|Journal of General Virology current issue|
Evidence is accumulating that one or more beta-retrovirus is associated with human breast cancer. Retroviruses can exist as an infectious (exogenous) virus or as a part of the genetic information of cells due to germline integration (endogenous). An exogenous virus with a genome that is highly homologous to mouse mammary tumour virus is gaining acceptance as possibly being associated with human breast cancer, and recently furnished evidence is discussed in this article, as is the evidence for involvement of an endogenous human beta-retrovirus, HERV-K. Modes of interaction are also reviewed and linkages to the APOBEC3 family are suggested.
Most viruses express one or several proteins that counter the antiviral defences of the host cell. This is the task of non-structural protein NS1 in influenza viruses. Absent in the viral particle, but highly expressed in the infected cell, NS1 dramatically inhibits cellular gene expression and prevents the activation of key players in the IFN system. In addition, NS1 selectively enhances the translation of viral mRNAs and may regulate the synthesis of viral RNAs. Our knowledge of the virus and of NS1 has increased dramatically during the last 15 years. The atomic structure of NS1 has been determined, many cellular partners have been identified and its multiple activities have been studied in depth. This review presents our current knowledge, and attempts to establish relationships between the RNA sequence, the structure of the protein, its ligands, its activities and the pathogenicity of the virus. A better understanding of NS1 could help in elaborating novel antiviral strategies, based on either live vaccines with altered NS1 or on small-compound inhibitors of NS1.
The 2009 pandemic H1N1 influenza A virus spread across the globe and caused the first influenza pandemic of the 21st century. Many of the molecular factors that contributed to the airborne transmission of this pandemic virus have been determined; however, the direct-contact transmission of this virus remains poorly understood. In this study, we report that a combination of two mutations (N159D and Q226R) in the haemagglutinin (HA) protein of the representative 2009 H1N1 influenza virus A/California/04/2009 (CA04) caused a switch in receptor binding preference from the aalpha;2,6-sialoglycan to the aalpha;2,3-sialoglycan receptor, and decreased the binding intensities for both glycans. In conjunction with a significantly decreased replication efficiency in the nasal epithelium, this limited human receptor binding affinity resulted in inefficient direct-contact transmission of CA04 between guinea pigs. Our findings highlight the role of the HA gene in the transmission of the influenza virus.
Interactions between the viral surface glycoprotein haemagglutinin (HA) and the corresponding receptors on host cells is one important aspect of influenza virus infection. Mutations in HA have been described to affect pathogenicity, antigenicity and the transmission of influenza viruses. Here, we detected polymorphisms present in HA genes of two pandemic 2009 H1N1 (H1N1pdm09) isolates, A/California/04/2009 (Ca/09) and A/Mexico/4108/2009 (Mx/09), that resulted in amino acid changes at positions 186 (S to P) and 194 (L to I) of the mature HA1 protein. Although not reported in the published H1N1pdm09 consensus sequence, the P186 genotype was more readily detected in primary infected and contact-naiiuml;ve pigs when inoculated with a heterogeneous mixed stock of Ca/09. Using reverse genetics, we engineered Ca/09 and Mx/09 genomes by introducing Ca/09 HA with two naturally occurring variants expressing S186/I194 (HA-S/I) and P186/L194 (HA-P/L), respectively. The Ca/09 HA with the combination of P186/L194 with either the Ca/09 or Mx/09 backbone resulted in higher and prolonged viral shedding in naiiuml;ve pigs. This efficiency appeared to be more likely through an advantage in cell surface attachment rather than replication efficiency. Although these mutations occurred within the receptor-binding pocket and the Sb antigenic site, they did not affect serological cross-reactivity. Relative increases of P186 in publicly available sequences from swine H1N1pdm09 viruses supported the experimental data, indicating this amino acid substitution conferred an advantage in swine.
Vaccination is the best measure to prevent influenza pandemics. Here, we studied the protective effect against heterologous influenza viruses, including A/reassortant/NYMC X-179A (pH1N1), A/Chicken/Henan/12/2004 (H5N1), A/Chicken/Jiangsu/7/2002 (H9N2) and A/Guizhou/54/89xA/PR/8/34 (A/Guizhou-X) (H3N2), in mice first vaccinated with a DNA vaccine of haemagglutinin (HA) or neuraminidase (NA) of A/PR/8/34 (PR8) and then infected with the homologous virus. We showed that PR8 HA or NA vaccination both protected mice against a lethal dose of the homologous virus; PR8 HA or NA DNA vaccination and then PR8 infection in mice offered poor or excellent protection, respectively, against a second, heterologous influenza virus challenge. In addition, before the second heterologous influenza infection, the highest antibody level against nucleoprotein (NP) and matrix (M1 and M2) proteins was found in the PR8 NA-vaccinated and PR8-infected group. The level of induced cellular immunity against NP and M1 showed a trend consistent with that seen in antibody levels. However, PR8 HA+NA vaccination and then PR8 infection resulted in limited protection against heterologous influenza virus challenge. Results of the present study demonstrated that infection of the homologous influenza virus in mice already immunized with a NA vaccine could provide excellent protection against subsequent infection of a heterologous influenza virus. These findings suggested that NA, a major antigen of influenza virus, could be an important candidate antigen for universal influenza vaccines.
Replicon-particle-based vaccines combine the efficacy of live-attenuated vaccines with the safety of inactivated or subunit vaccines. Recently, we developed Rift Valley fever virus (RVFV) replicon particles, also known as nonspreading RVFV (NSR), and demonstrated that a single vaccination with these particles can confer sterile immunity in target animals. NSR particles can be produced by transfection of replicon cells, which stably maintain replicating RVFV S and L genome segments, with an expression plasmid encoding the RVFV glycoproteins, Gn and Gc, normally encoded by the M-genome segment. Here, we explored the possibility to produce NSR with the use of a helper virus. We show that replicon cells infected with a Newcastle disease virus expressing Gn and Gc (NDV-GnGc) were able to produce high levels of NSR particles. In addition, using reverse genetics and site-directed mutagenesis, we were able to create an NDV-GnGc variant that lacks the NDV fusion protein and contains two amino acid substitutions in, respectively, Gn and HN. The resulting virus uses a unique entry pathway that facilitates the efficient production of NSR in a one-component system. The novel system provides a promising alternative for transfection-based NSR production.
We have previously documented the inhibitory activity of RNA aptamers to the RNA-dependent RNA polymerase of foot-and-mouth disease virus (3Dpol). Here we report their modification and use with a subgenomic replicon incorporating GFP (pGFP-PAC replicon), allowing replication to be monitored and quantified in real-time. GFP expression in transfected BHK-21 cells reached a maximum at approximately 8 h post-transfection, at which time change in morphology of the cells was consistent with a virus-induced cytopathic effect. However, transfection of replicon-bearing cells with a 3Dpol aptamer RNA resulted in inhibition of GFP expression and maintenance of normal cell morphology, whereas a control aptamer RNA had little effect. The inhibition was correlated with a reduction in 3Dpol (detected by immunoblotting) and shown to be dose dependent. The 3Dpol aptamers appeared to be more effective than 2'-C-methylcytidine (2'CMC). Aptamers to components of the replication complex are therefore useful molecular tools for studying viral replication and also have potential as diagnostic molecules in the future.
Previous studies have shown that hepatitis C virus (HCV) enters human hepatic cells through interaction with a series of cellular receptors, followed by clathrin-mediated, pH-dependent endocytosis. Here, we investigated the mechanisms of HCV entry into multiple HCV-permissive human hepatocyte-derived cells using trans-complemented HCV particles (HCVtcp). Knockdown of CD81 and claudin-1, or treatment with bafilomycin A1, reduced infection in Huh-7 and Huh7.5.1 cells, suggesting that HCV entered both cell types via receptor-mediated, pH-dependent endocytosis. Interestingly, knockdown of the clathrin heavy chain or dynamin-2 (Dyn2), as well as expression of the dominant-negative form of Dyn2, reduced infection of Huh-7 cells with HCVtcp, whereas infectious entry of HCVtcp into Huh7.5.1 cells was not impaired. Infection of Huh7.5.1 cells with culture-derived HCV (HCVcc) via a clathrin-independent pathway was also observed. Knockdown of caveolin-1, ADP-ribosylation factor 6 (Arf6), flotillin, p21-activated kinase 1 (PAK1) and the PAK1 effector C-terminal binding protein 1 of E1A had no inhibitory effects on HCVtcp infection into Huh7.5.1 cells, thus suggesting that the infectious entry pathway of HCV into Huh7.5.1 cells was not caveolae-mediated, or Arf6- and flotillin-mediated endocytosis and macropinocytosis, but rather may have occurred via an undefined endocytic pathway. Further analysis revealed that HCV entry was clathrin- and dynamin-dependent in ORL8c and HepCD81/miR122 cells, but productive entry of HCV was clathrin- and dynamin-independent in Hep3B/miR122 cells. Collectively, these data indicated that HCV entered different target cells through different entry routes.
Based on the hypothesis that interactions between virions and serum components may influence the outcome of dengue virus (DENV) infections, we decided to use affinity chromatography with domain III from the envelope (E) protein of DENV2 (DIIIE2) as a ligand to isolate virus-binding proteins from human plasma. This approach yielded serum amyloid P (SAP) and aalpha;2-macroglobulin (aalpha;2M) as novel viral interactors. After confirming the specific binding of both SAP and aalpha;2M to DIIIE2 by ELISA, the latter interaction was examined in greater detail. We obtain evidence suggesting that the binding species was actually the receptor-activated form of aalpha;2M (aalpha;2M*), that aalpha;2M* could bind monovalently to recombinant domain III from all four DENV serotypes with affinities in the micromolar range ranking as DENV4ggt;DENV1~DENV2ggt;DENV3 and that this interaction exhibited a strong avidity effect when multivalent binding was favoured (KD 8x10nndash;8 M for DIIIE2). We also showed that aalpha;2M* bound to DENV virions of the four serotypes, protecting the virus from temperature-induced inactivation in the absence of serum and enhancing infectivity. The latter effect exhibited an ED50 of 2.9x10nndash;8 M, also suggesting an avidity effect due to multivalent binding. These results will further contribute to the characterization of the virusnndash;host factor interaction network during human DENV infection.
The aim of this study was to investigate the infection and replication of swine-derived hepatitis E virus (HEV) in primary cultured human hepatocytes (PHCs). Hepatocytes were cultured from the resected normal livers of patients with metastatic tumours. These cultured hepatocytes were infected with swine-derived genotype 3 or 4 HEV. Viral replication was monitored using reverse transcriptase-quantitative PCR. The amount of HEV RNA increased in the culture media and cells following infection. Immunofluorescence staining implied that the spread of HEV infection in hepatocytes was attributed mainly to cell-to-cell transmission via the cell membrane. The sequences of the inoculated and propagated HEV were determined to examine whether sequence variation occurred during infection. Sequence analysis showed that there were no differences between inoculated and propagated HEV, demonstrating that in vitro infection and replication of swine HEV in PHCs occurred without sequence variation.
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay 32P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5' end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15 000 g. Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3'dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.
Classical swine fever virus (CSFV) has a tropism for vascular endothelial cells and immune system cells. The process and release of pro-inflammatory cytokines, including IL-1bbeta; and IL-18, is one of the fundamental reactions of the innate immune response to viral infection. In this study, we investigated the production of IL-1bbeta; from macrophages following CSFV infection. Our results showed that IL-1bbeta; was upregulated after CSFV infection through activating caspase-1. Subsequent studies demonstrated that reactive oxygen species may not be involved in CSFV-mediated IL-1bbeta; release. Recently, research has indicated a novel mechanism by which inflammasomes are triggered through detection of activity of viroporin. We further demonstrated that CSFV viroporin p7 protein induced IL-1bbeta; secretion which could be inhibited by the ion channel blocker amantadine and also discovered that p7 protein was a short-lived protein degraded by the proteasome. Together, our observations provided an insight into the mechanism of CSFV-induced inflammatory responses.
