|Journal of General Virology current issue|
Arthropod-borne viruses (arboviruses) pose a considerable threat to human and animal health, yet effective control measures have proven difficult to implement, and novel means of controlling their replication in arthropod vectors, such as mosquitoes, are urgently required. One of the most exciting approaches to emerge from research on arthropods is the use of the endosymbiotic intracellular bacterium Wolbachia to control arbovirus transmission from mosquito to vertebrate. These aalpha;-proteobacteria propagate through insects, in part through modulation of host reproduction, thus ensuring spread through species and maintenance in nature. Since it was discovered that Wolbachia endosymbiosis inhibits insect virus replication in Drosophila species, these bacteria have also been shown to inhibit arbovirus replication and spread in mosquitoes. Importantly, it is not clear how these antiviral effects are mediated. This review will summarize recent work and discuss determinants of antiviral effectiveness that may differ between individual Wolbachia/vector/arbovirus interactions. We will also discuss the application of this approach to field settings and the associated risks.
The drivers of influenza seasonality remain heavily debated, especially in tropical/subtropical regions where influenza activity can peak in winter, during the rainy season, or remain constant throughout the year. We compared the epidemiological and evolutionary patterns of seasonal influenza epidemics in Hong Kong and Shenzhen, two adjacent cities in subtropical southern China. This comparison represents a unique natural experiment, as connectivity between these two cities has increased over the past decade. We found that, whilst summer influenza epidemics in Shenzhen used to peak 1nndash;3 months later than those in Hong Kong, the difference decreased after 2005 (Pllt;0.0001). Phylogenetic analysis revealed that influenza isolates from Shenzhen have become genetically closer to those circulating in Hong Kong over time (P = 0.045). Furthermore, although Shenzhen isolates used to be more distant from the global putative source of influenza viruses than isolates from Hong Kong (Pllt;0.001), this difference has narrowed (P = 0.02). Overall, our study reveals that influenza activities show remarkably distinct epidemiological and evolutionary patterns in adjacent subtropical cities and suggests that human mobility patterns can play a major role in influenza dynamics in the subtropics.
In recent years, novel henipavirus-related sequences have been identified in bats in Africa. To evaluate the potential of African bat henipaviruses to spread in non-bat mammalian cells, we compared the biological functions of the surface glycoproteins G and F of the prototype African henipavirus GH-M74a with those of the glycoproteins of Nipah virus (NiV), a well-characterized pathogenic member of the henipavirus genus. Glycoproteins are central determinants for virus tropism, as efficient binding of henipavirus G proteins to cellular ephrin receptors and functional expression of fusion-competent F proteins are indispensable prerequisites for virus entry and cell-to-cell spread. In this study, we analysed the ability of the GH-M74a G and F proteins to cause cell-to-cell fusion in mammalian cell types readily permissive to NiV or Hendra virus infections. Except for limited syncytium formation in a bat cell line derived from Hypsignathus monstrosus, HypNi/1.1 cells, we did not observe any fusion. The highly restricted fusion activity was predominantly due to the F protein. Whilst GH-M74a G protein was found to interact with the main henipavirus receptor ephrin-B2 and induced syncytia upon co-expression with heterotypic NiV F protein, GH-M74a F protein did not cause evident fusion in the presence of heterotypic NiV G protein. Pulsenndash;chase and surface biotinylation analyses revealed delayed F cleavage kinetics with a reduced expression of cleaved and fusion-active GH-M74a F protein on the cell surface. Thus, the F protein of GH-M74a showed a functional defect that is most likely caused by impaired trafficking leading to less efficient proteolytic activation and surface expression.
