mice was conducted and suggested that current vaccines would afford some protection against BBLV although further studies are warranted to determine a serological cut-off for protection.
Persistent infection of hepatitis C virus (HCV) can lead to liver cirrhosis and hepatocellular carcinoma, which are currently diagnosed by invasive liver biopsy. Approximately 15nndash;20 % of cases of chronic liver diseases in India are caused by HCV infection. In North India, genotype 3 is predominant, whereas genotype 1 is predominant in southern parts of India. The aim of this study was to identify differentially regulated serum proteins in HCV-infected Indian patients (genotypes 1 and 3) using a two-dimensional electrophoresis approach. We identified eight differentially expressed proteins by MS. Expression levels of one of the highly upregulated proteins, retinol-binding protein 4 (RBP4), was validated by ELISA and Western blotting in two independent cohorts. We also confirmed our observation in the JFH1 infectious cell culture system. Interestingly, the HCV core protein enhanced RBP4 levels and partial knockdown of RBP4 had a positive impact on HCV replication, suggesting a possible role for this cellular protein in regulating HCV infection. Analysis of RBP4-interacting partners using a bioinformatic approach revealed novel insights into the possible involvement of RBP4 in HCV-induced pathogenesis. Taken together, this study provided information on the proteome profile of the HCV-infected Indian population, and revealed a link between HCV infection, RBP4 and insulin resistance.
An important step in poliovirus (PV) infection by the oral route in humans is replication of the virus in lymphatic tissues of the gastrointestinal (GI) tract, thought to be mainly in the Peyerrrsquo;s patches of the small intestine. No immunocompetent transgenic (tg) mice that express human PV receptor (CD155) under the control of different promoters can be infected orally. The mouse orthologue of human CD155 is Tage4, a protein expressed at the surface of enterocytes and in the Peyerrrsquo;s patches. We describe here the generation of a tg mouse model in which the Tage4 promoter was used to drive expression of the human PV receptor-coding region (Tage4-CD155tg mice). In this model, CD155 expression was observed by immunostaining in different regions in the Peyerrrsquo;s patches but not in their germinal centres. Although a similar pattern of staining was observed between 3- and 6-week-old Tage4-CD155tg mice, poliomyelitis was only seen in the younger mice after PV infection by the oral route. When compared with TgPVR21 mice that expressed CD155 driven by its human promoter, 3-week-old Tage4-CD155tg mice were more susceptible to gut infection and paralysis following feeding with PV. Also, Tage4-CD155tg mice exhibited higher susceptibility to poliomyelitis after parenteral inoculation of PV. Remarkably, the LD50 after intracerebral inoculation of PV was similar in both CD155 tg mouse strains. The CD155 tg mouse model reported here, although moderately susceptible to oral infection, may be suitable to study mechanisms of PV replication in the gastrointestinal tract and to dissect important aspects of PV neuroinvasiveness.
We characterized the full-length genomes of nine hepatitis C virus genotype 3 (HCV-3) isolates: QC7, QC8, QC9, QC10, QC34, QC88, NE145, NE274 and 811. To the best of our knowledge, NE274 and NE145 were the first full-length genomes for confirming the provisionally assigned subtypes 3d and 3e, respectively, whereas 811 represented the first HCV-3 isolate that had its extreme 3' UTR terminus sequenced. Based on these full-length genomes, together with 42 references representing eight assigned subtypes and an unclassified variant of HCV-3, and 10 sequences of six other genotypes, a timescaled phylogenetic tree was reconstructed after an evolutionary analysis using a coalescent Bayesian procedure. The results indicated that subtypes 3a, 3d and 3e formed a subset with a common ancestor dated to ~202.89 [95 % highest posterior density (HPD): 160.11, 264.6] years ago. The analysis of all of the HCV-3 sequences as a single lineage resulted in the dating of the divergence time to ~457.81 (95 % HPD: 350.62, 587.53) years ago, whereas the common ancestor of all of the seven HCV genotypes dated to ~780.86 (95 % HPD: 592.15, 1021.34) years ago. As subtype 3h and the unclassified variant were relatives, and represented the oldest HCV-3 lineages with origins in Africa and the Middle East, these findings may indicate the ancestral origin of HCV-3 in Africa. We speculate that the ancestral HCV-3 strains may have been brought to South Asia from Africa by land and/or across the sea to result in its indigenous circulation in that region. The spread was estimated to have occurred in the era after Vasco da Gama had completed his expeditions by sailing along the eastern coast of Africa to India. However, before this era, Arabians had practised slave trading from Africa to the Middle East and South Asia for centuries, which may have mediated the earliest spread of HCV-3.
Hepatitis E virus (HEV), a major cause of acute viral hepatitis across the world, is a non-enveloped, plus-strand RNA virus. Its genome codes three proteins, pORF1 (multifunctional polyprotein), pORF2 (capsid protein) and pORF3 (multi-regulatory protein). pORF1 encodes methyltransferase, putative papain-like cysteine protease, helicase and replicase enzymes. Of these, the protease domain has not been characterized. On the basis of sequence analysis, we cloned and expressed a protein covering aa 440nndash;610 of pORF1, expression of which led to cell death in Escherichia coli BL-21 and Huh7 hepatoma cells. Finally, we expressed and purified this protein from E. coli C43 cells (resistant to toxic proteins). The refolded form of this protein showed protease activity in gelatin zymography. Digestion assays showed cleavage of both pORF1 and pORF2 as observed previously. MS revealed digestion of capsid protein at both the N and C termini. N-terminal sequencing of the ~35 kDa methyltransferase, ~35 kDa replicase and ~56 kDa pORF2 proteins released by protease digestion revealed that the cleavage sites were alanine15/isoleucine16, alanine1364/valine1365 in pORF1 and leucine197/valine198 in pORF2. Specificity of these cleavage sites was validated by site-directed mutagenesis. Further characterization of the HEV protease, carried out using twelve inhibitors, showed chymostatin and PMSF to be the most efficient inhibitors, indicating this protein as a chymotrypsin-like protease. The specificity was further confirmed by cleavage of the chymotrypsin-specific fluorogenic peptide N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Mutational analysis of the conserved serine/cysteine/histidine residues suggested that H443 and C472/C481/C483 are possibly the active site residues. To our knowledge, this is the first direct demonstration of HEV protease and its function.
Non-primate hepacivirus (NPHV), equine pegivirus (EPgV) and Theilerrrsquo;s disease associated virus (TDAV) are newly discovered members of two genera in the Flaviviridae family, Hepacivirus and Pegivirus respectively, that include human hepatitis C virus (HCV) and human pegivirus (HPgV). To investigate their epidemiology, persistence and clinical features of infection, large cohorts of horses and other mammalian species were screened for NPHV, EPgV and TDAV viraemia and for past exposure through serological assays for NPHV and EPgV-specific antibodies. NPHV antibodies were detected in 43 % of 328 horses screened for antibodies to NS3 and core antibodies, of which three were viraemic by PCR. All five horses that were stablemates of a viraemic horse were seropositive, as was a dog on the same farm. With this single exception, all other species were negative for NPHV antibodies and viraemia: donkeys (n = 100), dogs (n = 112), cats (n = 131), non-human primates (n = 164) and humans (n = 362). EPgV antibodies to NS3 were detected in 66.5 % of horses, including 10 of the 12 horses that had EPgV viraemia. All donkey samples were negative for EPgV antibody and RNA. All horse and donkey samples were negative for TDAV RNA. By comparing viraemia frequencies in horses with and without liver disease, no evidence was obtained that supported an association between active NPHV and EPgV infections with hepatopathy. The study demonstrates that NPHV and EPgV infections are widespread and enzootic in the study horse population and confirms that NPHV and potentially EPgV have higher frequencies of viral clearance than HCV and HPgV infections in humans.
Dengue virus (DENV) infection in humans can cause flu-like illness, life-threatening haemorrhagic fever or even death. There is no specific anti-DENV therapeutic or approved vaccine currently available, partially due to the possibility of antibody-dependent enhancement reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are vulnerable to degradation by serum nucleases and rapid renal excretion due to their small size and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that cationic AuNPnndash;siRNA complexes could enter Vero cells and significantly reduce DENV serotype 2 (DENV-2) replication and infectious virion release under both pre- and post-infection conditions. In addition, RNase-treated AuNPnndash;siRNA complexes could still inhibit DENV-2 replication, suggesting that AuNPs maintained siRNA stability. Collectively, these results demonstrated that AuNPs were able to efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy.
The importance of innate immunity to rotaviruses is exemplified by the range of strategies evolved by rotaviruses to interfere with the IFN response. We showed previously that rotaviruses block gene expression induced by type I and II IFNs, through a mechanism allowing activation of signal transducer and activator of transcription (STAT) 1 and STAT2 but preventing their nuclear accumulation. This normally occurs through activated STAT1/2 dimerization, enabling an interaction with importin aalpha;5 that mediates transport into the nucleus. In rotavirus-infected cells, STAT1/2 inhibition may limit the antiviral actions of IFN produced early in infection. Here we further analysed the block to STAT1/2 nuclear accumulation, showing that activated STAT1 accumulates in the cytoplasm in rotavirus-infected cells. STAT1/2 nuclear accumulation was inhibited by rotavirus even in the presence of the nuclear export inhibitor Leptomycin B, demonstrating that enhanced nuclear export is not involved in STAT1/2 cytoplasmic retention. The ability to inhibit STAT nuclear translocation was completely conserved amongst the group A rotaviruses tested, including a divergent avian strain. Analysis of mutant rotaviruses indicated that residues after amino acid 47 of NSP1 are dispensable for STAT inhibition. Furthermore, expression of any of the 12 Rhesus monkey rotavirus proteins did not inhibit IFN-stimulated STAT1 nuclear translocation. Finally, co-immunoprecipitation experiments from transfected epithelial cells showed that STAT1/2 binds importin aalpha;5 normally following rotavirus infection. These findings demonstrate that rotavirus probably employs a novel strategy to inhibit IFN-induced STAT signalling, which acts after STAT activation and binding to the nuclear import machinery.
Vaccines against porcine circovirus 2 (PCV2) are now widely used to control the diseases caused by the virus. Although the vaccines protect pigs against the disease, they do not lead to sterilizing immunity and therefore infections with PCV2 continue in farms. It is expected that, due to its high evolutionary rate, PCV2 can adapt quickly to environmental pressures such as vaccination. The goal of this study was to elucidate the molecular variation of PCV2 in relation to vaccination. PCV2 variability was investigated from samples of infected pigs from five farms where vaccination had never been applied and two farms where pigs had been vaccinated for at least 2 years. For the genetic analysis, full PCV2 genomes were amplified and subsequently pooled by vaccination status from serum of eight vaccinated, infected pigs and 16 non-vaccinated, infected pigs. Variability of viral populations was quantified using next-generation sequencing and subsequent bioinformatics analysis. The number of segregating sites was similar in the non-vaccinated (n = 109) and vaccinated pools (n = 96), but the distribution of these sites in the genome differed. Most notably, in the capsid gene, the number of segregating sites was observed only in the non-vaccinated population. Based on the structural analysis, it is expected that some low-frequency amino acids result in biologically low-fit viruses. On the contrary, D294 in replicase represents a novel amino acid which was dominant and unique in the vaccinated pool. This work showed that variable PCV2 populations were circulating in commercial farms, and that this variability was different in samples obtained from vaccinating and non-vaccinating farms.
Adenoviruses are non-enveloped DNA viruses that replicate in the nucleus of infected cells. One of the core proteins, named pVIII, is a minor capsid protein connecting the core with the inner surface of the capsid. Here, we report the characterization of minor capsid protein pVIII encoded by the L6 region of bovine adenovirus (BAdV)-3. Anti-pVIII serum detected a 24 kDa protein at 12nndash;48 h post-infection and an additional 8 kDa protein at 24nndash;48 h post-infection. While the 24 kDa protein was detected in empty capsids, only the C-terminal-cleaved 8 kDa protein was detected in the mature virion, suggesting that amino acids147nndash;216 of the conserved C-terminus of BAdV-3 pVIII are incorporated in mature virions. Detection of hexon protein associated with both precursor (24 kDa) and cleaved (8 kDa) forms of pVIII suggest that the C-terminus of pVIII interacts with the hexon. The pVIII protein predominantly localizes to the nucleus of BAdV-3-infected cells utilizing the classical importin aalpha;/bbeta; dependent nuclear import pathway. Analysis of mutant pVIII demonstrated that amino acids 52nndash;72 of the conserved N-terminus bind to importin aalpha;-3 with high affinity and are required for the nuclear localization.
Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCPnndash;GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present nndash; indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCPnndash;GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCPnndash;His6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCPnndash;GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCPnndash;GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCPnndash;MCP interaction.
Kaposi's sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is a lytic structural protein expressed on the envelope of mature virions and on the membrane of cells supporting lytic infection. In addition to this viral glycoproteinrrsquo;s interaction with integrins via its RGD (Arg-Gly-Asp) motif, KSHV gB possesses a disintegrin-like domain (DLD), which binds integrins as well. Prior to this study, there has been minimal research involving the less common integrin-binding motif, DLD, of gB as it pertains to herpesvirus infection. By using phage display peptide library screening and molecular biology techniques, the DLD of KSHV gB was shown to interact specifically with non-RGD binding aalpha;9bbeta;1 integrins. Similarly, monitoring wild-type infection confirmed aalpha;9bbeta;1:DLD interactions to be critical to successful KSHV infection of human foreskin fibroblast (HFF) cells and human dermal microvascular endothelial cells (HMVEC-d) compared with 293 cells. To further demonstrate the importance of the DLD of gB in KSHV infection, two recombinant virus constructs were generated using a bacterial artificial chromosome (BAC) system harbouring the KSHV genome (BAC36): BAC36D-KSHV (lacking a functionally intact DLD of gB and containing an introduced tetracycline cassette) and BAC36.T-KSHV (containing an intact DLD sequence and an introduced tetracycline cassette). Accordingly, BAC36D-KSHV presented significantly lower infection rates in HFF and HMVEC-d cells compared with the comparable infection rates achieved by wild-type BAC36-KSHV and BAC36.T-KSHV. Thus, the present report has delineated a critical role for the DLD of gB in KSHV infection, which may lead to a broader knowledge regarding the sophisticated mechanisms utilized by virus-encoded structural proteins in KSHV entry and infection.
Cytotoxic T-lymphocytes (CTLs) are associated with protective immunity against disease caused by equid herpesvirus type 1 (EHV-1). However, the EHV-1 target proteins for CTLs are poorly defined. This limits the development of vaccine candidates designed to stimulate strong CTL immunity. Here, classical CTL assays using lymphocytes from horses of three defined MHC class I types that experienced natural infection with EHV-1 and a modified vaccinia virus construct containing an EHV-1 gene encoding the immediate-early (IE) protein are reported. Horses homozygous for the equine leukocyte antigen (ELA)-A2 haplotype, but not the ELA-A5 haplotype, produced MHC-restricted CTL responses against the IE protein. Previously, horses homozygous for the ELA-A3 haplotype also mounted CTL responses against the IE protein. Both haplotypes are common in major horse breeds, including the Thoroughbred. Thus, the IE protein is an attractive candidate molecule for future studies of T-cell immunity to EHV-1 in the horse.
The genus Macavirus of the subfamily Gammaherpesvirinae includes viruses that infect lymphoid cells of domestic and wild ruminants and swine, causing asymptomatic latent infections in reservoir hosts. Here, we describe the genome of bovine herpesvirus 6 (BoHV-6), a macavirus ubiquitous in healthy cattle populations. The BoHV-6 genome exhibited architecture conserved in macaviruses, including a repetitive H-DNA region and unique 141 kbp L-DNA region predicted to encode 77 genes. BoHV-6 encoded, in variable genomic regions, a novel complement of genes relative to other characterized macaviruses, probably contributing to distinctive aspects of BoHV-6 infection biology and host range. Most notably, BoHV-6 encoded the first herpesviral protein (Bov2.b2) similar to cellular ornithine decarboxylase, an enzyme that catalyses the first and rate-limiting step in the biosynthesis of polyamines. Bov2.b2 conceivably mediates a novel mechanism by which BoHV-6 promotes cell-cycle-dependent viral replication.
White spot syndrome virus (WSSV) is a large enveloped virus which has caused severe mortality and huge economic losses in the shrimp farming industry. The enveloped virus must be combined with the receptors of the host cell membrane by the virus envelope proteins. In the case of WSSV, binding of envelope proteins with receptors of the host cell membrane was discovered in a number of previous studies, such as VP53A and 10 other proteins with chitin-binding protein (CBP), VP28 with Penaeus monodon Rab7, VP187 with bbeta;-integrin, and so on. WSSV envelope proteins were also considered capable of forming a protein complex dubbed an llsquo;infectomerrsquo;. In this study, the research was focused on the role of CBP in the WSSV infection process, and the relationship between CBP and the envelope proteins VP24, VP28, VP31, VP32 VP39B, VP53A and VP56. The results of the reverse transcription-PCR analyses showed that CBP existed in a variety of shrimp. The speed of WSSV infection could be slowed down by inhibiting CBP gene expression. Far-Western blot analysis and His pull-down assays were conducted, and a protein complex was found that appeared to be composed of a llsquo;linkerrrsquo; protein consisting of VP31, VP32 and VP39B together with four envelope proteins, including VP24, VP28, VP53A and VP56. This protein complex was possibly another part of the infectome and the possible binding region with CBP. The findings of this study may have identified certain points for further WSSV research.
HIV-1 mediates pro-survival signals and prevents apoptosis via the phosphatidylinositol-3-kinase (PI3K) pathway. This pathway, however, also affects phosphorylation of serine-arginine (SR) proteins, a family of splicing regulatory factors balancing splice site selection. We now show that pharmacologic inhibition of PI3K signalling alters the HIV-1 splicing pattern of both minigene- and provirus-derived mRNAs. This indicates that HIV-1 might also promote PI3K signalling to balance processing of its transcripts by regulating phosphorylation of splicing regulatory proteins.
The aim of this study was to generate maraviroc (MVC)-resistant viruses in vitro using a human immunodeficiency virus type 1 subtype B clinical isolate (HIV-1KP-5) to understand the mechanism(s) of resistance to MVC. To select HIV-1 variants resistant to MVC in vitro, we exposed high-chemokine (C-C motif) receptor 5 (CCR5)-expressing PM1/CCR5 cells to HIV-1KP-5 followed by serial passage in the presence of MVC. We also passaged HIV-1KP-5 in PM1 cells, which were low CCR5 expressing to determine low-CCR5-adapted substitutions and compared the Env sequences of the MVC-selected variants. Following 48 passages with MVC (10 mmicro;M), HIV-1KP-5 acquired a resistant phenotype [maximal per cent inhibition (MPI) 24 %], whilst the low-CCR5-adapted variant had low sensitivity to MVC (IC50 ~200 nM), but not reduction of the MPI. The common substitutions observed in both the MVC-selected and low-CCR5-adapted variants were selected from the quasi-species, in V1, V3 and V5. After 14 passages, the MVC-selected variants harboured substitutions around the CCR5 N-terminal-binding site and V3 (V200I, T297I, K305R and M434I). The low-CCR5-adapted infectious clone became sensitive to anti-CD4bs and CD4i mAbs, but not to anti-V3 mAb and autologous plasma IgGs. Conversely, the MVC-selected clone became highly sensitive to the anti-envelope (Env) mAbs tested and the autologous plasma IgGs. These findings suggest that the four MVC-resistant mutations required for entry using MVC-bound CCR5 result in a conformational change of Env that is associated with a phenotype sensitive to anti-Env neutralizing antibodies.
Recently, we immunized different mammalian species (goats, mice, rats, rabbits, guinea pigs and hamsters) with the recombinant ectodomain of the transmembrane envelope (TM) protein p15E of porcine endogenous retrovirus (PERV). In all cases, neutralizing immune sera were induced, which recognized epitopes in the fusion peptide proximal region and the membrane proximal external region of p15E. In order to analyse whether pigs are also able to produce such antibodies, and whether such antibodies can be used to study the involvement of the TM protein in placental development (as was shown for endogenous retroviruses of other species), German landrace pigs were immunized with PERV p15E. No binding and neutralizing antibodies were produced as shown in three Western blot analyses and in a neutralization assay, indicating that pigs are tolerant to their endogenous retroviruses, at least for the ectodomain of the TM protein.
Bovine leukemia virus (BLV) induces abnormal B-cell proliferation and B-cell lymphoma in cattle, where the BLV provirus is integrated into the host genome. BLV-infected B-cells rarely express viral proteins in vivo, but short-term cultivation augments BLV expression in some, but not all, BLV-infected B-cells. This observation suggests that two subsets, i.e. BLV-silencing cells and BLV-expressing cells, are present among BLV-infected B-cells, although the mechanisms of viral expression have not been determined. In this study, we examined B-cell markers and viral antigen expression in B-cells from BLV-infected cattle to identify markers that may discriminate BLV-expressing cells from BLV-silencing cells. The proportions of IgMhigh B-cells were increased in blood lymphocytes from BLV-infected cattle. IgMhigh B-cells mainly expressed BLV antigens, whereas IgMlow B-cells did not, although the provirus load was equivalent in both subsets. Several parameters were investigated in these two subsets to characterize their cellular behaviour. Real-time PCR and microarray analyses detected higher expression levels of some proto-oncogenes (e.g. Maf, Jun and Fos) in IgMlow B-cells than those in IgMhigh B-cells. Moreover, lymphoma cells obtained from the lymph nodes of 14 BLV-infected cattle contained IgMlow or IgMnndash; B-cells but no IgMhigh B-cells. To our knowledge, this is the first study to demonstrate that IgMhigh B-cells mainly comprise BLV-expressing cells, whereas IgMlow B-cells comprise a high proportion of BLV-silencing B-cells in BLV-infected cattle.
Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the nasal mucosa of sheep and goats and is associated with enzootic nasal tumour virus (ENTV). As ENA is a common disease in North America and there are no vaccines against ENTV-1, diagnostic tests that can identify infected animals and assist with eradication and disease surveillance efforts are greatly needed. In this study, we endeavoured to develop a novel, non-invasive diagnostic tool that could be used not only to validate clinical signs of ENA but also to detect ENTV-1 infection prior to the onset of disease signs (i.e. pre-clinical diagnosis). Cytology, serology and reverse transcription (RT)-PCR-based diagnostic methods were investigated. Although the cytology-based assay was able to detect ENTV-1 infection in some animals, it had poor sensitivity and specificity and thus was not developed further as an ante-mortem diagnostic method. Three different assays, including ELISA, Western blotting and virus neutralization, were developed to detect the presence of ENTV-1-specific antibodies in sheep serum. Whilst a surprisingly large number of sheep mounted an antibody-mediated immune response against ENTV-1, and in some cases neutralizing, correlation with disease status was poor. In contrast, RT-PCR on RNA extracted from nasal swabs reliably detected exogenous ENTV-1 sequences, did not amplify endogenous ovine betaretroviral sequences, demonstrated high concordance with immunohistochemical staining for ENTV-1 envelope protein, and had perfect sensitivity and specificity. This report describes a practical and highly specific RT-PCR technique for the detection of clinical and pre-clinical ENA that may prove beneficial in future control or eradication programmes.
Bovine spongiform encephalopathy (BSE) in cattle and variant Creutzfeldtnndash;Jakob disease in humans have previously been shown to be caused by the same strain of transmissible spongiform encephalopathy agent. It is hypothesized that the agent spread to humans following consumption of food products prepared from infected cattle. Despite evidence supporting zoonotic transmission, mouse models expressing human prion protein (HuTg) have consistently shown poor transmission rates when inoculated with cattle BSE. Higher rates of transmission have however been observed when these mice are exposed to BSE that has been experimentally transmitted through sheep or goats, indicating that humans may potentially be more susceptible to BSE from small ruminants. Here we demonstrate that increased transmissibility of small ruminant BSE to HuTg mice was not due to replication of higher levels of infectivity in sheep brain tissue, and is instead due to other specific changes in the infectious agent.
|Journal of General Virology Publish Ahead of Print|
We describe here the metagenomics-derived feline enteric virome in the feces of 25 cats from a single shelter in California. More than 90% of the recognizable viral reads were related to mammalian viruses and the rest to bacterial viruses. Eight viral families were detected: Astroviridae, Coronaviridae, Parvoviridae, Circoviridae, Herpesviridae, Anelloviridae, Caliciviridae, and Picobirnaviridae. Six previously known viruses were also identified: feline coronavirus type 1, felid herpes 1, feline calicivirus, feline norovirus, feline panleukopenia virus and picobirnavirus. Novel species of astroviruses and bocaviruses and the first genome of a cyclovirus in a feline were characterized. The RNA dependent RNA polymerase region from four highly divergent partial viral genomes in the order Picornavirales were sequenced. The detection of such a diverse collection of viruses shed within a single shelter suggests that such animals experience robust viral exposures. This study also increases our understanding of the viral diversity in cats facilitating future evaluation of their pathogenic and zoonotic potentials.