A new nodavirus, named covert mortality nodavirus (CMNV), is associated with covert mortality disease of shrimp which has caused serious loss in China since 2009. Histopathological examination of shrimp suffering the disease revealed coagulative necrosis of striated muscle similar to typical histopathology features of infectious myonecrosis virus (IMNV), Penaeus vannamei nodavirus (PvNV) and Macrobrachium rosenbergii nodavirus (MrNV). However, shrimp suffering this disease tested negative for IMNV, MrNV and PvNV by reverse transcription (RT)-PCR. Additionally, eosinophilic inclusions were found in epithelium of the tubules in the hepatopancreas and lymphoid organ, and mass karyopyknotic nuclei existed in the muscle and lymphoid organ. The tubular epithelium of the hepatopancreas showed significant atrophy. A cDNA library was constructed from total RNA of infected shrimp. Sequencing and alignment analysis showed that one clone with an 1185 bp insert (designated CMNV-7) shared 54 , 53 and 39 % identity with the amino acid sequences of RNA-dependent RNA polymerase from Flock House virus, black beetle virus and MrNV. The results of fluorescence in situ hybridization showed that the hepatopancreas, striated muscle and lymphoid organ were positively reacting tissues. The mean size of negative-stained virus particles was 32 nm. In addition, a nested RT-PCR assay was developed for CMNV, and the RT-PCR detection results revealed that Fenneropenaeus chinensis, Litopenaeus vannamei and Marsupenaeus japonicus suffering from this disease were CMNV-positive.
The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain did not react with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis.
Previous studies of duck hepatitis A virus infection have focused only on the pathogenicity and host response of one strain. Here, we show that the virulent SH strain and the attenuated FC64 strain induced varied pathogenicity, apoptosis and immune responses in the livers of 1-day-old ducklings. SH infection caused apoptosis and visible lesions in the liver; serum aspartate aminotransferase, alanine transaminase, alkaline phosphatase, -glutamyltransferase and total bilirubin activities were markedly upregulated; and ducklings died at 36 h post-infection (p.i.). However, FC64 infection did not induce significant symptoms or impair liver function, and all of the infected ducklings remained healthy. In addition, both virus strains replicated well in the liver, spleen and intestine, whilst the SH strain replicated more efficiently than FC64. IFN-, IL-2, inducible nitric oxide synthase and nitric oxide were strongly induced by SH infection, and may be associated with the pathogenicity of the SH strain. IFN-aalpha;, IFN-bbeta;, IFN-stimulated transmembrane protein 1, IFN-stimulated gene 12, 2',5'-oligoadenylate synthetase-like and IL-6 were moderately induced by SH infection at 24 h p.i., and dramatically induced by FC64 infection at 36 h p.i. The intensive induction of cytokines by FC64 may be involved in restriction of virus replication and stimulation of adaptive immune responses. Ducklings inoculated with FC64 produced high levels of antiviral antibodies within 45 days p.i. The low virulence and strong immune response of FC64 rendered this strain a good vaccine candidate, as confirmed by a protective assay in this study.
During a hospital-based diarrhoeal disease study conducted in Ho Chi Minh City, Vietnam from 2009 to 2010, we identified four symptomatic children infected with G26P rotavirus (RV) nndash; an atypical variant that has not previously been reported in human gastroenteritis. To determine the genetic structure and investigate the origin of this G26P strain, the whole genome of a representative example was characterized, revealing a novel genome constellation: G26nndash;Pnndash;I5nndash;R1nndash;C1nndash;M1nndash;A8nndash;N1nndash;T1nndash;E1nndash;H1. The genome segments were most closely related to porcine (VP7, VP4, VP6 and NSP1) and Wa-like porcine RVs (VP1nndash;3 and NSP2nndash;5). We proposed that this G26P strain was the product of zoonotic transmission coupled with one or more reassortment events occurring in human and/or animal reservoirs. The identification of such strains has potential implications for vaccine efficacy in south-east Asia, and outlines the utility of whole-genome sequencing for studying RV diversity and zoonotic potential during disease surveillance.
Rats are known as reservoirs and vectors for several zoonotic pathogens. However, information on the viruses shed by urban wild rats that could pose a zoonotic risk to human health is scare. Here, intestinal contents from 20 wild Norway rats (Rattus norvegicus) collected in the city of Berlin, Germany, were subjected to metagenomic analysis of viral nucleic acids. The determined faecal viromes of rats consisted of a variety of known and unknown viruses, and were highly variable among the individuals. Members of the families Parvoviridae and Picobirnaviridae represented the most abundant species. Novel picornaviruses, bocaviruses, sapoviruses and stool-associated circular ssDNA viruses were identified, which showed only low sequence identity to known representatives of the corresponding taxa. In addition, noroviruses and rotaviruses were detected as potential zoonotic gastroenteritis viruses. However, partial-genome sequence analyses indicated that the norovirus was closely related to the recently identified rat norovirus and the rotavirus B was closely related to the rat rotavirus strain IDIR; both viruses clustered separately from respective human virus strains in phylogenetic trees. In contrast, the rotavirus A sequences showed high identity to human and animal strains. Analysis of the nearly complete genome of this virus revealed the known genotypes G3, P and N2 for three of the genome segments, whereas the remaining eight genome segments represented the novel genotypes I20nndash;R11nndash;C11nndash;M10nndash;A22nndash;T14nndash;E18nndash;H13. Our results indicated a high heterogeneity of enteric viruses present in urban wild rats; their ability to be transmitted to humans remains to be assessed in the future.
Papillomaviruses are a family of slowly evolving DNA viruses and their evolution is commonly linked to that of their host species. However, whilst bovine papillomavirus-1 (BPV-1) primarily causes warts in its natural host, the cow, it can also cause locally aggressive and invasive skin tumours in equids, known as sarcoids, and thus provides a rare contemporary example of cross-species transmission of a papillomavirus. Here, we describe the first phylogenetic analysis of BPV-1 in equine sarcoids to our knowledge, allowing us to explore the evolutionary history of BPV-1 and investigate its cross-species association with equids. A phylogenetic analysis of the BPV-1 transcriptional promoter region (the long control region or LCR) was conducted on 15 bovine and 116 equine samples from four continents. Incorporating previous estimates for evolutionary rates in papillomavirus implied that the genetic diversity in the LCR variants was ancient and predated domestication of both equids and cattle. The phylogeny demonstrated geographical segregation into an ancestral group (African, South American and Australian samples), and a more recently derived, largely European clade. Whilst our data are consistent with BPV-1 originating in cattle, we found evidence of multiple, probably relatively recent, cross-species transmission events into horses. We also demonstrated the high prevalence of one particular sequence variant (variant 20), and suggest this may indicate that this variant shows a fitness advantage in equids. Although strong host specificity remains the norm in papillomaviruses, our results demonstrate that exceptions to this rule exist and can become epidemiologically relevant.
Vaccinia virus (VACV) is a large dsDNA virus encoding ~200 proteins, several of which inhibit apoptosis. Here, a comparative study of anti-apoptotic proteins N1, F1, B13 and Golgi anti-apoptotic protein (GAAP) in isolation and during viral infection is presented. VACVs strains engineered to lack each gene separately still blocked apoptosis to some degree because of functional redundancy provided by the other anti-apoptotic proteins. To overcome this redundancy, we inserted each gene separately into a VACV strain (vv811) that lacked all these anti-apoptotic proteins and that induced apoptosis efficiently during infection. Each protein was also expressed in cells using lentivirus vectors. In isolation, each VACV protein showed anti-apoptotic activity in response to specific stimuli, as measured by immunoblotting for cleaved poly(ADP ribose) polymerase-1 and caspase-3 activation. Of the proteins tested, B13 was the most potent inhibitor, blocking both intrinsic and extrinsic stimuli, whilst the activity of the other proteins was largely restricted to inhibition of intrinsic stimuli. In addition, B13 and F1 were effective blockers of apoptosis induced by vv811 infection. Finally, whilst differences in induction of apoptosis were barely detectable during infection with VACV strain Western Reserve compared with derivative viruses lacking individual anti-apoptotic genes, several of these proteins reduced activation of caspase-3 during infection by vv811 strains expressing these proteins. These results illustrated that vv811 was a useful tool to determine the role of VACV proteins during infection and that whilst all of these proteins have some anti-apoptotic activity, B13 was the most potent.
Human herpesvirus 6 (HHV-6) glycoprotein M (gM) is an envelope glycoprotein that associates with glycoprotein N (gN), forming the gM/gN protein complex, in a similar manner to the other herpesviruses. Liquid chromatography-MS/MS analysis showed that the HHV-6 gM/gN complex interacts with the v-SNARE protein, vesicle-associated membrane protein 3 (VAMP3). VAMP3 colocalized with the gM/gN complex at the trans-Golgi network and other compartments, possibly the late endosome in HHV-6-infected cells, and its expression gradually increased during the late phase of virus infection. Finally, VAMP3 was incorporated into mature virions and may be transported with the gM/gN complex.
Human immunodeficiency virus type 2 (HIV-2) infects about two million people worldwide. HIV-2 has fewer treatment options than HIV-1, yet may evolve drug resistance more quickly. We have analysed several novel drugs for anti-HIV-2 activity. It was observed that 5-azacytidine, clofarabine, gemcitabine and resveratrol have potent anti-HIV-2 activity. The EC50 values for 5-azacytidine, clofarabine and resveratrol were found to be significantly lower with HIV-2 than with HIV-1. A time-of-addition assay was used to analyse the ability of these drugs to interfere with HIV-2 replication. Reverse transcription was the likely target for antiretroviral activity. Taken together, several novel drugs have been discovered to have activity against HIV-2. Based upon their known activities, these drugs may elicit enhanced HIV-2 mutagenesis and therefore be useful for inducing HIV-2 lethal mutagenesis. In addition, the data are consistent with HIV-2 reverse transcriptase being more sensitive than HIV-1 reverse transcriptase to dNTP pool alterations.
Despite the success of combined antiretroviral therapy in controlling viral replication in human immunodeficiency virus (HIV)-infected individuals, HIV-associated neurocognitive disorders, commonly referred to as neuroAIDS, remain a frequent and poorly understood complication. Infection of CD8+ lymphocyte-depleted rhesus macaques with the SIVmac251 viral swarm is a well-established rapid disease model of neuroAIDS that has provided critical insight into HIV-1-associated neurocognitive disorder onset and progression. However, no studies so far have characterized in depth the relationship between intra-host viral evolution and pathogenesis in this model. Simian immunodeficiency virus (SIV) env gp120 sequences were obtained from six infected animals. Sequences were sampled longitudinally from several lymphoid and non-lymphoid tissues, including individual lobes within the brain at necropsy, for four macaques; two animals were sacrificed at 21 days post-infection (p.i.) to evaluate early viral seeding of the brain. Bayesian phylodynamic and phylogeographic analyses of the sequence data were used to ascertain viral population dynamics and gene flow between peripheral and brain tissues, respectively. A steady increase in viral effective population size, with a peak occurring at ~50nndash;80 days p.i., was observed across all longitudinally monitored macaques. Phylogeographic analysis indicated continual viral seeding of the brain from several peripheral tissues throughout infection, with the last migration event before terminal illness occurring in all macaques from cells within the bone marrow. The results strongly supported the role of infected bone marrow cells in HIV/SIV neuropathogenesis. In addition, our work demonstrated the applicability of Bayesian phylogeography to intra-host studies in order to assess the interplay between viral evolution and pathogenesis.