Picornaviruses infecting pigs, described for many years as llsquo;porcine enterovirusesrrsquo;, have recently been recognized as distinct viruses within three distinct genera (Teschovirus, Sapelovirus and Enterovirus). To better characterize the epidemiology and genetic diversity of members of the Enterovirus genus, faecal samples from pigs from four provinces in Vietnam were screened by PCR using conserved enterovirus (EV)-specific primers from the 5' untranslated region (5' UTR). High rates of infection were recorded in pigs on all farms, with detection frequencies of approximately 90 % in recently weaned pigs but declining to 40 % in those aged over 1 year. No differences in EV detection rates were observed between pigs with and without diarrhoea [74 % (n = 70) compared with 72 % (n = 128)]. Genetic analysis of consensus VP4/VP2 and VP1 sequences amplified from a subset of EV-infected pigs identified species G EVs in all samples. Among these, VP1 sequence comparisons identified six type 1 and seven type 6 variants, while four further VP1 sequences failed to group with any previously identified EV-G types. These have now been formally assigned as EV-G types 8nndash;11 by the Picornavirus Study Group. Comparison of VP1, VP4/VP2, 3Dpol and 5' UTRs of study samples and those available on public databases showed frequent, bootstrap-supported differences in their phylogenies indicative of extensive within-species recombination between genome regions. In summary, we identified extremely high frequencies of infection with EV-G in pigs in Vietnam, substantial genetic diversity and recombination within the species, and evidence for a much larger number of circulating EV-G types than currently described.
A novel virus was detected in a sample collected from a Swedish moose (Alces alces). The virus was suggested as a member of the Hepeviridae family, although it was found to be highly divergent from the known four genotypes (gt1nndash;4) of hepatitis E virus (HEV). Moose are regularly hunted for consumption in the whole of Scandinavia. Thus, the finding of this virus may be important from several aspects: (a) as a new diverged HEV in a new animal species, and (b) potential unexplored HEV transmission pathways for human infections. Considering these aspects, we have started the molecular characterization of this virus. A 5.1 kb amplicon was sequenced, and corresponded to the partial ORF1, followed by complete ORF2, ORF3 and poly(A) sequence. In comparison with existing HEVs, the moose HEV genome showed a general nucleotide sequence similarity of 37nndash;63 % and an extensively divergent putative ORF3 sequence. The junction region between the ORFs was also highly divergent; however, two putative secondary stemnndash;loop structures were retained when compared to gt1nndash;4, but with altered structural appearance. In the phylogenetic analysis, the moose HEV deviated and formed its own branch between the gt1nndash;4 and other divergent animal HEVs. The characterization of this highly divergent genome provides important information regarding the diversity of HEV infecting various mammalian species. However, further studies are needed to investigate its prevalence in the moose populations and possibly in other host species, including the risk for human infection.
The Middle East respiratory syndrome coronavirus (MERS-CoV) presents a novel emerging threat to public health worldwide. Several treatments for infected individuals have been suggested including IFN, ribavirin and passive immunotherapy with convalescent plasma. Administration of IFN-aalpha;2b and ribavirin has improved outcomes of MERS-CoV infection in rhesus macaques when administered within 8 h post-challenge. However, detailed and systematic evidence on the activity of other clinically available drugs is limited. Here we compared the susceptibility of MERS-CoV with different IFN products (IFN-aalpha;2b, IFN-, IFN-universal, IFN-aalpha;2a and IFN-bbeta;), as well as with two antivirals, ribavirin and mycophenolic acid (MPA), against MERS-CoV (Hu/Jordan-N3/2012) in vitro. Of all the IFNs tested, IFN-bbeta; showed the strongst inhibition of MERS-CoV in vitro, with an IC50 of 1.37 U mlnndash;1, 41 times lower than the previously reported IC50 (56.08 U mlnndash;1) of IFN-aalpha;2b. IFN-bbeta; inhibition was confirmed in the virus yield reduction assay, with an IC90 of 38.8 U mlnndash;1. Ribavirin did not inhibit viral replication in vitro at a dose that would be applicable to current treatment protocols in humans. In contrast, MPA showed strong inhibition, with an IC50 of 2.87 mmicro;M. This drug has not been previously tested against MERS-CoV and may provide an alternative to ribavirin for treatment of MERS-CoV. In conclusion, IFN-bbeta;, MPA or a combination of the two may be beneficial in the treatment of MERS-CoV or as a post-exposure intervention in high-risk patients with known exposures to MERS-CoV.