Monoclonal antibodies (MAbs) constitute an important biological tool for influenza hemagglutinin (HA) epitope mapping through generation of escape mutants. The latter could provide insights into immune evasion mechanisms and such knowledge may benefit the future development of vaccines. Several influenza A(H1N1) pandemic 2009 (pdm09) HA escape mutants have been recently described. However, the HA antigenic sites of the previous seasonal A/Brisbane/59/2007 (H1N1) (Bris07) virus remain poorly documented. Herein, we produced MAbs against pdm09 and Bris07 HA proteins expressed in human HEK293 cells. Escape mutants were generated using MAbs that exhibited HA inhibition and neutralizing activities. The resulting epitope mapping of the pdm09 HA protein revealed 11 escape mutations including 3 that were previously described (G172E, N173D and K256E) and 8 novel ones (T89R, F128L, G157E, K180E, A212E, R269K, N311T, G478E). Among the 6 HA mutations that were part of predicted antigenic sites (Ca1, Ca2, Cb, Sa or Sb), 3 (G172E, N173D and K180E) were within the Sa site. Eight escape mutations (H54N, N55D, N55K, L60H, N203D, A231T, V314I, K464E) were obtained for Bris07 HA, and all but one (N203D, Sb site) were outside the predicted antigenic sites. Our results suggest that the Sa antigenic site is immunodominant in pdm09 HA, whereas the N203D mutation (Sb site), present in 3 different Bris07 escape mutants, appear as an immunodominant epitope in that strain. The fact that some mutations were not part of predicted antigenic sites reinforces the necessity of further characterizing the HA of additional H1N1 strains.
We isolated and characterized a novel virulent bacteriophage IME-EFm1 specifically infecting multiple-drug resistant Enterococcus faecium. IME-EFm1 is morphologically similar to the family Siphoviridae. It was capable of lysing a wide range of our E. faecium collections, including two strains resistant to vancomycin. One-step growth tests revealed the host lysis activity of phage IME-EFm1, with a latent time of 30 min and a large burst size of 116 plaque-forming units (PFU)/cell. These biological characteristics suggested that IME-EFm1 hold the potential to be used as a therapeutic agent. The complete genome of IME-EFm1 is a 42597bp in length, linear, terminally non-redundant double-stranded DNA, with a G+C content of 35.2%. The termini of the phage genome were determined with next generation sequencing data and were further confirmed by nuclease digestion analysis. To our knowledge, this is the first report of a complete genome sequence of a bacteriophage infecting E. faecium. IME-EFm1 exhibited low similarity with other phages in terms of genome organization and structural protein amino acid sequences. The coding region corresponds to 90.7% of the genome. 70 putative open reading frames were deduced, and of these, 28 could be functionally identified based on their homology to previously characterized proteins. A predicted metallo-beta-lactamase gene was detected in the genome sequence. The identification of antibiotic resistance gene emphasizes the necessity of complete genome sequencing of a phage to ensure it free of any undesirable genes.
The avian-like swine influenza viruses emerged in 1979 in Belgium and Germany. Thereafter, they spread through many European swine-producing countries, replaced the circulating classical swine H1N1 influenza viruses and became endemic. Serological and subsequent molecular data indicated an avian source, but details remained obscure due to a lack of relevant avian influenza virus sequence data. Here, the origin of the European avian-like swine influenza viruses was analyzed using a collection of sixteen European swine H1N1 influenza viruses sampled in 1979-1981 in Germany, the Netherlands, Belgium, Italy and France as well as several contemporaneous avian influenza viruses of various serotypes. The phylogenetic trees suggest a triple reassortant with a unique genotype constellation. Time-resolved maximum clade credibility trees indicate times to the most recent common ancestors of 34-46 years (before 2008) depending on the RNA segment and the method of tree inference.
The order Nidovirales contains large, enveloped viruses with a non-segmented positive-stranded RNA genome. Nidoviruses were detected in man and various animal species, but not in reptiles yet. In the present study, we describe the detection, characterization, phylogenetic analyses and disease association of a novel divergent nidovirus in the lung of an Indian python (Python molurus) with necrotizing pneumonia. Characterization of the partial genome (ggt;33,000nt) of this virus revealed several genetic features that are distinct from other nidoviruses, including a very large polyprotein 1a, a putative ribosomal frameshift signal that was identical to the frameshift signal of astroviruses and retroviruses and an accessory ORF that showed some similarity with the hemagglutinin-neuraminidase of paramyxoviruses. Analysis of the genome organization and phylogenetic analysis of polyprotein 1ab suggests that this virus belongs to the subfamily Torovirinae. Results of this study provide novel insights into the genetic diversity of the Nidovirales.nnbsp;
Bone marrow stromal antigen 2 (BST-2) also known as Tetherin) is an interferon-inducible gene that functions to block the release of a range of nascent enveloped virions from infected host cells. However, the role of BST-2 in viral pathogenesis remains poorly understood. BST-2 plays a multifaceted role in innate immunity-as it hinders retroviral infection and possibly promotes infection with some rhabdo and orthomyxo viruses. This paradoxical role has probably hindered exploration of BST-2 antiviral function in vivo. We previously reported that BST-2 tethers Chikungunya virus-like particle on the cell plasma membrane. To explore the role of BST-2 in Chikungunya virus (CHIKV) replication and host protection, we utilized Chikungunya virus strain 181/25 to examine early events during CHIKV infection in BST-2-/- mouse model. We observed interesting dichotomy between wild-type (WT) and BST-2-/- mice. BST-2 deficiency increased inoculation site viral load, culminating in higher systemic viremia and increased lymphoid tissues tropism. Suppressed inflammatory innate response demonstrated by impaired IFNaalpha;, IFN, and CD40 ligand (CD40L) expression was observed in BST-2-/- mice compared to the WT controls. These findings suggest that, in part, BST-2 protects lymphoid tissues from CHIKV infection and regulates CHIKV-induced inflammatory response by the host.
Reverse genetics is a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but methods based on the preparation of plasmid-based complete viral genomes are laborious and unpredictable. Here, both wild-type and genetically modified infectious RNA viruses were generated in days using the newly described ISA (Infectious-Subgenomic-Amplicons) method. This new versatile and simple procedure may enhance our capacity to obtain infectious RNA viruses from PCR-amplified genetic material.
The human herpesviruses (HHVs) are remarkably successful human pathogens, with some members of the family successfully establishing infection in the vast majority of humans in the world. Although many HHV infections result in asymptomatic infection or mild disease, there are rare cases of severe disease and death found with nearly every HHV. Many of the pathogenic mechanisms of these viruses are poorly understood, and in many cases effective anti-viral drugs are lacking. Only a single vaccine exists for the HHVs, and researchers have been unable to develop treatments to cure the persistent infections associated with HHVs. A major hindrance to HHV research has been the lack of suitable animal models, with the notable exception of the herpes simplex viruses. One promising area for HHV research is the use of humanized mouse models, in which human cells or tissues are transplanted into immunodeficient mice. Current humanized mouse models mostly transplant human hematopoietic stem cells (HSCs), resulting in the production of a variety of human immune cells. Although all HHVs are thought to infect human immune cells, the beta- and gammaherpesviruses extensively infect and establish latency in these cells. Thus, mice humanized with HSCs hold great promise to study these herpesviruses. In this review, we provide historical perspective on the use of both older and newer humanized mouse models to study HHV infections. The focus is on current developments in using humanized mice to study mechanisms of HHV-induced pathogenesis, human immune responses to HHVs, and effectiveness of anti-viral drugs.
Whole virus (WV) vaccines from influenza A/duck/Hokkaido/77 (H3N2) and its reassortant strains H3N4, H3N5, H3N7, which have the same hemagglutinin (HA) gene but different neuraminidase (NA) genes, were prepared from our influenza virus library. Mice were intranasally immunized with equivalent concentrations of each vaccine (1 to 0.01 mmicro;g/mouse). All of the mice that received the highest concentration of each vaccine (1 mmicro;g/mouse) showed equivalent high hemagglutinin inhibition (HI) antibody titers of over 1:40, and survived the H3N2 challenge viruses. However, mice that received lower concentrations of vaccine (0.1 or 0.01 mmicro;g/mouse) containing a heterologous NA had lower survival rates than those given the H3N2-based vaccine. The lungs of mice challenged with H3N2 virus showed a significantly higher virus clearance rate when the vaccine contained the homologous NA (N2) versus a heterologous NA, suggesting that NA contributes to the protection, especially when the HI antibody level is low. These results suggest that, even if vaccines prepared for a possible upcoming pandemic do not induce sufficient HI antibodies, WV vaccines can still be effective through other matched proteins such as NA.
Direct acting antivirals have significantly improved treatment outcomes in chronic hepatitis C (CHC), but side effects, drug resistance and cost mean that better treatments are still needed. Lipid metabolism is closely linked with hepatitis C virus (HCV) replication and endocannabinoids are major regulators of lipid homeostasis. The cannabinoid 1 (CB1) receptor mediates these effects in the liver. We have previously shown up-regulation of CB1 receptors in the livers of patients with CHC, and in a HCV cell culture model. Here we investigated whether CB1 blockade inhibits HCV replication. The antiviral effect of a CB1 antagonist, AM251 was examined in the JFH1 cell culture and subgenomic replicon models. The effects on the expression of genes involved in lipid metabolism were also measured. CB1 shRNA was used to confirm that the effects were specific for the cannabinoid receptor. Treatment with AM251 strongly inhibited HCV RNA (~70%), viral protein (~80%), the production of new virus particles (~70%), and virus infectivity (~90%). As expected, AM251 reduced the expression of pro-lipogenic genes (SREBP-1c, FASN, SCD1 and ACC1) and stimulated genes promoting lipid oxidation (CPT1 and PPARaalpha;). This effect was mediated by AMPK. Stable CB1 knockdown of cells infected with HCV showed reduced levels of HCV RNA, compared with controls. Reduced CB1 signalling inhibits HCV replication using either pharmacological inhibitors or CB1 shRNA. This may be due, at least in part, to reduced lipogenesis, mediated by AMPK activation. We suggest that CB1 antagonists may represent an entirely new class of drugs with activity against HCV.
In 2012, a mutant porcine circovirus type 2 (mPCV2) strain was identified in cases of porcine circovirus associated disease (PCVAD) in the United States. The mPCV2 has an additional amino acid, Lysine (K), in the capsid at position 234. The objectives of this study were to compare the pathogenicity of mPCV2, PCV2a and PCV2b in pigs using biologically pure infectious virus stocks derived from respective infectious DNA clones and to investigate the importance of genotype-specific open reading frames (ORF) 2 and presence of K at position 234 of the capsid. A total of 47, two-week-old, caesarian-derived, colostrum-deprived (CDCD) pigs were assigned to one of 7 groups. At 3 weeks of age, the pigs were experimentally inoculated with saline, PCV2a, PCV2b, mPCV2, PCV2b-234-K (Lysine addition in ORF2), chimeric PCV2b-ORF1/mPCV2-ORF2, or reciprocal chimeric mPCV2-ORF1/PCV2b-ORF2. All pigs were necropsied 21 days post infection (dpi). Gross lesions were limited to visible icterus and loss of body condition in a portion of the mPCV2 pigs. The amount of PCV2 DNA was significantly higher in pigs inoculated with mPCV2 compared to PCV2b in sera at 7 dpi and fecal swabs at 14 dpi. Based on lymphoid lesions, a higher prevalence of PCVAD was seen in pigs infected with PCV2's containing the additional 234-K (64.3%) compared to those infected with a PCV2 with the regular 233-bp ORF2 (40%). Results indicate that all PCV2 isolates were capable of inducing severe lesions and disease in the CDCD pig model, and there was no significant difference in virulence.