In the past decade, there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated llsquo;Nhumirim virusrrsquo;; NHUV) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential and 3' untranslated region (UTR) sequence homology with alternative flaviviruses, and evaluated the virusrrsquo;s capacity to suppress replication of representative Culex spp.-vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell lines were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3' UTR indicated NHUV to be most similar to viruses within the yellow fever serogroup and Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. NHUV was found to share the fewest conserved sequence elements when compared with traditional insect-specific flaviviruses. This suggests that, despite apparently being insect specific, this virus probably diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in a significant reduction in virus production of West Nile virus (WNV), St Louis encephalitis virus (SLEV) and Japanese encephalitis virus. The inhibitory effect was most effective against WNV and SLEV with over a 106-fold and 104-fold reduction in peak titres, respectively.
Aphid lethal paralysis virus (ALPV; family Dicistroviridae) was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi. ALPV-like virus sequences have been reported from many insects and insect predators. We identified a new isolate of ALPV (ALPV-AP) from the pea aphid, Acyrthosiphon pisum, and a new isolate (ALPV-DvV) from western corn rootworm, Diabrotica virgifera virgifera. ALPV-AP has an ssRNA genome of 9940 nt. Based on phylogenetic analysis, ALPV-AP was closely related to ALPV-AM, an ALPV isolate from honeybees, Apis mellifera, in Spain and Brookings, SD, USA. The distinct evolutionary branches suggested the existence of two lineages of the ALPV virus. One consisted of ALPV-AP and ALPV-AM, whilst all other isolates of ALPV grouped into the other lineage. The similarity of ALPV-AP and ALPV-AM was up to 88 % at the RNA level, compared with 78nndash;79 % between ALPV-AP and other ALPV isolates. The sequence identity of proteins between ALPV-AP and ALPV-AM was 98nndash;99 % for both ORF1 and ORF2, whilst only 85nndash;87 % for ORF1 and 91nndash;92 % for ORF2 between ALPV-AP and other ALPV isolates. Sequencing of RACE (rapid amplification of cDNA ends) products and cDNA clones of the virus genome revealed sequence variation in the 5' UTRs and in ORF1, indicating that ALPV may be under strong selection pressure, which could have important biological implications for ALPV host range and infectivity. Our results indicated that ALPV-like viruses infect insects in the order Coleoptera, in addition to the orders Hemiptera and Hymenoptera, and we propose that ALPV isolates be classified as two separate viral species.
The major envelope fusion protein F of the budded virus of baculoviruses consists of two disulfide-linked subunits: an N-terminal F2 subunit and a C-terminal, membrane-anchored F1 subunit. There is one cysteine in F2 and there are 15 cysteines in F1, but their role in disulfide linking is largely unknown. In this study, the inter- and intra-subunit disulfide bonds of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) F protein were analysed by site-directed mutagenesis. Results indicated that in a functional F protein, an inter-subunit disulfide bond exists between amino acids C108 (F2) and C241 (F1). When C241 was mutated, an alternative disulfide bond was formed between C108 and C232, rendering F non-functional. No inter-subunit bridge was observed in a double C232/C241 mutant of F1. C403 was not involved in the formation of inter-subunit disulfide bonding, but mutation of this amino acid decreased viral infectivity significantly, suggesting that it might be involved in intra-subunit disulfide bonds. The influence of reductant [tris(2-carboxyethyl) phosphine (TCEP)] and free-thiol inhibitors [4-acetamido-4'-maleimidylstilbene 2,2'-disulfonic acid (AMS) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] on the infectivity of HearNPV was tested. The results indicated that TCEP greatly decreased the infection of HzAm1 cells by HearNPV. In contrast, AMS and DTNB had no inhibitory effect on viral infectivity. The data suggested that free thiol/disulfide isomerization was not likely to play a role in viral entry and infectivity.
Viral suppressors of RNA silencing (VSRs) are critical for the success of virus infection and efficient accumulation of virus progeny. The chrysanthemum virus B p12 protein acts as a transcription factor to regulate cell size and proliferation favourable for virus infection. Here, we showed that the p12 protein suppressed RNA silencing and was able to complement a VSR-deficient unrelated virus. Moreover, p12 counter-silencing activity could be uncoupled from its function as a transcription factor in the nucleus. The altered p12 protein, which lacked a nuclear localization signal and was not imported into the nucleus, was able to suppress RNA silencing as efficiently as the native protein. The data revealed new aspects of p12 functioning and identified a novel role for this viral zinc-finger transcription factor. The results provided a general insight into one of the activities of the p12 protein, which appeared to possess more than one function.
|Journal of General Virology Publish Ahead of Print|
A fundamental aspect of herpesviruses is their ability to establish a life long latent infection within the host. Current research would suggest that small regulatory RNAs called microRNAs play a central role in all three aspects of virus latency and persistence. In this review we will discuss the role of miRNAs in virus persistence and latency, specifically focusing on herpesviruses. We will cover the current knowledge on the role of miRNAs in establishing and maintaining virus latency and promoting survival of infected cells through targeting of both viral and cellular transcripts, highlighting key publications in the field. We will also discuss potential areas of future research and how novel technologies may aid in determining how miRNAs shape virus latency in the context of herpesvirus infections.
The stability and conservation of the sequences of RNA viruses in the field and the high error rates measured in vitro are paradoxical. The field stability indicates that there are very strong selective constraints on sequence diversity. The nature of these constraints is discussed. Apart from constraints on variation in cis-acting RNA and the amino acid sequences of viral proteins, there are other ones relating to the presence of specific dinucleotides such CpG and UpA as well as the importance of RNA secondary structures and RNA degradation rates. Recent other constraints identified in other RNA viruses such as effects of secondary RNA structure on protein folding or modification of cellular tRNA complements are also discussed. Using the family Paramyxoviridae I show that the codon usage pattern (CUP) is (i) specific for each virus species and (ii) that it is markedly different from the host - it does not vary even in vaccine viruses that have been derived by passage in a number of inappropriate host cells. The CUP might thus be an additional constraint on variation and I propose the concept of codon constellation to indicate the informational content of the sequences of RNA molecules relating not only to stability and structure but also to the efficiency of translation of a viral mRNA resulting from the CUP and the numbers and position of rare codons.
Two strains of African swine fever virus (ASFV), the high virulence Lisboa60 (L60) and the low virulence NH/P68 (NHV), which have been used previously in effective immunization/protection studies, were sequenced. Both were isolated in Portugal in the 11-year period after the introduction of ASFV into the European continent in 1957. The predicted proteins coded by both strains were compared, and where differences were found, these were also compared to other strains of known virulence. This highlighted several genes with significant alterations in low virulence strains of ASFV that may constitute virulence factors, several of which still lacking knowledge regarding their function. Phylogenetic analysis grouped L60 and NHV closest with other P72 genotype I ASFV strains from Europe and West Africa, consistent with the assumed West-African origin of all European strains. Interestingly, a relatively lower genomic identity exists between L60 and NHV, both isolated in a similar geographical location 8-years apart, than with other European and West-African strains isolated later in time and in more distant locations. This may reflect the intensive passage in tissue culture during the early 60rrsquo;s of a Portuguese isolate to obtain an attenuated vaccine, which may have led to NHV. This study contributes to a better understanding of the evolution of ASFV and defines additional potential virulence genes for future studies of pathogenesis towards the development of effective vaccines.
Human Papillomaviruses (HPVs) have been detected in urban wastewaters, demonstrating that epitheliotropic viruses can find their way into sewage through the washing of skin and mucous membranes. Papillomavirus shedding through feces is still an unexplored issue. The objective of the present study was to investigate the presence of HPVs in stool samples. We analyzed 103 fecal specimens collected from hospitalized patients with diarrhea using validated primers able to detect aalpha;, bbeta; and HPVs. PCR products underwent sequencing analysis and sequences were aligned to reference genomes from the Papillomavirus Episteme database. A total of 15 sequences were characterized from the fecal samples. Thirteen samples (12.6%) were positive for 9 genotypes belonging to aalpha; and bbeta; genus: HPV32 (LR, aalpha;1), HPV39 (HR, aalpha;7), HPV44 (LR, aalpha;10), HPV8 (bbeta;1), HPV9, HPV23, HPV37, HPV38, and HPV120 (bbeta;2). Two putative novel genotypes of the bbeta; genus, species 1 and 2 were also detected. The tissue (s) of origin is unknown, since feces can collect HPVs originating from or passing through the entire digestive system. To our knowledge, this is the first investigation on the occurrence and diversity of HPVs in fecal samples. Results from this study demonstrate that HPVs can find their way into sewage as a result of shedding in the feces. This highlights the need of further studies aimed at understanding the prevalence of HPV in different water environments and the potential for waterborne transmission.
The unfolded protein response (UPR) is a cellular defense mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1aalpha; become activated. PERK phosphorylates eIF2aalpha; leading to a general inhibition of cellular translation, while the expression of transcription factor ATF4 is upregulated. Active IRE1aalpha; splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Herein we demonstrate that transient expression of chikungunya virus (CHIKV) (Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2aalpha; and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIK infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV infected cells. Individual expression of CHIKV non-structural proteins (nsP) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that render nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER, but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.
T-lymphotropic feline leukemia virus (FeLV-T), a highly pathogenic variant of FeLV, induces severe immunosuppression in cats. FeLV-T is fusion-defective because in its PHQ motif, a gammaretroviral consensus motif in the N-terminal of an envelope protein, histidine is replaced with aspartate. Infection by FeLV-T requires FeLIX, a truncated envelope protein encoded by an endogenous FeLV, for transactivation of infectivity and Pit1 for binding FeLIX. Although Pit1 is present in most tissues in cats, the expression of FeLIX is limited to certain cells in lymphoid organs. Therefore, the host cell range of FeLV-T was thought to be restricted to cells expressing FeLIX. However, because FeLIX is a soluble factor and expressed constitutively in lymphoid organs, we presumed it to be present in blood and evaluated its activities in sera of various mammalian species using a pseudotype assay. We demonstrated that cat serum has FeLIX activity at a functional level, suggesting that FeLIX is present in cats and FeLV-T may be able to infect cells expressing Pit1 regardless of the expression of FeLIX in vivo. In addition, FeLIX activities in sera were detected only in domestic cats but not in other feline species tested. To our knowledge, this is the first report to prove that a large amount of truncated envelope protein of endogenous retrovirus is circulating in the blood to facilitate the infection of a pathogenic exogenous retrovirus.
Neutralising antibodies (NAbs) are believed to comprise an essential component of the protective immune response induced by vaccines against FIV and HIV infections. However, relatively little is known about the role of NAbs in controlling FIV infection and subsequent disease progression. Here we present studies examining the neutralisation of HIV-luciferase pseudotypes bearing homologous and heterologous FIV Envs (n=278) by sequential plasma samples collected at 6 month intervals from naturally infected cats (n=38) over a period of 18 months. We evaluated the breadth of the NAb response against non-recombinant homologous and heterologous clade A and clade B viral variants as well as recombinants and assessed the results, testing for evidence of an association between the potency of the NAb response and the duration of infection, CD4 T lymphocyte numbers, health status and survival times of the infected cats. Neutralisation profiles varied significantly between FIV infected cats and strong autologous neutralisation, assessed using luciferase based in vitro assays, did not correlate with the clinical outcome. No association was observed between strong NAb responses and either improved health status or increased survival time of infected animals, implying that other protective mechanisms are likely to be involved. Similarly, no correlation was observed between the development of autologous NAbs and the duration of infection. Furthermore, cross-neutralising antibodies were evident in only a small proportion (13%) of cats.