Polymorphonuclear neutrophils (PMN) infiltrate the respiratory tract early after viral infection and can contribute to both host defence and pathology. Coronaviruses are important causes of respiratory tract infections, ranging from mild to severe depending on the viral strain. This study evaluated the role of PMN during a non-fatal pulmonary coronavirus infection in the natural host. Rat coronavirus (RCoV) causes respiratory disease in adult rats, characterized by an early PMN response, viral replication and inflammatory lesions in the lungs, mild weight loss and effective resolution of infection. To determine their role during RCoV infection, PMN were depleted and the effects on disease progression, viral replication, inflammatory response and lung pathology were analysed. Compared with RCoV infection in control animals, PMN-depleted rats had worsened disease with weight loss, clinical signs, mortality and prolonged pulmonary viral replication. PMN-depleted animals had fewer macrophages and lymphocytes in the respiratory tract, corresponding to lower chemokine levels. Combined with in vitro experiments showing that PMN express cytokines and chemokines in response to RCoV-infected alveolar epithelial cells, these findings support a role for PMN in eliciting an inflammatory response to RCoV infection. Despite their critical role in the protection from severe disease, the presence of PMN was correlated with haemorrhagic lesions, epithelial barrier permeability and cellular inflammation in the lungs. This study demonstrated that while PMN are required for an effective antiviral response, they also contribute to lung pathology during RCoV infection.
There are four dengue virus (DENV) serotypes. Primary infection with one does not confer protective immunity against the others. We have reported previously that the marmoset (Callithrix jacchus) is a useful primary DENV infection model. It has been reported that secondary DENV infection with a heterotypic serotype induces viraemia kinetics and antibody responses that differ from those in primary infection. Thus, it is important to determine the utility of the marmoset as a model for secondary DENV infection. Marmosets were infected with heterologous DENV by secondary inoculation, and viraemia kinetics and antibody responses were analysed. The marmosets consistently developed high levels of viraemia after the secondary inoculation with heterologous DENV serotypes. IgM responses were lower compared with primary inoculation responses, whilst IgG responses were rapid and high. Neutralizing activities, which possessed serotype cross-reactive activities, were detected as early as 4 days after inoculation. In addition, infectious viraemia titres were higher when assayed with Fc receptor-expressing baby hamster kidney (BHK) cells than when assayed with conventional BHK cells, suggesting the presence of infectious virusnndash;antibody immune complexes. After secondary infection with heterotypic DENV, the marmosets demonstrated viraemia kinetics, IgM and IgG responses, and high levels of serotype cross-reactive neutralizing antibody responses, all of which were consistent with secondary DENV infection in humans. The results indicate the marmoset as a useful animal for studying secondary, as well as primary, DENV infection.
The freshwater fish Lepomis macrochirus (bluegill) is common to North American waters, and important both ecologically and as a sport fish. In 2001 an unknown virus was isolated from bluegills following a bluegill fish kill. This virus was identified as a picornavirus [termed bluegill picornavirus (BGPV)] and a diagnostic reverse transcriptase PCR was developed. A survey of bluegills in Wisconsin waters showed the presence of BGPV in 5 of 17 waters sampled, suggesting the virus is widespread in bluegill populations. Experimental infections of bluegills confirmed that BGPV can cause morbidity and mortality in bluegills. Molecular characterization of BGPV revealed several distinct genome characteristics, the most unusual of which is the presence of a short poly(C) tract in the 3' UTR. Additionally, the genome encodes a polyprotein lacking a leader peptide and a VP0 maturation cleavage site, and is predicted to encode two distinct 2A proteins. Sequence comparison showed that the virus is most closely related to a phylogenetic cluster of picornaviruses that includes the genera Aquamavirus, Avihepatovirus and Parechovirus. However, it is distinct enough, for example sharing only about 38 % sequence identity to the parechoviruses in the 3D region, that it may represent a new genus in the family Picornaviridae.