Astroviruses infect human and many animal species and cause gastroenteritis. To extensively understand the distribution and genetic diversity of astrovirus in small mammals, we tested 968 anal swabs from 39 animal species, most of which were bats and rodents. We detected diverse astroviruses in 10 bat species, including known bat astroviruses and a large number of novel viruses. Meanwhile, novel groups of astroviruses were identified in three wild rodent species and a remarkably high genetic diversity of astrovirus was revealed in Eothenomys cachinus. We detected astroviruses in captive bred porcupines and nearly full-length genome sequence was determined for one strain. Phylogenetic analysis of complete ORF2 sequence suggested that it may share a common ancestor with porcine astrovirus type 2. Moreover, to our knowledge, this study reports the first discovery of astrovirus in shrews and pikas. Our results provide new insights for understanding these small mammals as natural reservoirs of astroviruses.
The objective of this study was to compare the virulence and pathogenicity of a combination of concurrent infections of two genotypes of porcine circovirus type 2 (PCV2) and two genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) in terms of PCV2 viremia, and PCV2-associated lesions and antigens in co-infected pigs. Co-infection induced significantly lower levels of anti-PCV2 and anti-PRRSV IgG antibodies than infection with one genotype alone, regardless of the genotype of the two viruses. Pigs with PCV2a (or 2b)/type 1 PRRSV had significantly (P llt; 0.05) higher levels of PCV2 viremia, more severe PCV2-associated lesions, and more PCV2 DNA within the lesions compared to pigs with PCV2a (or 2b)/type 1 PRRSV. However, there was no significant difference in these parameters in pigs with PCV2a/type 2 PRRSV or PCV2b/type 2 PRRSV. The results of this study demonstrate significant differences in the virulence and pathogenicity of type 1 and type 2 PRRSV but no significant differences in the virulence and pathogenicity of PCV2a and PCV2b with respect to the production of PCV2-associated lesions.
Begomoviruses are whitefly-transmitted, single-stranded DNA plant viruses and are among the most damaging pathogens causing epidemics in economically important crops worldwide. Wild/non-cultivated plants play a crucial epidemiological role, acting as begomovirus reservoirs and as 'mixing vessels' where recombination can occur. Previous work suggests a higher degree of genetic variability in begomovirus populations from non-cultivated hosts compared to cultivated hosts. To assess this supposed host effect on the genetic variability of begomovirus populations, cultivated (common bean, Phaseolus vulgaris, and lima bean, P. lunatus) and non-cultivated (Macroptilium lathyroides) legume hosts were intensively sampled from two regions across Brazil. A total of 212 full-length DNA-A genome segments were sequenced from samples collected between 2005 and 2012, and populations of the begomoviruses Bean golden mosaic virus (BGMV) and Macroptilium yellow spot virus (MaYSV) were obtained. We found, for each begomovirus species, similar genetic variation between populations infecting cultivated and non-cultivated hosts, indicating that the presumed genetic variability of the host did not a priori affect viral variability. The MaYSV population (N = 99) was more variable than the BGMV population (N = 147), which was explained by numerous recombination events in MaYSV. MaYSV and BGMV showed distinct distributions of genetic variation, with the BGMV population (but not MaYSV) being structured by both host and geography.
A high prevalence of the rtI187V polymerase substitution of hepatitis B virus (HBV) was detected in nucleos(t)ide analogs-naiiuml;ve and -treated chronic hepatitis B (CHB) patients. We aimed at assessing the replicative capacity and susceptibility of rtI187V alone or in conjunction with lamivudine (LAM) or adefovir (ADV) -resistant mutations to LAM and ADV in vitro. The reverse transcriptase region of HBV isolates was directly sequenced from a cohort of 300 CHB patients from China. Replication-competent HBV constructs containing rtI187V and combined with LAM-resistant (rtM204I, rtL180M/rtM204V) mutations were generated, and compared with wild-type (WT), LAM-resistant single (rtM204I) or double (rtL180M/rtM204V) and ADV-resistant (rtN236T) clones. In a Chinese cohort of 300 CHB patients, 8.7% (26/300) showed substitution in the rtI187 with V. Of note, the rtI187V prevalence in genotype B was significant higher than in genotype C (95.2 vs. 4.8%). In vitro phenotypic assays showed that the viruses bearing the rtI187V had impaired replication efficacy when compared to WT and the virus carrying the rtI187V combined with LAM-resistant single or double mutations showed even more significantly impaired replicative capacities. Furthermore, rtI187V remained susceptible towards treatment with LAM or ADV in vitro whereas the combination of rtI187V substitution with LAM-resistant mutations was resistant to LAM but still sensitive to ADV. Our study reveals that the rtI187V substitution in the HBV polymerase frequently occurred in CHB patients particular with genotype B. However, the emergence of rtI187V substitution significantly impair viral replication but without affecting drug sensitivity in vitro.
Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1 based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated sub-genomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted chimaeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full length and bipartite systems may provide further insight into this process.
Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrPSc) in the central nervous system. The pathological features and biochemical properties of PrPSc in macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt-Jakob disease (vCJD). Nonhuman primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method for in vitro amplification of cynomolgus macaque BSE PrPSc. This method involves amplifying PrPSc by protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrPC substrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSc contained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrPSc in the CSF by serial PMCA, and the CSF levels of PrPSc tended to increase with disease progression. In addition, PrPSc was detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrPSc in nonhuman primate models of CJD.
Hantaviruses are zoonotic viruses that cause life-threatening diseases when transmitted to man. Severe hantavirus infection is manifested by impairment of renal function, pulmonary edema, and capillary leakage. Both innate and adaptive immune responses contribute to the pathogenesis but the underlying mechanisms are not fully understood. Here we describe that galectin-3 binding protein (Gal-3BP) is upregulated as a result of hantavirus infection both in vitro and in vivo. Gal-3BP is a secreted glycoprotein found in human serum and increased Gal-3BP levels have been reported in chronic viral infections and in several types of cancer. Our in vitro experiments show that while Vero E6 cells (African green monkey kidney cell line) constitutively express and secrete Gal-3BP, this protein was detected in primary human cells only as a result of hantavirus infection. Analysis of Gal-3BP levels in serum samples of cynomolgus macaques experimentally infected with hantavirus indicated that hantavirus infection induces Gal-3BP also in vivo. Finally, the analysis of plasma samples collected from patients hospitalized because of acute hantavirus infection showed higher Gal-3BP levels during the acute than convalescent phase. Furthermore, the Gal-3BP levels in HFRS patients correlated with increased complement activation and with clinical variables reflecting the severity of acute hantavirus infection.
JS11C1, a member of putative new subgroup of avian leukosis virus different from all six known subgroups from chickens based on Gp85 amino acid sequence comparison, was isolated from Chinese native chicken breeds in 2012. In order to further study the genome structure, biological characteristics, and the evolutionary relationship of the virus with others of known subgroups infected chickens, we determined the complete genome sequence, constructed an infectious clone of ALV strain JS11C1, and performed comparison analysis using the whole genome sequence or elements with that of other ALSVs all available in GenBank. The results showed that the full-length sequence of the JS11C1 DNA provirus genome was 7707 bp, which is consistent with a genetic organization typical of a replication-competent type C retrovirus lacking viral oncogenes. The rescued infectious clone of JS11C1 showed similar growth rate and biological characteristics to its original virus. All the comparison analysis based on whole genomes support the opinion that the new isolates are relatively far related to any known subgroups of ALVs and might be classified as a new subgroup.
The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for the systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with an enhanced green fluorescent protein (EGFP) fused to the nucleocapsid to monitor the entry of virus into primary midgut epithelium cells ex vivo by confocal fluorescence microscopy. The EGFP-labeled virus showed the same BV virulence and ODV infectivity as wild type virus. The ability to bind and enter host cells was then visualized for wild type AcMNPVs and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, while PIF1 and PIF2 play important roles in entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.
Tick-borne encephalitis (TBE), a disease caused by tick-borne encephalitis virus (TBEV), represents the most important flaviviral neural infection in Europe and northeastern Asia. In the central nervous system (CNS), neurons are the primary target for TBEV infection; however, infection of non-neuronal CNS cells, such as astrocytes, is not well understood. In this study, we investigated the interaction between TBEV and primary human astrocytes. We report for the first time that primary human astrocytes are sensitive to TBEV infection, although the infection does not affect their viability. The infection induces a marked increase in the expression of glial fibrillary acidic protein, a marker of astrocyte activation. In addition, expression of matrix metalloproteinase 9 and several key proinflammatory cytokines/chemokines (e.g., tumor necrosis factor aalpha;, interferon aalpha;, interleukin (IL)-1bbeta;, IL-6, IL-8, interferon -induced protein 10, macrophage inflammatory protein, but not monocyte chemotactic protein 1) is upregulated. Moreover, we present a detailed description of morphological changes in TBEV-infected cells, as investigated using three-dimensional electron tomography. Several novel ultrastructural changes were observed, including the formation of unique tubule-like structures of 17.9 nm (pplusmn; 0.15 nm) diameter with associated viral particles and/or virus-induced vesicles and located in the rough endoplasmic reticulum of the TBEV-infected cells. This is the first demonstration that TBEV infection activates primary human astrocytes. The infected astrocytes might be a potential source of proinflammatory cytokines in the TBEV-infected brain, and might contribute to the TBEV-induced neurotoxicity and blood-brain barrier breakdown that occurs during TBE. The neuropathological significance of our observations is also discussed.
Laboratory animal models have provided valuable insight into foot-and-mouth disease virus (FMDV) pathogenesis in epidemiologically important target species. While not perfect, these models have delivered an accelerated time frame to characterize the immune responses in natural hosts and a platform to evaluate therapeutics and vaccine candidates at a reduced cost. Further expansion of these models in mice has allowed access to genetic mutations not available for target species, providing a powerful and versatile experimental system to interrogate the immune response to FMDV and to target more expensive studies in natural hosts. The purpose of this review is to describe commonly used FMDV infection models in laboratory animals and to cite examples when these models have failed or successfully provided insight relevant for target species, with an emphasis on natural and vaccine induced immunity.
The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the virus encoded 3C protease to VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210E) within the VP1 protein, close to the VP1/2A cleavage site, inhibited cleavage of this junction and produced "self-tagged" virus particles. A second site substitution (E83K) within VP1 was also observed within the rescued virus (Gullberg et al. (2013) J Virol 87, 11591nndash;11603). It is now shown that introduction of this E83K change alone, into a serotype O virus, resulted in the rapid accumulation of a second site substitution within the 2A sequence (L2P) which also blocked VP1/2A cleavage. This suggests a linkage between the E83K change in VP1 and cleavage of the VP1/2A junction. Cells infected with viruses containing the VP1 K210E or the 2A L2P substitutions contained the uncleaved VP1-2A protein; the 2A L2P substitution resulted in the VP1/2A junction being totally resistant to cleavage by the 3C protease and hence may be a preferred route for "tagging" virus particles.
Alternative methods to the standard hemagglutination inhibition (HI) and neutralization tests to probe the antigenic properties of the influenza virus hemagglutinin (HA) were developed in this study. Vaccinia virus recombinants expressing reference HAs were used to immunize rabbits from which polyclonal antibodies were obtained. These antibodies were subtype specific but showed limited intra-subtype strain specificity in ELISA. The discriminatory capacity of these antibodies was however markedly increased after adsorption to cells infected with heterologous influenza viruses, revealing antigenic differences that were otherwise undistinguishable by standard HI and neutralization tests. Furthermore, the unadsorbed antibodies could be used to select escape mutants of the reference strain which after sequencing unveiled amino acid changes responsible of the noted antigenic differences. These procedures therefore provide alternative methods for the antigenic characterization of influenza HA and might be useful in studies of HA antigenic evolution.
The family Hepeviridae consists of positive-stranded RNA viruses that infect a wide range of mammalian species, as well as chickens and trout. A subset of these viruses infects humans and can cause a self-limiting acute hepatitis that may become chronic in immunosuppressed individuals. Current published descriptions of the taxonomical divisions within the family Hepeviridae are contradictory in relation to the assignment of species and genotypes. Through analysis of existing sequence information, we propose a taxonomic scheme in which the family is divided into the genera Orthohepevirus (all mammalian and avian hepatitis E virus (HEV) isolates) and Piscihepevirus (cutthroat trout virus). Species within the genus Orthohepevirus are designated Orthohepevirus A (isolates from human, pig, wild boar, deer, mongoose, rabbit and camel), Orthohepevirus B (isolates from chicken), Orthohepevirus C (isolates from rat, greater bandicoot, Asian musk shrew, ferret and mink) and Orthohepevirus D (isolates from bat). Proposals are also made for the designation of genotypes within the human and rat HEVs. This hierarchical system is congruent with hepevirus phylogeny, and the three classification levels (genus, species and genotype) are consistent with, and reflect discontinuities in the ranges of pairwise distances between amino acid sequences. Adoption of this system would include the avoidance of host names in taxonomic identifiers and provide a logical framework for the assignment of novel variants.