Pleconaril is a capsid-inhibitor previously used to treat enterovirus infections. A pleconaril resistant Echovirus (E) 11 strain was identified before pleconaril treatment was given in an immune-compromised patient. The patient was also treated with immune-globulins (IVIG) for a long period, but remained unresponsive. The pleconaril resistant strains could not be neutralized in vitro confirming IVIG treatment failure. To identify the basis of pleconaril resistance genetic and structural analyses were conducted. Analysis of a modelled viral capsid indicated conformational changes in the hydrophobic pocket that could prevent pleconaril docking. Substitutions (V117I, V119M, and I188L) in the pleconaril resistant viruses were found in the pocket region of VP1. Modelling suggests V119M could confer resistance, most probably due to the protruding sulphate side chain of methionine. Although pleconaril resistance induced in vitro in a susceptible E11 clinical isolate was characterised by a different substitution (I183M), resistance is suggested to also results by a similar mechanism i.e. due to a protruding sulphate side chain of methionine. Our results show resistant strains that arise in vivo display different markers than those identified in vitro and suggest that multiple factors may play a role in pleconaril resistance in patient strains. Based on IVIG treatment failure, we predict one of these factors could be immune-related. Thus both IVIG and capsid-inhibitors target the viral capsid and can induce mutations that can be cross-reactive enabling escape from both IVIG and drug. This could limit treatment options and should be investigated further.
Salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared to terrestrial alphavirus replicons (Olsen et al., 2013a). In this study, we have compared several modifications to a SAV replicon construct and analyzed their influence on foreign gene expression. We found that with an insertion of a translational enhancer consisting of the N-terminal 102 nucleotides of the capsid gene together with a nucleotide sequence encoding the FMDV 2A peptide caused a significant increase of the EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 5rrsquo; end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at 3rrsquo; end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production which can be applied for immunization of salmonid fish in the future.
Crimean-Congo hemorrhagic fever virus (CCHFV) is a Nairovirus of the family Bunyaviridae, which can cause severe hemorrhagic fever in humans, with mortality rates above 30%. CCHFV is the most widespread of the tick-borne human viruses and it is endemic in areas of central Asia, the Middle East, Africa and southern Europe. Its viral genome consists of three negative sense RNA segments. The large segment (L) encodes a viral RNA-dependent RNA polymerase (L protein), the small segment (S) encodes the nucleocapsid protein (N) and the medium segment (M), encodes for the envelope proteins. The nucleocapsid protein of bunyaviruses binds genomic RNA forming the viral RiboNucleoProtein (RNP) complex. The L protein interacts with these RNP structures allowing the initiation of viral replication. The nucleocapsid protein also interacts with actin, although the regions and specific residues involved in these interactions have not been yet described. Here, by means of immunoprecipitation and immunofluorescence assays we identify the regions within the CCHFV nucleocapsid protein implicated in homo-oligomerization and actin binding. We describe the interaction of N with the CCHFV L protein, and identify the amino- and carboxy-terminal regions within the L protein that might be necessary for the formation of these N-L protein complexes. These results may guide the development of potent inhibitors of these complexes that could potentially block CCHFV replication.
Development of numerous advanced techniques in recent years have allowed detection of the pathological prion protein (PrPTSE), the unique marker of transmissible spongiform encephalopathies (TSEs, or prion diseases), in the blood of animals and humans; however, an ante-mortem screening test that can be used for the routine diagnosis of human prion diseases remains unavailable. A critical, analytical review of all the diagnostic assays developed to date will allow an evaluation of progress in this field and may facilitate the identification of the possible reason/s for this delay. Thus, in this review, I provide a detailed overview of the techniques currently available for detecting PrPTSE and other markers of the disease in blood, as well as an analysis of the significance, feasibility, reliability, and the application spectrum for these methods. I highlight that factors intrinsic and extrinsic to blood may interfere with the detection of PrPTSE/prions, and that this is not yet taken into account in current tests. This may inspire researchers in this field to not only aspire to increase test sensitivity but also to adopt other strategies in order to identify and overcome the limitations that hamper the development of a successful routine blood test for prion diseases.
In simian virus 40 (SV40) and several other polyomaviruses, the TATA box of the early promoter is embedded in an AT tract that is also an essential part of the replication origin. We generated an "AT trap", an SV40 genome lacking the AT tract and unable to grow in CV-1 monkey cells. Co-transfection of the AT trap with oligonucleotides containing AT tracts of human polyomaviruses, a poly (A:T) tract, or variants of the SV40 wild type sequence all restored infectious virus. In a transfection of the AT trap without suitable oligonucleotide, an AT-rich segment was incorporated, stemming either from bovine (calf serum) or monkey (host cell) DNA. Similarly, when cells were grown with human serum, a human DNA segment was captured by SV40 to substitute for the missing AT stretch. We conclude that the virus is quite opportunistic in accepting heterologous substitutes, and that even low-abundance DNA from serum can be incorporated into the viral genome.
In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2), consistently gained positively charged amino acids in the putative heparan sulfate-binding pocket (VP2 bbeta;E-bbeta;F loop, VP1 C terminus and VP3 bbeta;-B knob), surrounding the five-fold symmetry axis (VP1 bbeta;F-bbeta;G loop) and at other discrete sites on the capsid (VP3 bbeta;G-bbeta;H loop, VP1 C terminus, VP2 bbeta;C strand and VP1 bbeta;G-bbeta;H loop). A lysine insertion in the VP1 bbeta;F-bbeta;G loop of two of the BHK-21 adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line suggesting their possible role in ionic interaction with ubiquitous negatively charged cell surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared to the lower passage viruses with wild-type genotypes. Substitutions fixed in the VP1 bbeta;G-bbeta;H loop (-3, -2 and +2 'RGD' positions) or in the structural element known to be juxtaposed against that loop (VP1 bbeta;B-bbeta;C loop) suggest their possible role in modulating the efficiency and specificity of interaction of 'RGD' motif with aalpha;V-integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production.
Hepatitis E virus (HEV), the causative agent of hepatitis E, is a single-stranded positive-sense RNA virus belonging to the family Hepeviridae. At least four genotypes of the family infect humans: genotypes 1 and 2 are transmitted to humans through contaminated water, while genotypes 3 and 4 are zoonotic and have animal reservoirs. A novel strain of HEV recently identified in rabbits is a distant member of genotype 3, and thus poses a potential risk of zoonotic transmission to humans. The objective of this study was to construct and characterize an infectious cDNA clone of the rabbit HEV. Two full-length cDNA clones of rabbit HEV, pT7g-rabHEV and pT7-rabHEV, were constructed and their infectivity was tested by in vitro transfection of Huh7 human liver cells and by direct intrahepatic inoculation of rabbits with capped RNA transcripts. Results showed that positive signal for rabbit HEV protein was detected by an immunofluorescence assay with a HEV-specific antibody in Huh7 human liver cells transfected with capped RNA transcripts from the two full-length cDNA clones. Rabbits intrahepatically inoculated with capped RNA transcripts from each of the two clones developed active HEV infection as evidenced by seroconversion to anti-HEV antibodies, and detection of rabbit HEV RNA in sera and feces of inoculated animals. The availability of a rabbit HEV infectious cDNA clone now affords us the ability to delineate the mechanism of HEV replication and cross-species infection in a small animal model.
Shrews are small insectivorous mammals that are distributed worldwide. Similar to rodents, shrews live on the ground and are commonly found near human residences. In this study, we investigated the enteric virome of wild shrews in the genus Crocidurinae using a sequence-independent viral metagenomics approach. A large portion of the shrew enteric virome was composed of insect viruses, while novel viruses including cyclovirus, picornavirus and picorna-like virus were also identified. Several cycloviruses, including variants of human cycloviruses detected in cerebrospinal fluid (CSF) and stool, were detected in wild shrews at a high prevalence rate. The identified picornavirus is distantly related to human parechovirus, inferring the presence of a new genus in this family. The identified picorna-like viruses were characterized as different species of calhevirus 1, which was previously discovered in human stool. Complete or nearly complete genome sequences of these novel viruses were determined in this study and then were subjected to further genetic characterization. Our study provides an initial view of the diversity and distinctiveness of the shrew enteric virome and highlights unique novel viruses related to human stool-associated viruses.
Coronaviruses are enveloped RNA viruses that have evolved complex relationship with their host cells and modulate their lipid composition, lipid synthesis and signaling of host cell. Lipid rafts, enriched in sphingolipids, cholesterol and associated proteins, are special plasma membrane microdomains involved in several processes of viruses infections. The extraction of cholesterol leads to disorganization of lipid microdomains and to dissociation of proteins bound to the lipid rafts. Because of cholesterol-rich microdomains appear to be a general feature of the entry mechanism of noneneveloped viruses and of several coronaviruses, the purpose of this study was to analyze the contribution of lipids in the infectivity of canine coronavirus (CCoV). CCoV lifecycle is closely connected to plasma membrane cholesterol, from cell entry to viral particle production. The methyl-bbeta;-cyclodextrin (Mbbeta;CD) was employed to remove cholesterol and to disrupt the lipid rafts. Cholesterol depletion from cellular membrane resulted in a dose-dependent reduction but not in the abolishment of virus infectivity and at a concentration of 15 mM, the reduction of the infection rate was about 68%. Mbbeta;CD treatment to verify if cholesterol in the envelope was required for CCoV infection, resulted in a dose-dependent inhibitory effect and at a concentration of 9 mM Mbbeta;CD infectivity was reduced by about 73%. Since viral entry would constitute a target for antiviral strategies, inhibitory molecules interacting with viral and/or cellular membranes or interfering with the function of lipid metabolism, could offer strong antiviral potential. It will be interesting in future to analyze the membrane microdomains in CCoV envelope.
Several members of the Circoviridae family have been shown to encode proteins with apoptotic activity. For example, both Porcine Circovirus Type 2 (PCV2) and Chicken Anemia Virus (CAV) encode a third viral protein (VP3) that has been shown to be cytotoxic. Interestingly, in the case of the CAV protein (deemed Apoptin), apoptosis is specific to transformed cell types. Similarities in genome structure and organization suggest that PCV Type 1 (PCV1) may also contain a third open reading frame that codes for a protein with homologous activity. To investigate this, ORF prediction followed by gene expression analyses were conducted on a gene found to be homologous to CAV and PCV2 VP3. Our data presented herein elucidate a putative ORF3 that codes for a viral protein with functional similarity to that of Apoptin and PCV2 VP3. Unlike its homologs, sequence analysis revealed a highly hydrophobic, extended C terminal domain in PCV1 VP3, which harbors a strong nuclear export signal. Subcellular localization analysis demonstrated divergent PCV1 VP3 localization patterns compared to that of CAV VP3. Interestingly, cytotoxicity studies revealed evidence that apoptosis may be selective to transformed cell types, similar to Apoptin. These results indicate that nuclear localization of PCV1 VP3 is neither necessary for induction of apoptosis nor for transformed cell selectivity unlike CAV VP3, suggesting an alternate mechanism of action.
Coxsackievirus B4 (CV-B4) belongs to the genus Enterovirus within family Picornaviridae. To investigate target proteins recognised by T-cells in human enterovirus B infections, viral-encoded structural (VP0 [VP4 and VP2], VP1, VP3) and non-structural (2A, 2B, 2C, 3C and 3D) proteins were expressed and purified in E. coli. Peripheral blood of 19 healthy adult donors was used to create enterovirus-specific T-cell lines by repeated stimulation with CV-B4 cell lysate antigen. T-cell lines responded in individual patterns, and responses to all purified proteins were observed. The most often recognised enteroviral protein was VP0, which is the fusion between the most conserved structural proteins, VP4 and VP2. T-cell responses to VP0 were detected in 15 of 19 (79%) donor lines. Non-structural 2C protein was recognised in 11 of 19 (58%) lines, and 11 of 19 (58%) lines also had a response to 3D protein. Furthermore, responses to other non-structural proteins (2A, 2B and 3C) were also detected. T-cell responses did not correlate clearly to the individual HLA-DR-DQ phenotype or the history of past coxsackie B virus infections of the donors.