The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe pulmonary disease in humans and represents the second example of a highly pathogenic coronavirus (CoV) following severe acute respiratory syndrome coronavirus (SARS-CoV). Genomic studies revealed that two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), process the polyproteins encoded by the MERS-CoV genomic RNA. We previously reported that SARS-CoV PLpro acts as both deubiquitinase (DUB) and IFN antagonist, but the function of the MERS-CoV PLpro was poorly understood. In this study, we characterized MERS-CoV PLpro, which is a protease and can recognize and process the cleavage sites (CS) of nsp1-2, nsp2-3 and nsp3-4. The LXGG consensus cleavage sites in the N terminus of pp1a/1ab, which is generally essential for CoV PLpro-mediated processing, were also characterized in MERS-CoV. MERS-CoV PLpro, like human SARS-CoV PLpro and NL63-CoV PLP2, is a viral deubiquitinating enzyme. It acts on both K48- and K63-linked ubiquitination and ISG15-linked ISGylation. We confirmed that MERS-CoV PLpro acts as an IFN antagonist through blocking the phosphorylation and nuclear translocation of IFN regulatory factor 3 (IRF3). These findings indicate that MERS-CoV PLpro acts as a viral DUB and suppresses production of IFN-bbeta; by an interfering IRF3-mediated signalling pathway, in addition to recognizing and processing the CS at the N terminus of replicase polyprotein to release the non-structural proteins. The characterization of proteolytic processing, DUB and IFN antagonist activities of MERS-CoV PLpro would reveal the interactions between MERS-CoV and its host, and be applicable to develop strategies targeting PLpro for the effective control of MERS-CoV infection.
During the 2009 national group A rotavirus (RVA) surveillance, five unusual strains of the human G8P genotype were detected in Brazilian indian children with acute gastroenteritis. The aim of this study was to carry out sequence analysis of the two outer capsid proteins (VP4 and VP7) and the inner capsid protein (VP6) of the G8P strains detected in order to provide further information on the genetic relationship between human and animal RVA. A total of 68 stool samples, collected in Mato Grosso do Sul during 2009, were tested for RVA using ELISA, following by reverse transcriptase-PCR and sequencing. RVA infection was detected in 7.3 % of samples (5/68). The IAL-RN376 G8 sequence shares a clade with bovine and human strains, displaying highest nucleotide identity to African human strains DRC86 and DRC88, followed by African bovine strain NGRBg8. IAL-RN376 and IAL-RN377 P sequences showed highest identity to human strain R330 from Ireland, and a close genetic relationship to African fruit bat RVA strain KE4852/07. Strains IAL-RN376 and IAL-RN377 display genogroup I VP6 specificity and the I2 genotype, and share high nucleotide identities with human strains B1711, 272-BF and 06-242, and moderate identities with bovine (RUBV81, 86 and KJ9-1) and porcine (HP140) strains. This study suggested that a reassortment between bovine and bat RVA strains could have occurred in animal host(s) preceding the transmission to humans. In the indigenous population, zoonotic transmission is probably fairly frequent as the inhabitants live in close contact with animals under conditions of poor hygiene.
African horse sickness virus (AHSV) is an arbovirus capable of successfully replicating in both its mammalian host and insect vector. Where mammalian cells show a severe cytopathic effect (CPE) following AHSV infection, insect cells display no CPE. These differences in cell death could be linked to the method of viral release, i.e. lytic or non-lytic, that predominates in a specific cell type. Active release of AHSV, or any related orbivirus, has, however, not yet been documented from insect cells. We applied an integrated microscopy approach to compare the nanomechanical and morphological response of mammalian and insect cells to AHSV infection. Atomic force microscopy revealed plasma membrane destabilization, integrity loss and structural deformation of the entire surface of infected mammalian cells. Infected insect cells, in contrast, showed no morphological differences from mock-infected cells other than an increased incidence of circular cavities present on the cell surface. Transmission electron microscopy imaging identified a novel large vesicle-like compartment within infected insect cells, not present in mammalian cells, containing viral proteins and virus particles. Extracellular clusters of aggregated virus particles were visualized adjacent to infected insect cells with intact plasma membranes. We propose that foreign material is accumulated within these vesicles and that their subsequent fusion with the cell membrane releases entrapped viruses, thereby facilitating a non-lytic virus release mechanism different from the budding previously observed in mammalian cells. This insect cell-specific defence mechanism contributes to the lack of cell damage observed in AHSV-infected insect cells.