Influenza B viruses have become increasingly more prominent during influenza seasons. Influenza B species is typically considered a mild disease that receives less attention than influenza A species, but has been causing 20% to 50% of the total influenza incidence in several regions around the world. Although there is increasing evidence of mid to lower respiratory tract diseases such as bronchitis and pneumonia in influenza B patients, little is known about the pathogenesis of recent influenza B viruses. Here we investigated the clinical and pathological profiles of infection with strains representing the two current co-circulating B lineages (B/Yamagata and B/Victoria) in the ferret model. Specifically, we studied two B/Victoria (B/Brisbane/60/2008 and B/Bolivia/1526/2010) and two B/Yamagata (B/Florida/04/2006 and B/Wisconsin/01/2010) strain infections in ferrets and observed strain-specific but not lineage-specific pathogenicity. We found B/Brisbane/60/2008 caused the most severe clinical illness and B/Brisbane/60/2008 and the B/Yamagata strains instigated pathology in the middle to lower respiratory tract. Importantly, B/Brisbane/60/2008 established efficient lower respiratory tract infection with high viral burden. Phylogenetic analyses demonstrated profound reassortment among recent influenza B viruses which indicated the genetic make-up of B/Brisbane/60/2008 differed from the other strains and resembled the 2002-2003 influenza B global outbreak strain, B/Brisbane/32/2002. This may explain the pathogenicity difference post infection in ferrets. Our study characterized influenza B infections in ferrets and this model may aid in the future development of effective influenza therapeutic treatment.
The cell line IPLB-LD-652Y derived from the gypsy moth (Lymantria dispar, Linn.) is routinely used to study insect virus-host interactions. Here we report the full genome sequence and biological characteristics of a small RNA virus, designated Lymantria dispar iflavirus 1 (LdIV1), that was discovered persistently infecting this cell line. LdIV1 belongs to the genus Iflavirus. LdIV1 has icosahedral particles of approximately 30 nm in diameter and a 10,044 nucleotide polyadenylated, positive sense RNA genome encoding a predicted polyprotein of 2,980 amino acids. LdIV1 was induced by a viral suppressor of RNA silencing suggesting that acute infection is restricted by RNAi. We detected LdIV1 in all tested tissues of gypsy moth larvae and adults but the virus was absent from other L. dispar nndash; derived cell lines. We confirmed LdIV1 infectivity in two of these cell lines (IPLB-LD-652 and IPLB-LdFB). Our results provide a novel system to explore persistent infections in lepidopterans and a new model for the study of iflaviruses, a rapidly expanding group; many of which covertly infect their hosts.
Human Cytomegalovirus (HCMV) infection and reactivation is a major cause of morbidity in immuno-suppressed transplant patients and late stage AIDS sufferers. Interestingly, epidemiological studies have shown that the immuno-suppressive regimen used can have differential prognoses for patients regarding HCMV infection and disease. Specifically, patients administered Sirolimus (rapamycin) exhibit more favourable outcomes, suggesting that rapamycin has activity against HCMV in vivo. Given the relative lack of activity against lytic infection it has been postulated rapamycin may inhibit HCMV reactivation. Here we show that rapamycin given acutely or chronically has little impact on the induction of HCMV reactivation in experimentally latent dendritic cells or cells from naturally latent individuals. Furthermore, we extend these observations to include other inhibitors of mTORC1 and mTORC 2 which similarly have minimal effect on the induction of IE gene expression from latency. Taken together, these data suggest that favourable outcomes associated with sirolimus may be due to indirect effects that influence HCMV reactivation rather than a direct mechanistic action against HCMV itself.
The Orbivirus genus of the family Reoviridae is comprised of 22 virus species including the Changuinola virus (CGLV) serogroup. The complete genome sequences of 13 CGLV serotypes isolated between 1961 and 1988 from distinct geographic areas of the Brazilian Amazon region were obtained. All viral sequences were obtained from single-passaged CGLV strains grown in Vero cells. CGLVs are the only orbiviruses known to be transmitted by phlebotomine sandflies. Ultrastructure and molecular analyses by electron microscopy and gel electrophoresis, respectively revealed viral particles with typical orbivirus size and morphology, as well as the presence of a segmented genome with 10 segments. Full-length nucleotide sequencing of each of the ten RNA segments of the 13 CGLV serotypes provided basic information regarding the genome organization, encoded proteins and genetic traits. Segment 2 (encoding VP2) of the CGLV is uncommonly larger in comparison to those found in other orbiviruses and shows varying sizes even among different CGLV serotypes. Phylogenetic analyses support previous serologic findings, which indicated that CGLV constitutes a separate serogroup within the genus Orbivirus. In addition, 6 out of 13 analyzed CGLV serotypes show reassortment of their genome segments.
The mechanism used by Bluetongue Virus (BTV) to ensure the sorting and packaging of its 10 genomic segments is still poorly understood. In this study, we have investigated the packaging constraints for two BTV genomic segments from two different serotypes. Segment 4 (S4) of BTV serotype 9 was sequentially mutated; and packaging of mutant ssRNAs was investigated by two newly developed RNA packaging assay systems, one in vivo and the other in vitro. Modelling of the mutated ssRNA suggested that a conformational motif formed by interaction of the 5rrsquo; and the 3rrsquo;ends of the molecule is necessary and sufficient for packaging. A similar structural signal was also identified in segment 8 (S8) of serotype 1. Furthermore, the same conformational analysis of secondary structures for positive-sense ssRNAs was used to generate a chimeric segment that maintained the putative packaging motif but contained unrelated internal sequences. This chimeric segment was successfully packaged, confirming that the motif identified directs the correct packaging of the segment.
Porcine hemagglutinating encephalomyelitis virus (PHEV) is the main causative agent of porcine coronavirus-associated disease, which is characterized by encephalomyelitis and involves the central nervous system. However, little is known about the molecular mechanisms of brain injury. To gain insight into the interaction between the virus and host cells, global gene expression changes in the cerebral cortex of PHEV- or mock-infected mice were investigated using DNA microarray analysis and quantitative real-time PCR. The results of the microarray analysis showed that 365 genes on day 3 post infection and 781 genes on day 5 post infection are differentially expressed in response to PHEV infection in the cerebral cortex. The up-regulated genes are mainly involved in immune system processes, antigen processing and presentation, the Jak-STAT signaling pathway, the RIG-I-like receptor signaling pathway, Toll-like receptor signaling and apoptosis-related proteases, etc. However, significantly down-regulated genes are mainly involved in nervous system development, synaptic transmission, neuron projection development, the transmission of nerve impulse, the negative regulation of glial cell differentiation, etc. The differential expression of these genes suggests a strong host antiviral response, but may also contribute to the pathogenesis of PHEV resulting in encephalomyelitis.
Bungowannah virus is the most divergent pestivirus, and both origin and reservoir host have not been identified so far. We therefore performed in vitro tropism studies, which showed that Bungowannah virus differs remarkably from other pestiviruses. Interestingly, cell lines of vervet monkey, mouse, human and even of bat origin were susceptible. This broad in vitro-tropism was not observed for a chimeric bovine viral diarrhea virus expressing all structural proteins of Bungowannah virus. The viral envelope was not sufficient to completely transfer the cell tropism of Bungowannah virus to another pestivirus, and viral RNA replication was markedly reduced or even not detectable in a number of different cell lines for the tested BVDV strain and the chimera. We therefore suggest that the replication machinery together with the viral envelope is responsible for the unique broad cell tropism of Bungowannah virus.
Background: Chronic hepatitis C (HCV) results in progressive liver fibrosis leading to cirrhosis and liver cancer. The mechanism for this remains unclear but hepatocyte apoptosis is thought to play a major role. Methods: Hepatocyte apoptosis in human liver tissue was determined by immunohistochemistry for M30 cytoDEATH and cleaved PARP. In vitro studies were performed with replication-defective recombinant adenoviruses expressing HCV proteins (rAdHCV) to study the effects of HCV on cell death in Huh7 cells, and primary mouse (PMoH) and human hepatocytes (PHH). Cell viability and apoptosis were studied using crystal violet assays and Western immunoblots probed for cleaved caspase 3 and cleaved PARP, with and without treatment with Q-VD-Oph and necrostatin-1. Results: Liver tissue of HCV-infected patients expressed elevated levels of apoptotic markers compared to HCV-negative patients. rAdHCV infection reduced cell viability compared to uninfected controls and cells infected with control virus (rAdGFP). Huh7, PMoH and PHH infected with rAdHCV showed significantly increased levels of apoptotic markers compared to uninfected controls and rAdGFP-infected cells. In rAdHCV-infected Huh7, treatment with Q-VD-Oph and necrostatin-1 both improved cell viability. Q-VD-Oph also reduced cleaved PARP in rAdHCV-infected Huh7 and PMoH. Conclusions: Hepatocyte apoptosis is increased in the livers of HCV-infected patients. HCV promoted cell death in primary and immortalized hepatocytes and this was inhibited by Q-VD-Oph and necrostatin-1. These findings indicate that HCV-induced cell death occurs by both apoptosis and necroptosis and provide new insights into the mechanisms of HCV-induced liver injury.
This study examined the potential for cross-species transmission of influenza viruses by comparing the genetic and pathogenic characteristics of H1 avian influenza viruses (AIVs) with different host origins in Korea. Antigenic and phylogenetic analyses of H1 AIVs circulating in Korea provided evidence of genetic similarity between viruses that infect domestic ducks and those that infect wild birds, although there was no relationship between avian and swine viruses. However, there were some relationships between swine and human viral genes. The replication and pathogenicity of the H1 viruses was assessed in chickens, domestic ducks, and mice. Viral shedding in chickens was relatively high. Virus was recovered from both oropharyngeal and cloacal swabs up to 5-10 days post-inoculation (p.i.). The titers of domestic duck viruses in chickens were much higher than those of wild bird viruses. Both domestic duck and wild bird viruses replicated poorly in domestic ducks. None of the swine viruses replicated in chickens or domestic ducks; however, six viruses showed relatively high titers in mice, regardless of host origin, and induced clinical signs such as ruffled fur, squatting, and weight loss. Thus, although the phylogenetic and antigenic analyses showed no evidence of interspecies transmission between birds and swine, the results suggest that Korean H1 viruses have the potential to cause disease in mammals. Therefore, we should intensify continuous monitoring of avian H1 viruses in mammals and seek to prevent interspecies transmission.
Our previous studies indicated that hepatitis E virus (HEV) forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and requires the multivesicular body (MVB) pathway to release virus particles, and the released HEV particles with a lipid membrane retain the trans-Golgi network protein 2 on their surface. To examine whether HEV utilizes the exosomal pathway to release the virus particles, we analysed whether the virion release from PLC/PRF/5 cells infected with genotype 3 HEV (strain JE03-1760F) is affected by treatment with bafilomycin A1 or GW4869, or by the introduction of a small interfering RNA (siRNA) against Rab27A or Hrs. The extracellular HEV RNA titre was increased by treatment with bafilomycin A1, but was decreased by treatment with GW4869. The relative levels of virus particles released from cells depleted of Rab27A or Hrs were decreased to 16.1 % and 11.5%, respectively, of those transfected with negative control siRNA. Electron microscopic observations revealed the presence of membrane-associated virus-like particles with a diameter of approximately 50 nm within the MVB, which possessed internal vesicles in infected cells. Immunoelectron microscopy showed positive immunogold staining for the HEV ORF2 protein on the intraluminal vesicles within the MVB. Additionally, an immunofluorescence analysis indicated the triple co-localization of the ORF2, ORF3 and CD63 proteins in the cytoplasm, as specific loculated signals, supporting the presence of membrane-associated HEV particles within the MVB. These findings indicate that membrane-associated HEV particles are released together with internal vesicles through MVBs by the cellular exosomal pathway.