Feline rotaviruses, members of Rotavirus A, are an infrequent source of zoonotic infections, and were previously shown by RNA-RNA hybridization assays to possess two distinct genomic RNA constellations each of which was represented by strains FRV-1 and FRV64. Due to the lack of whole genome sequence information of FRV-1, human rotavirus strain AU-1 has been used as a surrogate for the genotype constellation of feline rotaviruses. The aim of this study was to determine the whole genome sequence of FRV-1 and FRV64 to help understand the genetic relationships among existing feline rotaviruses from the evolutionary perspective. The genotype constellation of FRV-1 and FRV64 were G3-P-I3-R3-C3-M3-A3-N3-T3-E3-H3 and G3-P-I3-R3-C2-M3-A9-N2-T3-E3-H6, respectively. FRV-1 has a genotype constellation identical with that of the AU-1 strain. Although for individual genes they shared lineages with the exception of genes encoding VP2, VP6 and VP7, the sequence identity between FRV-1 and AU-1 was considered to be sufficiently high for the AU-1 to be regarded as an example of the direct transmission of a feline rotavirus to a child. On the other hand, the FRV64 strain was not only similar in all the 11 genome segments to another feline rotavirus strain, Cat97, but also to canine rotavirus strains (K9 and CU-1) and feline/canine-like human rotavirus strains (Ro1845 and HCR3). In conclusion, this study revealed intermingled sharing of genotypes and lineages among feline rotaviruses, suggesting the occurrence of frequent reassortment events over the course of evolution to emerge in four genotype constellations represented by FRV-1, FRV64/Cat97, Cat2 and BA222 strains.
Retinoic acid-induced gene I (RIG-I), plays a crucial role in sensing viral RNA and facilitating the production of IFN-bbeta;. It varies in length and sequence among different species. The present study assessed the functional differences among RIG-I proteins derived from mammals and birds. The transfection of duck caspase recruitment domains (CARDs) and RIG-I (dCARDs and dRIG-I) and goose CARDs and RIG-I (gCARDs and gRIG-I) into DF-1 cells increased the production of IFN-bbeta; mRNA and IFN-stimulated genes and decreased influenza A virus (IAV) replication, whereas, human CARDs and RIG-I (hCARDs and hRIG-I) and mouse CARDs and RIG-I (mCARDs and mRIG-I) had no effect. In 293T and A549 cells, hCARDs had the strongest IFN-inducing activity, followed by mCARDs, dCARDs, and gCARDs. The IFN-inducing activity of hRIG-I was stronger than that of mRIG-I, dRIG-I, and gRIG-I, in that order. The results also showed that although the ability dCARDs to activate IFN was stronger than that of gCARDs in DF-1, 293T, and A549 cells, dRIG-I had a weaker ability to activate IFN than gRIG-I in DF-1 with or without IAV infection. Taken together, these data suggest that RIG-I protein has different amino acid sequences and functions among species. This genetic and functional diversity renders it flexible, adaptable, and capable of recognizing many viruses in different species.
Bacteriophages and their derivatives are continuously gaining impetus as viable alternative therapeutic agents to control harmful multi-drug resistant bacterial pathogens, particularly in the food industry. The reduced efficacy of conventional antibiotics has resulted in a quest to find novel alternatives in the war against infectious disease. This study describes the full genome sequence of Cronobacter phage Ss1, with subsequent cloning and expression of its endolysin, capable of hydrolysing Gram-negative peptidoglycan. Cronobacter phage Ss1 is composed of 42,205 bp of double stranded DNA with a GC content of 46.1%. 57 ORFs were identified of which 18 could be assigned a putative function based on similarity to characterised proteins. The genome of Cronobacter phage Ss1 showed little similarity to the any other bacteriophage genomes available in the database and thus was considered unique. In addition, functional analysis on the predicted endolysin (LysSs1) was also investigated. Zymographic experiments demonstrated the hydrolytic activity of LysSs1 against Gram-negative peptidoglycan, thus highlighting the potential use of this enzyme as an antibacterial against Gram-negative pathogens.
Human cytomegalovirus (HCMV) infected cells a dramatic remodeling of the nuclear architecture linked to the creation, utilization and manipulation of sub-nuclear structures. This review outlines the involvement of several viral and cellular sub-nuclear structures in areas of HCMV replication and virus-host interaction that include viral transcription, viral DNA synthesis and the production of DNA filled viral capsids. The structures discussed include those that promote or impede HCMV replication (such as viral replication compartments and PML nuclear bodies, respectively) and those whose role in the infected cell is unclear (for example, nucleoli and nuclear speckles). Viral and cellular proteins associated with sub-nuclear structures are also discussed. The data reviewed here highlights advances in our understanding of HCMV biology and emphasizes the complexity of HCMV replication and virus-host interaction in the nucleus.
Human herpesvirus-6A (HHV-6A) is rarer than HHV-6B in many infant populations. However, they are similarly prevalent as germline chromosomally integrated genomes (ciHHV-6A/B). This integrated form affects 0.1-1% human populations, where potentially virus gene expression could be in every cell, although virus relationships and health effects are not clear. In a Czech/German patient cohort ciHHV-6A was more common and diverse than ciHHV-6B. Quantitative PCR, nucleotide sequencing and telomeric integration site amplification characterised ciHHV-6 in 44 German myocarditis/cardiomyopathy and Czech malignancy/inflammatory disease (MI) patients plus donors. Comparisons were made to sequences from global virus reference strains, and blood DNA from childhood-infections from Zambia (HHV-6A mainly) and Japan (HHV-6B). The MI cohort were 95%(19/21) ciHHV-6A, the cardiac cohort 65%(13/20) ciHHV-6B, suggesting different disease links. Reactivation was supported by findings of 1) recombination between ciHHV-6A and HHV-6B genes in 20%(4/21) of the MI cohort; 2) expression in a subset, of early/late transcripts tested from encoded inflammatory mediator genes chemokine receptor U51 and chemokine U83, both identical to ciHHV-6A DNA sequences; and 3) superinfection shown by deep sequencing identifying minor virus-variants only in ciHHV-6A which expressed transcripts, indicating virus infection reactivates latent ciHHV-6A. Half the MI cohort had more than two copies/cell, median 5.2, indicative of reactivation. Remarkably, the integrated genomes encoded the secreted-active form of virus chemokines, rare in virus from childhood-infections. This shows integrated virus genomes can contribute new human genes with links to inflammatory pathology and supports ciHHV-6A reactivation as a source for emergent infection.
Part of the Togaviridae family, alphaviruses are arthropod-borne viruses that are widely distributed throughout the globe. Alphaviruses are able to infect a variety of vertebrate hosts, but in humans infection can result in extensive morbidity and mortality. Symptomatic infection can manifest as fever, an erythematous rash and/or significant inflammatory pathologies such as arthritis and encephalitis. Recent overwhelming outbreaks of alphaviral disease have highlighted the void in our understanding of alphavirus pathogenesis and the re-emergence of alphaviruses has given new impetus to anti-alphaviral drug design. In this review, the development of viable mouse models of Old Word and New World alphaviruses is examined. How mouse models that best replicate human disease have been used to elucidate the immunopathology of alphavirus pathogenesis and trial novel therapeutic discoveries is also discussed.
Summary: Chikungunya virus (CHIKV) has recently affected millions of people and with rare cases of encephalopathy and encephalitis essentially in neonates. We herein tested the capacity of mouse brain cells to control infection through innate immune antiviral responses. In vitro, CHIKV infected principally a subpopulation of mouse GFAP+ primary astrocytes. Oligodendrocytes and neurons could also be infected. An innate immune response was engaged by CHIKV-infected astrocytes and with elevated expression of mRNAs for IFN-aalpha;-bbeta;, inflammatory cytokines (e.g. IL-1bbeta;, IL-12, IL-10, IL-24), and proapoptotic factors (e.g. TNF-aalpha;, FasL, Lymphotoxin B). Programmed-cell death through the intrinsic caspase 9 pathway was observed by immunofluorescence in infected astrocytes, neurons but not in oligodendrocytes. Interestingly, microglia were not replicating CHIKV but responded as indicated by elevated MAPK activity. Intracerebroventricular injection of CHIKV in neonate mice led to the infection of astrocytes. The astrogliosis response was accompanied by a dendritic CD206+ cell mobilization restricted to the site of infection. Our results support the paradigm that a multifaceted innate immune response can be mobilized by both, professional immune and glial cells, to control CHIKV neuroinfection events in neonates.
Core plays a critical role during HCV assembly, not only as a structural component of the virion, but also as a regulator of the formation of assembly sites. In this study, we observed that core is expressed later than other HCV proteins in a single viral cycle assay, resulting in a relative increase of core expression during a late step of the viral life cycle. This delayed core expression results from an increase of core half-life, indicating that core is initially degraded and is stabilized at a late step of the HCV life cycle. A stabilization-mediated delayed kinetics of core expression was also observed using heterologous expression systems. Core stabilization did not depend on its interaction with non-structural proteins or lipid droplets but was correlated to its expression levels and its oligomerization status. Therefore in the course of a HCV infection, core stabilization likely occurs when the prior amplification of the viral genome during an initial replication step allows core to be synthesized at higher levels as a stable protein during the assembly step of the viral life cycle.
Vaccinia virus (VACV) is a large DNA virus that replicates in the cytoplasm and encodes about 200 proteins of which ~50% may be non-essential for virus replication. These proteins enable VACV to suppress transcription and translation of cellular genes, to inhibit the innate immune response, to exploit microtubule and actin based transport for virus entry and spread, and to subvert cellular metabolism for the benefit of the virus. VACV strain WR protein C16 induces stabilisation of the hypoxia inducible transcription factor (HIF)-1aalpha; by binding to the cellular oxygen sensor prolyl hydroxylase domain containing protein (PHD)2. Stabilisation of HIF-1aalpha; is induced by several virus groups, but the purpose and consequences are unclear. Here, 1H-NMR spectroscopy and liquid chromatography-mass spectrometry are used to investigate the metabolic alterations during VACV infection in HeLa and 2FTGH cells. The role of C16 in such alterations was examined by comparing infection with wild type VACV (strain WR) and a derivative virus lacking gene C16L (vC16). Compared to uninfected cells, VACV infection caused increased nucleotide and glutamine metabolism. In addition, there were increased concentrations of glutamine derivatives in cells infected with wild type VACV compared to vC16. This indicates that C16 contributes to enhanced glutamine metabolism, and this may help preserve tricarboxylic acid cycle activity. These data show that VACV infection reprograms cellular energy metabolism towards increased synthesis of the metabolic precursors utilised during viral replication, and that C16 contributes to this anabolic reprogramming of the cell, likely via the stabilisation of HIF-1aalpha;.