Echinoderms are important constituents of marine ecosystems, where they may influence the recruitment success of benthic flora and fauna, and are important consumers of detritus and plant materials. There are currently no described viruses of echinoderms. We used a viral metagenomic approach to examine viral consortia within three urchins nndash; Colobocentrotus atratus, Tripneustes gratilla and Echinometra mathaei nndash; which are common constituents of reef communities in the Hawaiian archipelago. Metagenomic libraries revealed the presence of bacteriophages and densoviruses (family Parvoviridae) in tissues of all three urchins. Densoviruses are known typically to infect terrestrial and aquatic arthropods. Urchin-associated densoviruses were detected by quantitative PCR in all tissues tested, and were also detected in filtered suspended matter (ggt;0.2 mmicro;m) from plankton and in sediments at several locations near to where the urchins were collected for metagenomic analysis. To the best of our knowledge, this is the first report of echinoderm-associated viruses, which extends the known host range of parvoviruses.
Despite a rigorous blockade of interferon- (IFN-) signalling in infected fibroblasts as a mechanism of immune evasion by human cytomegalovirus (HCMV), IFN- induced indoleamine-2,3-dioxygenase (IDO) has been proposed to represent the major antiviral restriction factor limiting HCMV replication in epithelial cells. Here we show that HCMV efficiently blocks transcription of IFN--induced IDO mRNA both in infected fibroblasts and epithelial cells even in the presence of a preexisting IFN-induced antiviral state. This interference results in severe suppression of IDO bioactivity in HCMV-infected cells and restoration of vigorous HCMV replication. Depletion of IDO expression nonetheless substantially alleviated the antiviral impact of IFN- treatment in both cell types. These findings highlight the effectiveness of this IFN- induced effector gene in restricting HCMV productivity, but also the impact of viral counter-measures.
Bovine herpesvirus type 5 (BoHV-5) and bovine herpesvirus 1 (BoHV-1) are two closely related viruses. However, BoHV-5 is responsible for fatal meningitis in calves, while BoHV-1 is associated with infectious rhinotracheitis in cattle, and the mechanism by which the two viruses cause different symptoms is not well understood. In this study, we identified 11 microRNA (miRNA) genes, encoded by the BoHV-5 genome, that were processed into 16 detectable mature miRNAs in productive infection as determined by deep sequencing. We found that 6 out of 16 miRNA genes were present as two copies in the internal repeat and terminal repeat regions, resulting in a total of 17 miRNA-encoding loci distributed in both DNA strands. Surprisingly, BoHV-5 shared only one conservative miRNA with BoHV-1, which was located upstream of the origin of replication. Furthermore, in contrast to BoHV-1, no miRNAs were detected in the unique short region and locus within or near the bovine infected-cell protein 0 and latency-related genes. Variations in both the 5' and 3' ends of the reference sequence were observed, resulting in more than one isoform for each miRNA. All of the 16 miRNAs were detectable by stemnndash;loop reverse transcriptase-PCR. The miRNAs with high read numbers were subjected to Northern blot analysis, and all pre-miRNAs and one mature miRNA were detected. Collectively, the data suggest that BoHV-5 encodes a different pattern of miRNAs, which may regulate the life cycle of BoHV-5 and might account for the different pathogenesis of this virus compared with BoHV-1.