The flavivirus West Nile virus (WNV) is an emerging pathogenic arbovirus responsible for outbreaks of encephalitis around the world. Whilst no vaccines are currently available to prevent WNV infection of humans, the use of flavivirus cDNA genomes with large internal deletions within capsid (C) appears promising. C-deleted vaccines are able to replicate and secrete large amounts of non-infectious immunogenic subviral particles (SVPs) from transfected cells. We have previously generated a WNV DNA vaccine candidate pKUNdC/C where C-deleted WNV cDNA was placed under the control of one copy of the cytomegalovirus promoter (CMV) and C gene was placed under the control of a second copy of CMV promoter in the same plasmid DNA. This DNA was shown to generate single-round infectious particles (SRIPs) capable of delivering self-replicating C-deleted RNA producing SVPs to surrounding cells thus enhancing the vaccine potential. However, the amounts of both SRIPs and SVPs produced from pKUNdC/C DNA were relatively low. In this investigation we aimed at increasing SRIP production by optimizing trans-C expression via incorporating different forms of C and the use of a more powerful promoter. The EF1aalpha; promoter encoding an extended form of C was demonstrated to produce the highest titers of SRIPs and was immunogenic in mice. Additionally, SRIP and SVP titers were further improved via incorporation of a glycosylation motif in E protein. The optimized DNA yields ~100-fold greater titers of SRIPs than the original construct, thus providing a promising candidate for further vaccine evaluation.
Dengue virus belongs to Flavivirus and contains a positive-stranded RNA genome Binding of dengue virus to host cells was mediated through domain III of the viral envelope protein. Many therapeutic monoclonal antibodies (mAbs) against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 amino acids (V365NIEAEPPFG374) from domain III of the envelope protein in dengue virus serotype 2 (DENV2) was selected to design the primers for LAE technique. A DNA fragment encoding 10 directed repeats of these 10 amino acids for producing the tandem repeated peptides was obtained and fused it with GST-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from E. coli and used as antigen for immunizing rabbits to obtain polyclonal antibody. Furthermore, this EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using immunoblot and immunofluorescence assays. Most importantly, this antibody was also capable of detecting DENV2 virions by ELISA assay and could block viral entry into BHK-21 cells as shown by immunofluorescence and qRT-PCR assays. Taken together, LAE technique could be applied for production of antibody against antigen with low antigenicity successfully and carries a high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic application in the future.
Virions of the RPV strain of Cereal yellow dwarf virus (CYDV-RPV) were purified from infected oat tissue and analyzed by mass spectrometry. Two conserved residues, K147 and K181, residing in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional data are available for K147, K181 lies within an interfacial region critical for virion assembly and stability. The signature immonium ion at m/z 126.0919 demonstrated the presence of N-acetyllysine, and the sequence fragment ions enabled an unambiguous assignment of the epsilon-N-acetyl modification on K181. We hypothesize that selection favors acetylation of K181 in a fraction of coat protein monomers to stabilize the capsid by promoting intermonomer salt bridge formation.
Alphaviruses including Barmah Forest virus (BFV) and Ross River virus (RRV) cause arthritis, arthralgia and myalgia in humans. The rheumatic symptoms in BFV are very similar to RRV. Although RRV disease has been extensively studied, little is known about the pathogenesis of BFV infection. We sought to establish a mouse model for BFV to facilitate our understanding of BFV infectivity, tropism and pathogenesis and identify key pathological and immunological mechanisms of BFV infection that may distinguish between infections with BFV and RRV. Herein to our knowledge we report the first study assessing the virulence and replication of several BFV isolates in a mouse model. We infected newborn Swiss outbred mice with BFV and established that the BFV2193 prototype was the most virulent strain. BFV2193 infection resulted in the highest mortality among all BFV variant isolates, comparable to RRV. In comparison to RRV, C57BL/6 mice infected with BFV showed delayed onset, moderate disease scores and early recovery of the disease. BFV replicated poorly in muscle and did not cause the severe myositis seen in RRV-infected mice. The mRNAs for the inflammatory mediators TNF-aalpha;, IL-6, CCL2 and arginase-1 were highly upregulated in RRV- but not BFV-infected muscle. To our knowledge this is the first report of a mouse model of BFV infection, which we have used to demonstrate differences between BFV and RRV infections and to further understanding disease pathogenesis. With an increasing number of BFV cases occurring annually a better understanding of the disease mechanisms is essential for future therapeutic development.
Infection of macaques with live-attenuated SIV usually results in long-lasting efficient protection against infection with pathogenic immunodeficiency viruses. However, attenuation by deletion of regulatory genes such as nef is not complete, leading to a high viral load and fatal disease in some animals. To characterize immunological parameters and polymorphic host factors we studied 17 rhesus macaques infected with attenuated SIVmac239NU. Eight animals were able to control viral replication whereas the remaining animals (non-controllers) displayed variable set-point viral loads. Peak viral load at 2 weeks post infection correlated significantly with set-point viral load (pllt;0.0001). CD4+ T cell frequencies differed significantly soon after infection between controllers and non-controllers. Abnormal B-cell activation previously ascribed to Nef-function can be observed in non-controllers already 8 weeks after infection despite the absence of Nef. Two non-controllers developed an AIDS-like disease within 102 weeks post infection. Virus from these animals transmitted to naiiuml;ve animals replicated at low levels and the recipients did not develop immunodeficiency. This suggests that host factors determined differential viral load and subsequent disease course. Known MHC class I alleles associated with disease progression in SIV wild type infection only marginally influenced the viral load in nef-infected animals. Protection from SIVmac251 was associated with homozygosity for Mhc class II in conjunction with a TLR7 polymorphism and showed a trend with initial viral replication. We speculate that host factors whose effects are usually masked by Nef are responsible for the different disease courses in individual animals upon infection with nef-deleted viruses.
During infection the influenza A virus NS1 protein interacts with a diverse range of viral and cellular factors to antagonize host antiviral defences and promote viral replication. Here, I review the structural basis for some of these functions, and discuss the emerging view that NS1 cannot simply be regarded as a rrsquo;staticllsquo; protein with a single structure. Rather, the dynamic property of NS1 to adopt various quaternary conformations is critical for its multiple activities. Understanding NS1 plasticity, and the mechanisms governing this, will be essential for assessing both fundamental protein function and the consequences of strain-dependent polymorphisms in this important virulence factor.
Coxsackievirus B3 (CVB3) infection can trigger myocarditis and, ultimately, dilated cardiomyopathy. It has been known that CVB3-induced T cell infiltration into heart tissue is one of pathological factors for cardiomyocyte injury by inflammation. However, the underlying mechanism for this remains unclear. We investigated the mechanism of T cell infiltration by two types of CVB3 stains: the H3 wild-type and YYFF attenuated strains. T cell activation was confirmed by the morphological change in lymphocyte function-associated antigen-1 (LFA-1). Finally, we identified which viral gene was responsible for LFA-1 activation. CVB3 can infect and activate T cells in vivo and in vitro, and activated T cells were detected in CVB3-infected mouse hearts. LFA-1 on the surface of these T cells had been activated through the cAMP and Rap1 pathway. Recombinant lentivirus expressing VP2 of CVB3 can also induce LFA-1 activation via an increase of cAMP, while VP2 of YYFF did not. These results indicate that CVB3 infection increased cAMP, and then activated Rap1 in T cells. In particular, VP2, among the CVB3 proteins, may be a critical factor for this activation. This VP2-cAMP-Rap-1-LFA-1 axis may be a potential therapeutic target for CVB3-induced myocarditis.
The molecular basis for the increased resistance of astrocytes to a nonneuropathogenic strain of WNV, WNV-MAD78, compared to the neuropathogenic strain WNV-NY remains unclear. In this report, we demonstrate that the reduced susceptibility of astrocytes to WNV-MAD78 is due to a combination of both cellular activities as well as viral determinants. Analyses of the viral particle indicated that astrocyte-derived WNV-MAD78 particles are less infectious than those of WNV-NY. Additionally, inhibition of cellular furin-like proteases increased WNV-MAD78 infectious particle production in astrocytes, suggesting that high levels of furin-like protease activity within these cells acts in a cell- and strain-specific manner to inhibit WNV-MAD78 replication. Moreover, analysis of recombinant viruses indicated that the structural proteins of WNV-MAD78 were responsible for decreased particle infectivity and the corresponding reduction in infectious particle production compared to WNV-NY. Thus, the composition of the WNV virion is also a major determinant for viral fitness within astrocytes and may contribute to WNV propagation within the CNS. Whether the WNV-MAD78 structural genes reduce virus replication and particle infectivity through the same mechanism as the cellular furin-like protease activity, or whether these two determinants function through distinct pathways, remains to be determined.
Low-level HCV RNA may persist in peripheral blood mononuclear cells (PBMC) after successful treatment of chronic hepatitis C, but the consequences of this phenomenon are unclear. Forty-nine patients who achieved sustained virological response (SVR) after pegylated interferon and ribavirin therapy were analyzed after 52-66 months. HCV RNA was detected in PBMC from 18 (47.4%) and two patients stained positive for NS3 protein. Quantitation of various cytokine and chemokine transcripts in PBMC (IL-1aalpha;, IL-1bbeta;, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12 IL-15, IL-16, IL-18, TNF-aalpha;, HLA-DRA, IFN-aalpha;, IFN-bbeta;, IP-10/CXCL10, GM-CSF, M-CSF, MCP-1/CCL2, MCP-2/ CCL8, MIP-1aalpha;/CCL3, MIP-1bbeta;/ CCL4, RANTES/CCL5, TGF-bbeta; and myxovirus resistance protein A/MxA) revealed that IL-6, IL-8, IL-12, TNF-aalpha; and MIP-1bbeta; were significantly higher in HCV positive patients. In conclusion, it seems that persistence of HCV RNA in PBMC in patients with SVR is associated with immune activation.
We previously demonstrated that immunization with a DNA vaccine expressing the JEV envelope (E) protein conferred a high level of protection through a poorly neutralizing antibody response. In this report, we further investigated the role of IgG subclass in this antibody-dependent protection using cytokine co-immunization and cytokine-deficient mice. A significant difference in IgG2a/c but not IgG1 was observed between mice that survived or died following a lethal challenge. Correspondingly, the IgG2a/c response and protection increased in IL-4-deficient mice but decreased in IFN--deficient mice, highlights the importance of IgG2a/c. In addition, the restoration of protection and E-specific IgG2a/c production in IFN--deficient mice by a Th1-biased intramuscular immunization suggests that IgG2a/c but not IFN- is the major component for protection. The failure of protection against a direct intracranial challenge indicated that IgG2a/c-mediated protection is restricted to outside the CNS. Consistent with this conclusion, passive transfer of E-specific antisera conferred protection only pre-exposure to JEV. Therefore, our data here provide evidence that IgG subclass plays an important role in the protection against JEV, particular in poorly neutralizing E-specific antibodies; and Th1-biased IgG2a/c confers a better protection than Th2-biased IgG1 to against JEV.
Virologic surveillance is a critical component of measles surveillance because one of the criteria for verification of elimination of endemic measles is genetic analysis of wild-type viruses to demonstrate lack of an indigenous genotype. Measles has not yet been eliminated in China, and genotype H1 has been detected continuously since virologic surveillance was initiated in 1993. Virologic surveillance has been very strong in China and this provided a unique opportunity to conduct a detailed study of the evolution of a single, endemic genotype over a time span of nearly 20 years. Phylogenetic analysis performed on the 450 nucleotides coding for the COOH terminal 150 amino acids of the nucleoprotein (N-450), the fusion (F) gene and the hemagglutinin (H) gene confirmed the continued circulation of genotype H1 viruses for 19 years. No evidence was found for selective pressure on the H protein. The substitution rates ranged from 0.75 x 10-3 substitutions/site/year for H to 1.65 x10-3 substitutions/site/year for N-450. An estimate of the time of the most recent common ancestor for genotype H1 was approximately 1985 (95% Highest Probability Density = 1979-1989). Finally, the overall diversity of the measles sequences from China decreased from 2005 to 2012 which is coincident with a substantial decrease in measles cases. Overall, the results suggest that detailed evolutionary analyses will help to document the eventual elimination of measles in China, and the molecular approaches used in this study can be applied in other countries that are approaching measles elimination.