Introductions of H7 Influenza A virus (IAV) from wild birds into poultry have been documented worldwide, resulting in varying degrees of morbidity and mortality. H7 IAV infection in domestic poultry has served as a source of human infection and disease. We report the detection of H7N9 subtype IAV in Minnesota turkey farms during 2009 and 2011. The full-genome was sequenced from eight isolates as well as the hemagglutinin (HA) and neuraminidase (NA) gene segments of H7 and N9 virus subtypes for 108 isolates from North American wild birds between 1986 and 2012. Through maximum likelihood and coalescent phylogenetic analyses, we identified the recent H7 and N9 IAV ancestors of the turkey-origin H7N9 IAV, estimated the time and geographic origin of the ancestral viruses, and determined the relatedness between the 2009 and the 2011 turkey-origin H7N9 IAV. Analyses supported that the 2009 and the 2011 viruses were distantly related genetically, suggesting that the two outbreaks arose from independent introduction events from wild birds. Our findings further support that the 2011 MN turkey-origin H7N9 virus was closely related to H7N9 IAV isolated in poultry in Nebraska during the same year. Although the precise origin of the wild-bird donor of the turkey-origin H7N9 IAV could not be determined, our findings suggest that, for both the NA and HA gene segments, the MN turkey-origin H7N9 viruses were related to viruses circulating in wild birds between 2006 and 2011 in the Mississippi flyway.
In this study, we identified a novel virus from gentian (Gentiana triflora) that caused ring-spots on ovaries. Pollen grains carrying the virus were used to infect host plants by hand-pollination. Furthermore, the virus causes unusual symptoms, ring-spots that appear specifically on the outer surface of the ovarian wall after pollination. RNA extracted from the purified virions indicated that the virus has two segments, RNA1 and RNA2. The full-length cDNA sequence indicated that RNA1 had two ORFs: ORF1 had methyltransferase and helicase motifs, and ORF2 had an RNA-dependent RNA polymerase motif. RNA2 had five ORFs encoding a coat protein, triple gene block proteins 1-3 and a cysteine-rich protein. The length of RNA1 was 5519 bases and that of RNA2 was 3810 bases not including a polyU/polyA region between the first and second ORFs. Viral RNA does not have a polyA tail at the 3' end. Sequence homology and phylogenetic analysis suggested that the virus is closely related to Pecluvirus and Hordeivirus but was distinct from them. These combined results suggest that the causal agent inducing ring-spot symptoms on gentian ovaries is a new virus belonging to the family Virgaviridae but not to any presently known genus. We tentatively name the virus Gentian ovary ring-spot virus.
The dengue virus (DENV) envelope protein domain 3 (ED3) is the target of potent virus neutralizing antibodies. The DENV-2 ED3 contains adjacent type-specific and DENV complex-reactive antigenic sites that are composed of a small number of residues that were previously demonstrated to be critical for antibody binding. Site-directed mutagenesis of a DENV-2 16681 infectious clone was used to mutate critical residues in the DENV-2 type-specific (K305A and P384A) and DENV complex-reactive (K310A) antigenic sites. The K305A mutant virus multiplied like the parent virus in mosquito and mammalian cells, as did the P384A mutant virus, which required a compensatory mutation (G330D) for viability. However, the K310A mutant virus could not be recovered. The DENV-2 type-specific critical residue mutations K305A and P384A+G330D reduced the ability of DENV-2 type-specific, but not DENV complex-reactive, monoclonal antibodies (mAbs) to neutralize virus infectivity and this was directly correlated with mAb binding affinity to the rED3 mutants.
H9N2 avian influenza viruses are enzootic around the world, and can infect many different avian and mammalian hosts, including humans. Unlike the H9N2 viruses, which mainly originated in other countries and possess an non-structural protein 1( NS1) of 230 amino acids (aa), 98% of the H9N2 viruses isolated in China lack the 13 aa at the C-terminus of NS1 (217 aa in total). The biological significance of NS1 elongation remains elusive. In the current study, reverse genetics was used to generate a wild-type avian influenza H9N2 virus containing a 217 aa NS1 (H9N2NS1217) and two mutant viruses with elongated NS1s of 230 aa and 237 aa (H9N2NS1230 and H9N2NS1237), in order to examine the effect NS1 C-terminal elongation in the influenza virus. The C-terminal elongation of NS1 did not have significant impact on virus replication in MDCK or DF-1 cells. Although the three variants exhibited similar replicability in mice, the H9N2NS1230 and H9N2NS1237 up-regulated the level of inflammatory cytokines. In addition, both the H9N2NS1230 and H9N2NS1237 viruses increased replication and induced a high level of inflammatory cytokines and transmission in chickens, compared to the wild-type virus. These findings suggest that the NS1 extension conferred a gain of fitness to some extent.
The pattern of flavivirus infection in mosquitoes belonging to the genus Aedes and Culex collected in two regions of north-eastern Italy (Trentino and Veneto) was assessed. Mosquitoes were collected during 2012 and screened for Flavivirus using a generic RT-nested-PCR targeted on a region of the nonstructural 5 (NS5) gene. The phylogenetic analysis was performed on a fragment of around 1000 bp. Virus isolation was attempted in C6/36 insect cell lines and the infected cell cultures were studied by electron microscopy. We detected a wide distribution of Aedes Flavivirus (AeFV) in Aedes (Ae.) albopictus, with higher infection prevalence in Trentino than in Veneto. In Culex (Cx.) pipiens collected in Veneto, we detected a new sequence of an insect-specific flavivirus and one of Usutu virus (USUV). Interestingly, we detected AeFV in Cx. Pipiens for the first time in both regions. Viral isolation in cell culture was successful for AeFV. AeFV sequences found in Veneto showed a high percentage of similarity to those detected in Trentino and to those previously reported in other areas of northern Italy. Co-infections with different flaviviruses were not detected.
Human monkeypox is a viral zoonosis caused by monkeypox virus, an orthopoxvirus (OPXV). The majority of human monkeypox cases have been reported in moist forested regions in West and Central Africa, particularly in the Democratic Republic of the Congo (DRC). In this study we investigated zoonotic OPXV infection among wild animals in Zambia, which shares a border with DRC, to assess geographical distribution of OPXV. We screened for OPXV antibodies in sera from non-human primates (NHPs), rodents, and shrews by ELISA, and performed real-time PCR to detect OPXV DNA in spleen samples. Serological analysis indicated that 38 of 259 (14.7%) rodents, 14 of 42 (33.3%) shrews, and four of 188 (2.1%) NHPs had antibodies against OPXV. The OPXV DNA could not be detected in spleens from any animals tested. Our results indicated that wild animals living in rural human habitation areas of Zambia have been infected with OPXV.
The comparative long-term kinetics of human cytomegalovirus (HCMV) load and HCMV-specific antibody responses in the immunocompetent and immunocompromised solid-organ transplanted host during primary HCMV infection was investigated. On the whole, 40 immunocompetent subjects and 17 transplanted patients were examined for viral load as well as for IgG antibody responses to HCMV glycoproteins gH/gL/pUL128L, gH/gL and gB, and neutralizing antibodies in ARPE-19 epithelial cells and human fibroblasts. In parallel, the CD4+ and CD8+ HCMV-specific T-cell responses were determined by cytokine flow cytometry. Transplanted patients reached significantly higher viral DNA peaks, which persisted longer than in immunocompetent subjects. The ELISA-IgG responses to the pentamer, gH/gL and gB were significantly higher in primary infections of the immunocompetent until six months after onset, then the two antibody levels overlapped from six to 12 months. Antibody levels neutralizing infection of epithelial cells were significantly higher in transplanted patients after six months, persisting up to a year after transplantation. This trend was not observed for antibodies neutralizing infection of human fibroblasts, which showed higher titers in the immunocompetent over the entire 1-year follow-up. In conclusion, in immunocompromised patients the viral load peak was much higher, while the neutralizing antibody response exceeded that detected in the immunocompetent host starting six months after onset of follow-up, often concomitantly with a lack of specific CD4+ T-cells. In these setting, the elevated antibody response occurred in the presence of differentiated follicular helper T-cells in blood, which decreased in number as did antibody titers upon reappearance of HCMV-specific CD4+ T-cells.
PRD1 is a gram-negative bacteria infecting complex tailless icosahedral virus with an inner membrane. This type virus of Tectiviridae family contains at least 18 structural protein species of which several are membrane associated. Vertices of PRD1 virion consist of complexes recognizing the host cell except one special vertex through which the genome is packaged. Despite the extensive knowledge of the overall structure of PRD1 virion and several individual proteins at the atomic details, the locations and interactions of various integral membrane proteins and membrane-associated proteins still remains a mystery. Here, we demonstrate that blue native polyacrylamide gel electrophoresis can be used to probe protein-protein interactions in complex membrane-containing viruses. Using this technique and PRD1 as a model, we identified the known PRD1 multiprotein vertex structure composed of penton protein P31, spike protein P5, receptor binding protein P2 and stabilizing protein P16 linking the vertex to the internal membrane. Our results also indicate that two transmembrane proteins P7 and P14, involved in the viral nucleic acid delivery, make a complex. In addition, we performed a zymogram analysis using mutant particles devoid of special vertex indicating that the lytic enzyme P15 of PRD1 is not part of the packaging vertex thus contradicting previously published results.
We investigated the infectivity and transmissibility of the human seasonal H3N2, pandemic (pdm) H1N1 (2009), and B influenza viruses in dogs. Dogs inoculated with human seasonal H3N2 and pdm H1N1 influenza viruses exhibited nasal shedding and were seroconverted against the viruses; this did not occur in the influenza B virus-inoculated dogs. Transmission of human H3N2 virus between dogs was demonstrated by observing nasal shedding and seroconversion in naiiuml;ve dogs after contact with inoculated dogs. The seroprevalence study offered evidence of human H3N2 infection in dogs since 2008. Further, serological evidence of pdm H1N1 influenza virus infection alone and in combination with canine H3N2 virus was found in the serum samples collected from field dogs during 2010 and 2011. Our results suggest that dogs may be hosts for human seasonal H3N2 and pdm H1N1 influenza viruses.
Graft rejection in transplant patients is managed clinically by suppressing T-cell function with immunosuppressive drugs such as prednisolone and methylprednisolone. In such immunocompromised hosts, human cytomegalovirus (HCMV) is an important opportunistic pathogen and can cause severe morbidity and mortality. Currently, the effect of glucocorticosteroids on the HCMV life-cycle remains unclear. Previous reports showed enhanced lytic replication of HCMV in vitro in the presence of GCSs. In the present study, we explored the implications of steroid exposure on latency and reactivation. We observed a direct effect of several GCSs used in the clinic on the activation of a quiescent viral major immediate-early promoter in stably transfected THP-1 monocytic cells. This activation was prevented by the glucocorticoid receptor (GR) antagonist Ru486 and by shRNA-mediated knockdown of the GR. Consistent with this observation prednisolone treatment of latently infected primary monocytes resulted in HCMV reactivation. Analysis of the phenotype of these cells showed that reactivation of latent virus by GCSs was correlated with differentiation to an anti-inflammatory macrophage-like cell type. On the basis that these observations may be pertinent to HCMV reactivation in post-transplant settings, we retrospectively evaluated the incidence, viral kinetics and viral load of HCMV in liver transplant patients in the presence or absence of GCS treatment. We observed that combination therapy of baseline prednisolone and augmented methylprednisone, upon organ rejection, significantly increased the incidence of HCMV infection in the intermediate risk group where donor and recipient are both HCMV seropositive (D+R+) to levels comparable with the high risk D+R- group.
In contrast to the current wealth of structural information concerning dicistrovirus particle structure very little is known about their morphogenetic pathways. Here we describe the expression of the two open reading frames encoded by Triatoma virus (TrV) genome. TrV, a member of the Cripavirus genus of the Dicistroviridae family, infects blood-sucking insects belonging to the Triatominae subfamily that act as vectors for the transmission of Trypanosoma cruzi, the etiological agent of the Chagas disease. We have established a baculovirus-based model for the expression of the NS (non structural) and P1 (structural) polyproteins. A preliminary characterization of the proteolytic processing of both polyprotein precursors has been performed using this system. We show that the proteolytic processing of the P1 polyprotein is strictly dependent upon the coexpression of the NS polyprotein, and that NS/P1 coexpression leads to the assembly of virus like particles (VLPs) exhibiting a morphology and a protein composition akin to natural TrV empty capsids. Remarkably, the unprocessed P1 polypeptide assembles into quasi-spherical structures conspicuously larger than VLPs produced in NS/P1-coexpressing cells likely representing a previously undescribed morphogenetic intermediate. This intermediate has not been found in members of the related Picornaviridae family currently used as a model for dicistrovirus studies, thus suggesting the existence of major differences in the assembly pathways of these two virus groups.