Bluetongue is a disease in ruminants caused by the bluetongue virus (BTV), and is spread by Culicoides biting midges. Bluetongue outbreaks cause huge economic losses and death in sheep in several parts of the world. The most effective measure to control BTV is vaccination. However, both commercially available vaccines and recently developed vaccine candidates have several shortcomings. Therefore we generated and tested next-generation vaccines for Bluetongue based on the backbone of a laboratory-adapted strain of BTV1, avirulent BTV6 or virulent BTV8. All vaccine candidates were serotyped with VP2 of BTV8 and did not express NS3/NS3a proteins, due to induced deletions in the NS3/NS3a open reading frame. Sheep were vaccinated once with one of these vaccine candidates and were challenged with virulent BTV8 three weeks after vaccination. The NS3/NS3a knockout mutation caused complete avirulence for all three BTV backbones, including for virulent BTV8, indicating that safety is associated with the NS3/NS3a knockout phenotype. Viraemia of vaccine virus was not detected using sensitive PCR diagnostics. Apparently, the vaccine viruses replicated only locally, which will minimalize spread by the insect vector. Especially the vaccine based on the BTV6 backbone protected against disease and prevented viraemia of challenge virus, showing the efficacy of this vaccine candidate. The lack of NS3/NS3a expression potentially enables the differentiation of infected from vaccinated animals, which is important for monitoring of virus spread in vaccinated livestock. The here presented Disabled Infectious Single Animal (DISA) vaccine for Bluetongue is very promising and will be subject of future studies.
The genus Flavivirus includes some of the most important human viral pathogens and its members are found in all parts of the populated world. The temporal origin of diversification of the genus has for long been debated due to the inherent problems with dating deep RNA virus evolution. A generally accepted hypothesis suggests that Flavivirus emerged within the last 10,000 years. However, it has been argued that the tick-borne Powassan flavivirus was introduced into North America some time between the opening and closing of the Beringian land bridge that connected Asia and North America between 15,000nndash;11,000 years ago, indicating a yet even older origin for Flavivirus. To determine the temporal origin of Flavivirus, we performed Bayesian relaxed molecular clock dating on a data set with high coverage of the presently available Flavivirus diversity by combining tip date calibrations and internal node calibration, based on the Powassan virus and Beringian land bridge biogeographical event. Our analysis suggests that Flavivirus originated approximately 85,000 (64,000nndash;110,000) or 120,000 (87,000nndash;159,000) years ago, depending on the circumscription of the genus. This is significantly older than previously estimated. In light of our results, we propose that it is likely that modern humans have come in contact with several Flavivirus members much earlier than previously suggested, and that it is possible that the spread of several flaviviruses coincided with, and was facilitated by, the migration and population expansion of modern humans out of Africa.
Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants and the elderly, and no vaccine against this virus has yet been licensed. In this report, a recombinant PR8 influenza virus with the RSV fusion protein epitopes of the subgroup A gene inserted into the influenza virus nonstructural (NS) gene (rFlu/RSV/F) was generated as an RSV vaccine candidate. The rescued viruses were assessed by microscopy and Western blotting. The proper expression of NS1, the NS gene product, and the nuclear export protein (NEP) of rFlu/RSV/F were also investigated using an immunofluorescent assay. The rescued virus replicated well in the MDCK kidney cell line, A549 lung adenocarcinoma cell line, and the CNE-2Z nasopharyngeal carcinoma cell line. BALB/c mice immunized intranasally with rFlu/RSV/F had specific hemagglutination inhibition antibody responses against the PR8 influenza virus and RSV NT proteins. Furthermore, intranasal immunization with rFlu/RSV/F elicited Th1-dominant cytokine profiles against the RSV-A2 virus. Taken together, our findings suggest that rFlu/RSV/F is immunogenic in vivo, and warrants further development as a promising candidate vaccine.
Ubiquitylation is a covalent post-translational modification that regulates protein stability and is involved in many biological functions. Proteins may be modified with mono-ubiquitin or ubiquitin chains. Viruses have evolved multiple mechanisms to perturb the cell ubiquitin system and manipulate it to their own benefit. Here we report ubiquitylation of vaccinia virus (VACV) protein N1. N1 is an inhibitor of NF-B and apoptosis that contributes to virulence, has a Bcl-2-like fold and is highly conserved amongst orthopoxviruses. The interaction between N1 and ubiquitin occurs at endogenous protein levels, during VACV infection and following ectopic expression of N1. Biochemical analysis demonstrated that N1 is covalently ubiquitylated and heterodimers of ubiquitylated and non-ubiquitylated N1 monomers were identified, suggesting that ubiquitylation does not inhibit N1 dimerisation. Studies with other VACV Bcl-2 proteins, such as C6 or B14, revealed that although these proteins also interact with ubiquitin, these interactions are non-covalent. Finally, mutagenesis of N1 showed that ubiquitylation occurs in a conventional lysine-dependent manner at multiple acceptor sites because only an N1 allele devoid of lysine residues remained unmodified. Taken together, we describe a previously uncharacterised modification of the VACV protein N1 that provides a new layer of complexity to the biology of this virulence factor and provides another example of the intricate interplay between poxviruses and the host ubiquitin system.
While chickens are an important reservoir for emerging pathogens such as avian influenza viruses, little is known about the diversity of picornaviruses in these poultry animals. We discover a previously unknown diversity of picornaviruses in chickens in Hong Kong. picornaviruses were detected in 87 cloacal and 7 tracheal samples from 93 of 900 chickens by RT-PCR, with their partial 3Dpol gene sequences forming five distinct clades (I to V) among known picornaviruses. Analysis of eight genomes from different clades revealed seven different picornavirus species, including six novel picornavirus species (ChPV1 from clade I, ChPV2 and ChPV3 from clade II, ChPV4 and ChPV5 from clade III, ChGV1 from clade IV) and one existing species (avian encephalomyelitis virus from clade V). The six novel chicken picornavirus genomes exhibited distinct phylogenetic positions and genome features different from related picornaviruses, supporting their classification as separate species. Moreover, ChPV1 may potentially belong to a novel genus, with low sequence homologies to related picornaviruses especially in the P1 and P2 regions including the predicted L and 2A proteins. Nevertheless, these novel picornaviruses were most closely related to picornaviruses of other avian species (ChPV1 related to Passerivirus A, ChPV2 and ChPV3 to Avisivirus A and duck hepatitis A virus, ChPV4 and ChPV5 to Melegrivirus A, ChGV1 to Gallivirus A). Furthermore, ChPV5 represented a potential recombinant picornavirus, with its P2 and P3 regions possibly originating from Melegrivirus A. Chickens are an important reservoir for diverse picornaviruses which may cross avian species barriers through mutation or recombination.
Group A rotaviruses, members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The rotavirus genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. It has been shown that some rearranged segments are preferentially encapsidated into viral progenies after serial passages in cell culture. Based on this characteristic, a reverse genetics system was previously used to introduce exogenous segment 7 rearrangements into an infectious rotavirus. This study extends this reverse genetics system to RNA segments 5 and 11. Transfection of exogenous rotavirus rearranged RNA segments 5 or 11 into cells infected with a wild-type helper rotavirus (bovine strain RF), resulted in subsequent gene rearrangements in the viral progeny. Whilst recombinant viruses were rescued with an exogenous rearranged segment 11, the exogenous segment was modified by a secondary rearrangement. The occurrence of spontaneous rearrangements of wild-type or exogenous segments is a major hindrance to the use of this reverse genetics approach.
Ancient endogenous retroviruses (ERVs) designated as the EAVs are present in all Gallus species including the chicken and resemble the modern avian sarcoma and leukosis viruses (ASLVs). The EAVs comprise several distinct retroviruses, including EAV-0, EAV-E51 and EAV-HP, as well as a putative member previously coined the avian retrotransposon of chickens (ART-CH). Thus far, only the EAV-HP elements have been well characterized. In this report we determined sequences of representative EAV-0 and EAV-E51 proviruses by cloning and data mining of the 2011 assembly of the G. gallus genome. Although the EAV-0 elements are primarily deleted in the env region, we identified two complete EAV-0 env genes within the G. gallus genome and prototype elements sharing identity to an EAV-E51-related clone previously designated EAV-E33. Prototype EAV-0, EAV-E51 and EAV-E33 gag, pol and env gene sequences used for phylogenetic analysis of deduced proteins showed that the EAVs form three distinct clades, with EAV-0 sharing the last common ancestor with the ASLVs. The EAV-E51 clade showed the greatest level of divergence compared to other EAVs or ASLVs suggesting that these ERVs represent exogenous retroviruses that evolved and integrated into the germ line over a long period of time. Moreover, the degree of divergence between the chicken and red jungle fowl EAV-E51 sequences suggests that they are more ancient than the other EAVs and may have diverged through mutations that accumulated post- integration. Finally, we show that the ART-CH elements are chimeric defective ERVs comprising portions of EAV-E51 and EAV-HP rather than authentic retrotransposons.
Nitric oxide (NO) is an important defensive signal in plants but its effects on virus infection are not well understood. Administration of NO-releasing compounds immediately before inoculation of tobacco leaves with potato virus X and tobacco mosaic virus decreased the accumulation of virus, indicating that NO can induce resistance rapidly. Resistance induction was inhibited by co-administration with a NO-scavenging compound or when experiments were done in transgenic tobacco plants expressing increased alternative respiratory pathway capacity due to constitutive expression of the plant mitochondrial enzyme, alternative oxidase (AOX). These results indicate that NO, which inhibits electron transport chain activity, is triggering defensive signalling by inducing changes in mitochondrial reactive oxygen species levels that are in turn regulated by AOX. Experiments using NahG-transgenic plants, which cannot accumulate the defensive plant hormone salicylic acid (SA) showed that NO rapidly induces resistance to virus infection independently of SA. However, this initial state of resistance may be transient. Subsequently, by five days post-treatment, NO had caused an increase in pathogenesis-related protein 1 (PR1) expression (a proxy for increased SA biosynthesis), which correlated with a longer-term state of resistance to virus infection. The induction by NO of PR1 accumulation was modified in AOX-transgenic plants. This indicates that the influence of NO on defensive gene expression is in part mediated through its effects on mitochondria.
Monitoring serum antibodies against natural infections or after immunizations has been a standard clinical diagnostic procedure. However, collecting blood samples requires trained personnel and may cause discomfort and increase the risk of complications. In this study, we investigated whether tear samples could serve as a surrogate for serum samples to measure specific antibody. A widely-used preclinical Cottontail Rabbit Papillomavirus (CRPV)/ rabbit model has been a surrogate model for high-risk Human papillomavirus (HPV) infections. New Zealand White rabbits either naturally infected with CRPV or immunized with two clinically available HPV vaccines (Gardasil and Cervarix) were examined for antibody generation in both tear and serum samples. We demonstrated that antibodies were detectable in tears from both naturally infected as well as vaccinated animals. Overall, the antibody levels in tears were about 10 fold lower than those from the corresponding serum samples but background noise was lower in tear samples. The isotypes of antibody in tears were predominately IgA and IgG. These findings clearly showed that tears could be a surrogate for serum samples for monitoring antibody responses. Because collecting tears causes no discomfort and poises no risk to patients, it represents a novel and promising method for monitoring future HPV epidemiologic studies as well in clinical practice.
Herein, we report the isolation and functional characterization of monoclonal antibodies (mAbs) against two murine norovirus (MNV) strains MNV-1 and WU20, which were isolated following oral infection of mice. The mAbs were screened for reactivity against the respective homologous and heterologous MNV strain by enzyme-linked immunosorbent assay (ELISA). Selected mAbs were of IgA, IgG1, IgG2a, or IgG2b isotype and showed a range of Western blot reactivities from non-binding to strongly binding, suggesting recognition of conformational and linear epitopes. Some of the anti-MNV-1 antibodies neutralized both MNV-1 and WU20 infections in culture and in mice but none of the anti-WU20 mAbs neutralized either virus. The non-neutralizing anti-MNV-1 IgG2b 5C4.10 was mapped to the S domain of the MNV-1 capsid, while the epitopes of the neutralizing anti-MNV-1 IgA antibodies 2D3.7 and 4F9.4 were mapped to the P domain. Generation of neutralization escape viruses showed that two mutations (V339I and D348E) in the Crrsquo;Drrsquo; loop of the MNV-1 P domain mediated escape from mAb 2D3.7 and 4F9.4 neutralization. These findings broaden the known neutralizing epitopes of MNV to the main surface exposed loops of the P domain. In addition, the current panel of antibodies provides valuable reagents for studying norovirus biology and development of diagnostic tools.
Human cardioviruses or Saffold viruses (SAFV) of the family Picornaviridae are newly emerging viruses whose genetic and phenotypic diversity are poorly understood. We report here the full genome sequence of eleven genotypes of SAFV from Pakistan and Afghanistan, along with a re-evaluation of their genetic diversity and recombination. We detected 88 SAFV from stool samples of 943 acute flaccid paralysis cases by RT-PCR targeting the 5rrsquo;-UTR. Further characterization based on complete VP1 revealed 71 SAFV belonging to eleven genotypes, including three new genotypes. SAFV showed high genetic diversity and recombination based on the phylogenies, pairwise distance distributions and recombination mapping analyses. Phylogenies based on non-structural and UTRs were highly incongruent indicating frequent recombination events among SAFV. We improved the SAFV genotyping classification criteria by determining new VP1 thresholds based on the principles used for the classification of enteroviruses. For genotype assignment, we propose a threshold of 23% and 10% divergence for VP1 nucleotide and amino acid sequences, respectively. Other members of Theilovirus, such as Thera virus and Theilerrrsquo;s murine encephalomyelitis virus, are difficult to classify in the same species as SAFV, because they are genetically distinct from SAFV, with 41-56% amino acid pairwise distances. The new genetic information obtained in this study will improve our understanding of the evolution and classification of SAFV.
The Epstein-Barr virus (EBV) infects most of the world's adult population. In most cases primary infection and virus persistence are asymptomatic, the virus having evolved a sophisticated strategy to exist long-term in the B cell pool. However, EBV can contribute to the development of several human B-cell lymphomas, which include Hodgkin's lymphoma (HL), Burkitt's lymphoma, and a subset of diffuse large B cell lymphomas. EBV potently transforms resting B cells in vitro (Young aamp; Murray, 2003; Young aamp; Rickinson, 2004; Oyama et al., 2003; Oyama et al., 2007). Two questions central to our understanding of the origins of EBV-associated B cell lymphomas are; 1) how the host and virus interact to allow benign persistent latent infection, and 2) how perturbation of this normal homeostasis leads to neoplastic transformation. This review will summarise current knowledge of how the EBV life cycle is regulated in the B cells of the asymptomatic host. It will also discuss how the disruption of normal B cell homeostasis can contribute to the development of B cell lymphomas, focussing on several novel pathogenic mechanisms in EBV-associated HL which include the suppression of the virus lytic cycle and the activation of collagen receptor signalling.
Toll-like receptors (TLR) play an important role in innate immunity to virus infections. We investigated the role of TLR3 in the pathogenesis of H5N1 and pandemic H1N1 (pH1N1) influenza virus infection in mice. The results of TLR3 was compared to that of wild type (WT) mice. WT mice and those defective in TLR3 were infected with influenza A/HK/486/97 (H5N1) or A/HK/415742/09 pH1N1 virus. As a comparison, mice with a defective MyD88 gene were also infected with the viruses. Survival and body weight loss were monitored for 14 days, and lung pathology, immune cells profile, viral load and cytokine responses were studied. Compared to wild-type mice, TLR3-/- mice have a survival advantage, a significantly faster regain of body weight, lower lung viral titer and less lung pathology following challenge with H5N1 virus. Such a survival advantage is not seen in pH1N1 infected TLR3-/- mice. On the contrary, MyD88-/- mice have an increased viral titer and decreased leukocyte infiltration in the lungs after infection with H5N1 virus and a poorer survival in pH1N1 infection. In conclusion, TLR3 has a detrimental effect on the pathogenesis of H5N1 infection but not in pH1N1 infection highlighting the differences in the pathogenesis of these two viruses and in the role of TLR3 in such pathogenesis.
The genus Negevirus consists of insect-only viruses isolated from mosquitoes and sandflies. Here, we report the successful construction of a full-length infectious cDNA clone of Negev virus strain M30957. Viral RNA was transcribed in vitro and virus was readily rescued with or without the use of a cap analog. These results strongly suggest that Negev virus, and likely other members within the genus, is a non-segmented, single stranded, positive sense RNA virus.
White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNv). The N-terminal end of the MrNv capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutageneses revealed that the RNA-binding region is located at positions 20 to 29, where 80% of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA binding region still assembled into virus-like particles (VLPs) suggesting that this region is not a part of the capsid assembly domain. This paper is the first to report the specific RNA binding region of MrNv capsid protein.
To initiate infections, many coronaviruses use sialic acids, either as receptor determinants or as attachment factors helping the virus finding its receptor underneath the heavily glycosylated mucus layer. In the present study, the role of sialic acids in serotype I feline enteric coronavirus (FECV) infections was studied in feline intestinal epithelial cell cultures. Treatment of cells with neuraminidase (NA) enhanced infection efficiency, showing that terminal sialic acid residues on the cell surface are not receptor determinants and even hamper efficient virus-receptor engagement. Knowing that NA treatment of coronaviruses can unmask viral sialic acid binding activity, replication of untreated and NA-treated viruses was compared, showing that NA treatment of the virus enhanced infectivity in untreated cells but was detrimental in NA-treated cells. By using sialylated compounds as competitive inhibitors, it was demonstrated that sialyllactose (aalpha;2,6-linked ggt; aalpha;2,3-linked) especially reduced infectivity of NA-treated viruses, whereas bovine submaxillary mucin (BSM) inhibited both treated and untreated viruses. In desialylated cells on the other hand, viruses were less prone to competitive inhibition with sialylated compounds. In conclusion, this study demonstrates that FECV has a sialic acid binding capacity, which is partially masked by virus-associated sialic acids, and that attachment to sialylated compounds can facilitate enterocyte infections. However, this sialic acid binding is not a prerequisite for the initiation of infection, and virus-receptor engagement is even more efficient after desialylation of cells, showing that FECV requires sialidases for efficient enterocyte infections.
Difficulties associated with efficient delivery and targeting of miRNAs to cells is hampering the real world application of miRNA technology. This study utilised an influenza A based delivery system to express miR-155 in order to knockdown SOCS1 mRNA. Using qPCR and dual luciferase technology we show that miR-155 delivery resulted in a significant increase in cellular miR-155 that facilitated a down regulation of SOCS1 gene expression and a functional increase in IL-6 and IFN-bbeta; cytokines.
Poxvirus vectors represent promising HIV vaccine candidates and were a component of the only successful HIV vaccine efficacy trial to date. We tested the immunogenicity of a novel recombinant Capripoxvirus vector, Lumpy Skin Disease Virus (LSDV) in combination with Modified Vaccinia Ankara (MVA), both expressing genes from HIV-1. Here, we demonstrate that the combination regimen was immunogenic in rhesus macaques, inducing high magnitude, broad and balanced CD4+ and CD8+ T cell responses, and transient activation of the immune response. These studies support further development of LSDV as a vaccine vector.
Several baculoviruses can induce host cell aggregation during infection; however, the molecular basis remains unknown. The Rho family of small GTPases, including Rho1, Racs and Cdc42, plays important roles in cell migration and cell-cell contact. Activated GTPases target actin polymerization to discrete sites on the plasma membrane, thereby inducing membrane protrusions. In this study, we demonstrated that Spodoptera litura nucleopolyhedrovirus (SpltNPV) infection induced the amoeboid movement and aggregation of SpLi-221 cells in vitro. The amount of Rho1-GTP increased in the infected cells, which suggests that Rho1 was activated upon infection. RNA interference and superinfection of dominant-negative recombinants revealed that the SpltNPV-induced SpLi-221 cell aggregation was dependent on the Rho1, but not Racs or Cdc42, signaling pathway. Inhibition of Rho-associated protein kinase (Rok) activity by the inhibitor Y-27632 significantly reduced SpLi-221 cell aggregation. Silencing Rho1 expression with RNA interference decreased SpltNPV propagation by approximately 40% in vitro, when SpLi-221 cells were infected at a low, but not high, multiplicity of infection, suggesting that the SpltNPV-induced cell aggregation may benefit SpltNPV spread.
Wheat dwarf virus (WDV) is a DNA virus belonging to the genus Mastrevirus of the family Geminiviridae. In this study, we report that the Rep protein encoded by WDV is a RNA silencing suppressor (RSS) as determined by coinfiltration assays using transgenic Nicotiana benthamiana line 16c carrying the GFP reporter gene. The Rep protein was shown to inhibit both local and systemic RNA silencing of the GFP gene as well as the spread of systemic GFP RNA silencing signals. Gel mobility shift assays showed that the Rep protein binds 21 nt and 24 nt small interfering RNA (siRNA) duplexes and single-stranded (ss)-siRNA. To our knowledge, this is the first identification of an RSS encoded by Mastreviruses. Further deletion mutagenesis indicates that both the N and C terminal regions of the Rep protein are not critical for silencing suppression and self-interaction, but the N-termini of Rep is necessary for its pathogenicity.
Cholesterol is a critical component of the Hepatitis C virus (HCV) life cycle as demonstrated by its accumulation within infected hepatocytes and lipoviral particles. To cope with excess cholesterol, hepatic enzymes ACAT1 and ACAT2 produce cholesteryl esters which are destined for storage in lipid droplets or for secretion as apolipoproteins. Here we demonstrate in vitro that cholesterol accumulation following HCV infection induces up-regulation of the ACAT genes and increases cholesteryl ester synthesis. Analysis of human liver biopsy tissue showed increased ACAT2 mRNA expression in liver infected with HCV genotype 3, compared to genotype 1. Inhibiting cholesterol esterification using the potent ACAT inhibitor TMP-153, significantly reduced production of infectious virus, but did not inhibit virus RNA replication. Density gradient analysis showed that TMP-153 treatment caused a significant increase in lipoviral particle density, suggesting reduced lipidation. These data suggest that cholesterol accumulation following HCV infection stimulates the production of cholesteryl esters, a major component of lipoviral particles. Inhibition of cholesteryl ester synthesis reduces HCV particle density and infectivity, suggesting that cholesteryl esters are required for optimal infection of hepatocytes.
The Gag polyprotein of feline immunodeficiency virus (FIV) assembles at the plasma membrane of the infected cells. Since the FIV Gag domains whose interaction promotes Gag multimerization have not been identified, we generated a series of Gag subdomains which were tested for their ability to associate with full-length Gag and be recruited into extracellular virus-like particles (VLPs). Removal from FIV Gag of the C-terminal 37 residues as well as deletion of the amino and central regions of the nucleocapsid (NC) domain attenuated but did not abrogate association with wild-type Gag, whereas a Gag mutant encompassing the matrix (MA) and capsid (CA) domains interacted poorly with full-length Gag. Association with wild-type Gag was abolished by deleting most of the NC together with the N-terminal 40 residues of the MA, which most likely reflects the inability of this Gag mutant to bind RNA. Of note, the CA-NC Gag subdomain associated with wild-type Gag and was recruited into particles in a proportion close to 50% of the total Gag-related protein mass of VLPs. Moreover, both a Gag protein lacking the C-terminal p2 peptide and a nonmyristoylated version of the polyprotein exhibited a transdominant negative effect on the assembly of wild-type Gag. Analysis of Gag mutants carrying internal deletions within the CA revealed that N-terminal as well as C-terminal domains of the CA are necessary for assembly. Our results demonstrate that the FIV CA-NC region constitutes the principal self-interaction domain of Gag and that the RNA-binding capacity of Gag is necessary for its multimerization.
Key virus traits such as virulence and transmission strategies rely on genetic variation that result in functional changes in the interactions between hosts and viruses. Here comparative genomic analyses of seven isolates of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) with differing phenotypes were employed to pinpoint candidate genes that may be involved in host-virus interactions. These isolates obtained after vertical or horizontal transmission of infection in insects differed in virulence. Apart from one genome containing a piggyBac transposon, all European SeMNPV isolates have similar genome size and content. Complete genome analyses of single nucleotide polymorphisms, insertions and deletions identified mutations in 48 ORFs that could result in functional changes. Among these, 13 ORFs could be correlated with particular phenotypic characteristics of SeMNPV isolates. Mutations were found in all gene functional classes and most of the changes we highlighted could potentially be associated with differences in transmission. The regulation of DNA replication (helicase, lef-7) and transcription (lef-9, p47) might be important for the establishment of sublethal infection prior to and following vertical transmission. Virus-host cells interactions also appear instrumental in the modulation of viral transmission as significant mutations were detected in virion proteins involved in primary (AC150), or secondary infections (ME35), and in apoptosis inhibition (IAP2, AC134). Baculovirus populations naturally harbor high genomic variation located in genes involved at different levels of the complex interactions between virus and host during the course of an infection. The comparative analyses performed here suggest the differences in baculovirus virulence and transmission phenotypes involve multiple molecular pathways.
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.