Porcine sapovirus (PSaV) of the Caliciviridae, is the only member of the Sapovirus genus with cell culture and reverse genetics systems. When combined with the piglet model, these approaches provide a system to understand the molecular basis of sapovirus pathogenesis. The replication of PSaV in cell culture is however restricted, displaying an absolute requirement for bile acids and producing lower levels of infectious virus than other caliciviruses. The effect of bile acids has previously been linked to the reduction in STAT1-mediated signaling pathway. In the current study, we observed that even in the presence of bile acids, PSaV replication in cell culture was restricted by soluble factors produced from infected cells. This effect was at least partially due to secreted interferon because treatment of cells with recombinant porcine IFN-bbeta; resulted in significantly reduced viral replication. Moreover, IFN-mediated signaling pathways (IFN, STAT1 and OAS1) were activated during PSaV infection. Characterization of PSaV growth in cell lines deficient in their ability to induce or respond to interferon, resulted in a 100-150 fold increase in infectious virus production, indicating that the primary role of bile acids is not the inactivation of the innate immune response. Furthermore, the use of IFN-deficient cell lines enabled more efficient recovery of PSaV from cDNA constructs. Overall, the highly efficient cell culture and reverse genetics system established here for PSaV highlights the key role of the innate immune response in the restriction of PSaV infection and should greatly facilitate further molecular studies on sapovirus host-cell interactions.
Torquetenovirus (TTV) is increasingly considered a universal marker of global immune function. The virus is supposed to replicate in lymphocytes, but poor information is available about fluctuations of viremia after administration of anti-lymphocyte agents. We studied TTV kinetics in a cohort of 70 kidneypplusmn;pancreas recipients receiving one of two different anti-T cell induction immunosuppressants. During the first 30 days after anti-T cell antibody administration, we report kinetics of TTV viremia compatible with replication in T-lymphocytes, and highly dependent on the potency of the anti-T cell drug administered.
Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving the templated conversion of the normal cellular prion protein (PrPC) to a pathogenic misfolded conformation. Templated conversion has been modeled in several in vitro assays, including serial protein misfolding amplification (sPMCA), amyloid seeding, and real time quaking induced conversion (RT-QuIC). Because RT-QuIC measures formation of amyloid fibrils in real time, it can be used to estimate the rate of seeded conversion. Here we use samples from deer infected with chronic wasting disease (CWD) in RT-QuIC to show that serial dilution of prion seed is linearly related to the rate of amyloid formation over a range of 10-3 to 10-8 mmicro;g. We then used an amyloid formation rate standard curve derived from a bioassayed reference sample (CWD+ brain homogenate) to estimate the prion seed concentration and infectivity in tissues, body fluids and excreta. Using these methods we estimate that urine and saliva from CWD-infected deer contain between 1 and 5 LD50 per 10 ml, respectively. Thus, over the 1 to 2 year course of infection, a substantial environmental reservoir of CWD prion contamination accumulates.
Rotaviruses of group A (RVA) are enteric pathogens with well documented zoonotic transmissions to humans. The segmented genome of the virus enables reassortment events which might alter host susceptibility and/or disease course. Genetic analysis of rotavirus in dogs so far only revealed RVAs with the VP7 and VP4 genome constellation G3P. RVA G3P have also been found in cats, humans, monkeys and bats. In the present study an unusual RVA of genotype G8P is described which was isolated from an asymptomatically infected young dog. The dog did not show signs of diarrhoea. Analysis of full length segments of VP2, VP6 and VP7 as well as NSP1 to NSP5 revealed a typical bovine-like genotype constellation G8-P-I2-Rx-C2-Mx-A3-N2-T6-E2-H3. Phylogenetic analysis supports the hypothesis of an interspecies transmission from a bovine/artiodactyl species or from humans to the young dog. The isolate is likely to represent a multiple reassortant virus.
Human papillomavirus (HPV) has a well-recognized etiological role in the development of cervical cancer and other anogenital tumors. Recently, an association between colorectal cancer and HPV infection has been suggested, although still controversial. This study aimed at detecting and characterizing HPV infection in fifty-seven paired biopsies from colorectal cancers and adjacent intact tissues using a degenerate PCR approach. All amplified fragments were genotyped by means of sequencing. Overall, HPV prevalence was 12.3%. In particular, 15.8% of tumor tissues and 8.8% of noncancerous tissue samples were HPV DNA positive. 85.7% of these samples were successfully genotyped, with 41.7% of sequences identifying four genotypes comprised in the HR-clade Group 1; the remaining 58.3% of HPV-genotyped specimens had an unclassified beta-HPV. Examining additional cases and analyzing whole genomes will help to outline the significance of this finding.
A common critical cellular event many human enveloped viruses share is the requirement for proteolytic cleavage of the viral glycoprotein by furin in the host secretory pathway. For example, the furin-dependent proteolytic activation of highly pathogenic (HP) influenza A (infA) H5 and H7 hemagglutinin precursor (HA0) subtypes is critical for yielding fusion-competent infectious virions. In this study, we hypothesize that viral hijacking of the furin pathway by HP infA viruses to permit cleavage of HA0 could represent a novel molecular mechanism controlling the dynamic production of fusion-competent infectious virus particles during the viral lifecycle. We explore the biological role in this process of a newly identified furin-directed human microRNA, miR-24, as a potential post-transcriptional regulator of the furin-mediated activation of HA0 and production of fusion-competent virions in the host secretory pathway. We report that miR-24 and furin are differentially expressed in human A549 cells infected with HP avian-origin infA H5N1. Using miR-24 mimics, we demonstrate a robust decrease in both furin mRNA level and intracellular furin activity in A549 cells. Importantly, pretreatment of A549 cells with miR-24 mimics results in a robust decrease in the production of H5N1 infectious virions and a complete block of H5N1 virus spread that was not observed in A549 cells infected with low-pathogenicity swine-origin infA H1N1 virus. Our results suggest that viral-specific down-regulation of furin-directed microRNAs such as miR-24 during the lifecycle of HP infA viruses may represent a novel regulatory mechanism that governs furin-mediated proteolytic activation of HA0 glycoproteins and production of infectious virions.
Human T-cell leukemia virus type-1 (HTLV-1) infection causes adult T-cell leukemia (ATL). The viral protein HTLV-1 bZIP factor (HBZ) is constitutively expressed in ATL cells, suggesting that HBZ plays a major role in HTLV-1 pathogenesis. Here, we identified centromere protein B (CENP-B) as a novel interacting partner with HBZ. HBZ and CENP-B associate with their central regions in cells. Furthermore, overexpression of HBZ abrogated the DNA-binding activity of CENP-B to the aalpha;-satellite DNA region containing the CENP-B box motif, which in turn inhibited the CENP-B-mediated trimethylation of histone H3K9 in T-cells.
Grapevine Vein Clearing Virus (GVCV) is a newly discovered DNA virus in grapevine that is closely associated with grapevine vein clearing syndrome observed in vineyards in Missouri and surrounding states. The genome sequence of GVCV indicates that it belongs to the genus Badnavirus in the family Caulimoviridae. To identify the GVCV promoter, we cloned portions of the GVCV large intergenic region in front of a GFP gene present in an Agrobacterium tumefaciens binary vector. GFP expression was assessed by ELISA three days after agroinfiltration of Nicotiana benthamiana leaves. We found that the GVCV DNA segment between nucleotides 7,332 and 7,672 directed expression of GFP and it was stronger than the Cauliflower mosaic virus 35S promoter. 5rrsquo; and 3rrsquo; RACE revealed that transcription was initiated predominantly at nucleotide 7,571 and terminated at nucleotide 7,676.
Invertebrate iridescent virus 6 (IIV-6) is a nucleocytoplasmic virus with a 212 kb-long linear double-stranded DNA genome that encodes 215 putative open reading frames. The IIV-6 virion-associated proteins consist of at least 54 virally-encoded proteins. One of our previous findings showed that most of these proteins are encoded by genes from the early transcriptional class. This indicates that these structural proteins may not only function in the formation of the virion, but also in the initial stage of viral infection. In the current study, we followed the protein expression profile of IIV-6 over time in Drosophila S2 cells by label-free quantitation using a proteomic approach. A total of 95 viral encoded proteins were detected in infected cells, of which 37 are virion proteins. The expressed IIV-6 virion proteins could be categorized into three main clusters based on their expression profiles. These clusters were: 1) proteins with stably low or 2) exponentially increasing expression levels during infection, and 3) proteins that were initially highly abundant, but showed slightly reduced levels after 48 hours (h) post infection (p.i.). Here, we provide novel information on the kinetics of virion and infected cell-specific protein levels that assists in understanding gene regulation in this lesser known DNA virus model.
Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-type chickens that had developed myeloid leukosis. However, ALV-J infections associated with hemangiomas have occurred in layer flock in China. In this study, we identified an ALV-J layer isolate (HLJ13SH01) as a recombinant of ALV-J and a Rous sarcoma virus Schmidt Ruppin B strain (RSV-SRB), which contained the RSV-SRB 5rrsquo;-LTR region and the other genes of ALV-J. Replication kinetic testing indicated that the HLJ13SH01 strain replicated faster than other ALV-J layer isolates in vitro. Sequence analysis indicated that the main difference between the two isolates was the 5rrsquo;-LTR sequences, particularly the U3 sequences. A 19-nt insertion was uniquely found in the U3 region of the HLJ13SH01 strain. The results of the Dual-Glo Luciferase revealed that the 19-nt insertion in the HLJ13SH01 strain increased the enhancer activity of the U3 region. Moreover, an additional CCAAT/enhancer element was found in the 19-nt insertion, and the luciferase assay indicated that this element played a key role in increasing the enhancer activity of the 5rrsquo;-U3 region. To confirm the potentiation effect of the 19-nt insertion and the CCAAT/enhancer element on virus replication, three infectious clones with 5rrsquo;-U3 region variations were constructed and rescued. Replication kinetic testing of the rescued viruses demonstrated that the CCAAT/enhancer element in the 19-nt insertion enhanced the replication capacity of the ALV-J recombinant in vitro.
The outbreak of human infections caused by the novel avian-origin H7N9 influenza viruses in China since March 2013 underscores the urgent need to find an effective treatment strategy against H7N9 infection in humans. In this study, we assessed the effectiveness of combinations of oseltamivir and two immunomodulators, simvastatin and fenofibrate, against H7N9 infection in a mouse model. Mice treated with oseltamivir plus fenofibrate exhibited the longest mean survival time, the largest reduction of viral titer in lung tissue, the highest levels of CD4+ and CD8+ T lymphocytes, and the greatest decrease in pulmonary inflammation. Thus, the combination of oseltamivir plus fenofibrate improved the outcomes of mice infected with H7N9 virus by simultaneously reducing viral replication and normalizing the aberrant immune response. This drug combination should be considered in randomized controlled trials of treatments for H7N9 patients.
Antibody response plays a crucial role against hepatitis C virus (HCV) infection and our understanding of this intricate progress in vivo is far from complete. We previously reported a novel and robust technique based on a large combinatorial viral antigen library displayed on the surface of the yeast Saccharomyces cerevisiae, allowing comprehensive profiling of polyclonal antibody responses in vivo in both qualitative and quantitative terms. Here, we report the generation and application of a combinatorial library of HCV JFH1 envelope glycoprotein to profile antibody response in four HCV chronically infected individuals. Systematic analysis of the location and frequency of antigenic fragments along the JFH1 envelope glycoprotein, we showed that the major binding antibody response was targeted to E2 (80.9-99.8%) while that against E1 was relatively small (0.3-19.0%). A total of five major antigenic domains (D1-D5) were identified; one was within the E1 and additional four within the E2, despite substantial variability among the different individuals. However, serum absorption with the yeast clones containing the antigenic domain D1 resulted in more reduction in neutralizing antibody activity against pseudotyped HCV than those in E2, suggesting that the E1 contained additional neutralizing epitopes. Our results have provided additional insghts into HCV-specific antibody response in human and should assist our better understanding of protective antibody immunity and to guide development of effective vaccines and therapeutics against HCV infection.
Newcastle disease virus (NDV) has been developed as a vector for vaccine and gene therapy purposes. However, the optimal insertion site for foreign gene expression remained to be determined. In the present study, we inserted the green fluorescence protein (GFP) gene into five different intergenic regions of the enterotropic NDV VG/GA vaccine strain using reverse genetics technology. The rescued recombinant viruses retained lentogenic pathotype and displayed delayed growth dynamics, particularly when the GFP gene was inserted between the NP and P genes of the virus. The GFP mRNA level was most abundant when the gene was inserted closer to the 3rrsquo; end and gradually decreased as the gene was inserted closer to the 5rrsquo; end. Measurement of the GFP fluorescence intensity in recombinant virus-infected cells demonstrated that the noncoding region between the P and M genes is the optimal insertion site for foreign gene expression in the VG/GA vaccine vector.
Patients' biopsies show that hepadnaviral core proteins and capsids - collectively called core - are found in the nucleus and in the cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viremia while nuclear core is associated with high viral load. In order to better understand the molecular interactions leading to core localization, we analyzed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids while expression of core in the viral context resulted in a predominantly cytoplasmic stain. Analyzing which viral partner was responsible for the cytoplasmic retention we found the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that not only the polymerase but also , an RNA structure to which the viral polymerase binds, was essential. Furthermore we showed that core protein phosphorylation at serine 164 was essential for the cytoplasmic phenotype of core, which likely explains differences between individual cells.
Infection of astrocytes by the neuropathogenic mutant of Moloney murine leukemia virus (MuLV), ts1, exhibits increased levels of reactive oxygen species (ROS) and signs of oxidative stress compared to uninfected astrocytes. Previously we have demonstrated that ts1 infection caused two separate events of ROS upregulation. The first upregulation occurs during early viral establishment in host cells and the second during the virus-mediated apoptotic process. In this study, we show that virus-mediated ROS upregulation activates ATM kinase, which in turn phosphorylates the serine 15 site on p53. This activation of p53 however, is unlikely associated with ts1-induced cell death. Rather p53 appears to be involved in suppressing intracellular ROS levels in astrocytes under oxidative stress. The activated p53 appears to delay retroviral gene expression by suppressing NADPH oxidase, a superoxide-producing enzyme. These results suggest that p53 plays a role as a retrovirus-mediated oxidative stress modulator.
Coronaviruses have been studied for over 60 years, but have only recently gained notoriety as deadly human pathogens with the emergence of severe respiratory syndrome coronavirus and Middle East respiratory syndrome virus. The rapid emergence of these viruses has demonstrated the need for good models to study severe coronavirus respiratory infection and pathogenesis. There are, currently, different methods and models for the study of coronavirus disease. The available genetic methods for the study and evaluation of coronavirus genetics are reviewed here. There are several animal models, both mouse and alternative animals, for the study of severe coronavirus respiratory disease that have been examined, each with different pros and cons relative to the actual pathogenesis of the disease in humans. A current limitation of these models is that no animal model perfectly recapitulates the disease seen in humans. Through the review and analysis of the available disease models investigators can employ the most appropriate available model to study coronavirus various aspects of pathogenesis and evaluate potential antiviral treatments that may potentially be successful in future treatment and prevention of severe coronavirus respiratory infections.
In December 1983 a seminal paper appeared on the overexpression of human interferon-bbeta; in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produce massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes are dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years major improvements have been achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells, and the scale-up of the cell culture process. To date, thousands of recombinant proteins have been produced in this successful expression system, including several protein-based human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted baculovirus expression vectors are used to deliver and express heterologous genes in mammalian cells and may find applications for gene therapy and cancer treatment in the future. The purpose of this paper is to highlight the 30-years 'anniversary' of this expression system by summarizing the fundamental research that allowed the development of this expression system and by indicating the major technological advances since 1983. Finally, attention will be paid to the future challenges to further optimize this amazing technology.
Chikungunya virus (CHIKV) is a mosquito-borne virus that has been responsible for over 2 million human infections during the past decade. This virus, which previously had a geographic range primarily restricted to Sub-Saharan Africa, the Indian subcontinent, and Southeast Asia, has recently moved to subtropical latitudes as well as the Western Hemisphere. This expansion into novel habitats brings unique risks associated with further spread of the virus and the disease it causes.
Equine herpesvirus type 1 (EHV-1) replicates in the epithelial cells of the upper respiratory tract and disseminates through the body via a cell-associated viremia in monocytic cells, despite the presence of neutralizing antibodies. However, the mechanism by which EHV-1 hijacks immune cells and uses them as llsquo;Trojan horsesrrsquo; in order to disseminate inside its host is still unclear. Here, we hypothesized that EHV-1 delays its replication in monocytic cells in order to avoid recognition by the immune system. We compared replication kinetics in vitro of EHV-1 in RK-13, a cell line fully susceptible to EHV-1 infection, and primary horse cells from the myeloid lineage (CD172a+). We found that EHV-1 replication was restricted to 4% of CD172a+ compared to 100% in RK-13. In susceptible CD172a+, the expression of immediate-early (IEP) and early (EICP22) proteins was delayed in the cell nuclei by 2-3 hpi compared to RK-13, and the formation of replicative compartments by 15 hpi. The viral production in CD172a+ was significantly lower (from 101.7 to 3.1 TCID50/105 inoculated cells) than in RK-13 (from 105 to 5.7 TCID50/105 inoculated cells). Less than 0.02% of inoculated CD172a+ produced and transmitted infectious virus to neighbor cells. Pretreatment of CD172a+ with inhibitors of HDAC activity increased and accelerated viral protein expression at very early time of infection and induced productive infection in CD172a+. Our results demonstrated that the restriction and delay of EHV-1 replication in CD172a+ is part of an immune evasive strategy and involves silencing of EHV-1 gene expression associated with histone deacetylases.
MicroRNAs (miRNAs) are a class of short endogenous RNA molecules with the ability to control development, autophagy, apoptosis and the stress response in eukaryotes by pairing with partially complementary sites in the 3' untranslated regions (UTRs) of targeted genes. Recent studies have demonstrated that miRNAs serve as critical effectors in intricate networks of host-pathogen interactions. Notably, we found that Bos taurus bta-miR-29b (referred to as miR-29b herein) was significantly upregulated more than 2.3-fold in bovine viral diarrhea virus (BVDV) NADL-infected Madin-Darby bovine kidney (MDBK) cells 6 h post-infection (pi) compared to normal MDBK cells. However, the roles of miR-29b in BVDV infection and pathogenesis remain unclear. Here, we discover the inhibitory effects of miR-29b on BVDV NADL replication and viral infection-related autophagy. miR-29b overexpression mediated by miRNA precursor-expressing lentivirus resulted in the attenuation of BVDV NADL infection-related autophagy by directly downregulating the intracellular expression levels of two key autophagy-associated proteins, ATG14 and ATG9A. Moreover, ATG14 and ATG9A overexpression rescue not only reversed miR-29b-inhibited autophagy but also increased BVDV NADL replication. In previous studies, we found that the early stages of autophagy contribute to BVDV NADL replication in MDBK cells and that the inhibition of autophagy represses BVDV NADL replication, which was also proved in the present study. Collectively, our results establish a novel link between miR-29b and viral replication and they provide a new avenue for the intimate interaction between host cells and pathogens.
Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) genome segment 4 (S4) showed that it consists of 3410 nucleotides with a single ORF of 1110 amino acids which could encode a protein of ~ 127 kDa (p127). Bioinformatics analysis showed the presence of 5rrsquo; RNA triphosphatase (RTPase) domain (LRDR), S-adenosyl-L-methionine (SAM) binding (GxGxG) motif and K-D-K-E tetrad of 2rrsquo;-O-methyltransferase (MTase), which suggests that S4 may encode RTPase and MTase. The ORF of S4 was expressed in E. coli as His-tagged fusion protein and purified by Ni-NTA affinity chromatography. Biochemical analysis of recombinant p127 showed its 5rrsquo; RTPase as well as SAM dependent guanine N-7 and ribose 2rrsquo;-O-MTase activity. MTase assay using in vitro transcribed AmCPV S2 RNA having 5rrsquo; G*pppG end showed that guanine N-7- methylation occurs prior to the ribose 2rrsquo;-O-methylation to yield m7GpppG/ m7GpppGm RNA cap.. Mutagenesis of the SAM binding (GxGxG) motif (G831A) completely abolished N-7 and 2rrsquo;-O-MTase activity indicating importance of these residues for capping. From the kinetic analysis the Km of N-7-MTase for SAM and RNA was calculated as 4.41 and 0.39 mmu;M, respectively. These results suggest that AmCPV S4 encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5rrsquo; end of viral RNA.
Bats have been found to harbor a number of new emerging viruses with zoonotic potential and there has been a great deal of interest in identifying novel bat pathogens to determine risk to human and animal health. Many groups have identified novel viruses in bats by detection of viral nucleic acid, however virus isolation is still a challenge and there are few reports of viral isolates from bats. In recent years, our group has developed optimized procedures for virus isolation from bat urine, including the use of primary bat cells. In previous reports we have described the isolation of Hendra virus, Menangle virus and Cedar virus, in Queensland, Australia. Here, we report the isolation of four additional novel bat paramyxoviruses from urine collected from beneath pteropid bat (flying fox) colonies in Queensland and New South Wales during 2009-2011.
The interferon (IFN) immune system plays an essential role in protecting the host against most viral infections. In order to explore the interactions between the IFN pathway and Respiratory syncytial virus (RSV) infection, and to identify potential IFN-stimulated genes (ISGs) that may be involved in suppressing the replication of RSV, we utilized an IFN pathway-specific microarray to study the effects of RSV infection on the IFN pathway in HeLa cells. We showed that RSV infection enhanced the expression of a series of ISGs, including oligoadenylate synthetase 2 (OAS2), interferon-induced transmembrane protein 1 (IFITM1) and myxovirus-resistance 2 (Mx2). Our results also showed that the IFITM proteins potently inhibited RSV infection mainly by interfering with both virus entry and the subsequent replication steps, but not the attachment process. The anti-viral effect of IFITM3 protein was not affected by ubiquitination modification. Furthermore, knocking down the endogenous and IFN-induced expressions of IFITM1 and IFITM3 proteins facilitated RSV infection. Expression of the IFITM proteins was found to delay the phosphorylation of interferon regulatory factor-3 (IRF3) through interfering with the detection of viral RNA by the melanoma differentiation-associated gene 5 (MDA5) and the retinoic acid-inducible gene I (RIG-I). These results demonstrated that the restriction of RSV infection by the IFITM proteins was achieved through the inhibition of virus entry and replication, and they provide further insight for exploring the mechanism of IFITM proteins-mediated virus restriction.