Rana grylio virus (RGV) is a pathogenic iridovirus that has resulted in high mortality in cultured frog. Here, an envelope protein gene, 2L, was identified from RGV and its possible role in virus infection was investigated. Database searches found that RGV 2L had homologues in all sequenced iridoviruses and is a core gene of iridoviruses. Western blotting detection of purified RGV virions confirmed that 2L protein was associated with virion membrane. Fluorescence localization revealed that 2L protein co-localized with viral factories in RGV infected cells. In co-transfected cells, 2L protein co-localized with two other viral envelope proteins, 22R and 53R. However, 2L protein did not co-localize with the major capsid protein of RGV in co-transfected cells. Meanwhile, fluorescence observation showed that 2L protein co-localized with endoplasmic reticulum, but did not co-localize with mitochondria and Golgi apparatus. Moreover, a conditional lethal mutant virus containing the lac repressor/operator system was constructed to investigate the role of RGV 2L in virus infection. The ability to form plaques and the virus titres were strongly reduced when expression of 2L was repressed. Therefore, the current data showed that 2L protein is essential for virus infection. Our study is the first report, to our knowledge, of co-localization between envelope proteins in iridovirus and provides new insights into the understanding of envelope proteins in iridovirus.
The genetic diversity of avian leukosis virus subgroup J (ALV-J) is determined not only by the env gene, but also by its 3' UTR and 3' LTR. They all play important roles in extending the host range and tumour development. In the present study, one ALV-J strain (ZB110604-6) from Black-Bone Silky Fowl (BSF) and three ALV-J strains (ZB110604-3/4/5) from grey partridge (GP), which bore multiple tumours and breed in one house of Farm A, were demonstrated extending their host to GP, while two other ALV-J strains (LC110515-3/4) from BSF of Farm B could not infect the embryo fibroblast of GP. The BSF is a unique species of chicken in China, while the GP is a close relative of the pheasant that previously demonstrated resistance to ALV-J. Histopathology showed that various tumours were induced by ALV-J in the two species. Phylogenetic tree analysis showed that the isolates from Farms A and B, rather than species, belong to two different clusters of ALV-J. Genetic mutations analysis revealed that the isolates obtained from Farm A showed a higher frequency of mutation in the hypervariable region 2 domain than in other variable regions of the gp85 gene. From the nucleotide alignment of the 3' UTR and 3' LTR gene, and the spectrum of tumours observed in this study, we speculate that the deletions or mutations in the redundant transmembrane region, E element and U3 (CAAT boxes, CArG box and Y box) might associate with tumour formation and development. The extension of the host range of ALV-J to the GP suggested that housing different species together provides more opportunities for ALV-J to evolve rapidly.
Following infection with Human immunodeficiency virus 1 (HIV-1) there is a remarkable variation in virus replication and disease progression. Both host and viral factors have been implicated in the observed differences in disease status. Here, we focus on understanding the contribution of HIV-1 viral protein R (Vpr) by evaluating the disease-associated Vpr polymorphism and its biological functions from HIV-1 positive rapid progressor (RP) and long-term nonprogressor (LTNP) subjects. Results presented here show distinct variation in phenotypes of Vpr alleles from LTNP and RP subjects. Most notably, the polymorphism of Vpr at R36W and L68M associated with RP shows higher levels of oligomerization, and increased virus replication, whereas R77Q exhibits poor replication kinetics. Interestingly, we did not observe correlation with cell cycle arrest function. Together these results indicate that polymorphisms in Vpr in part may contribute to altered virus replication kinetics leading to the observed differences in disease progression in LTNP and RP groups.
In this study, sequences of small RNA (sRNA) libraries derived from the insect vector Laodelphax striatellus were assembled into contigs and used as queries for database searches. A large number of contigs were highly homologous to the genome sequence of an insect dicistrovirus, himetobi P virus (HiPV). Interestingly, HiPV-derived sRNAs had a wide size distribution, and were relatively abundant throughout the 18nndash;30 nt size range with only a slight peak at 22 nt. HiPV sRNAs had a strong bias towards the sense strand, whilst the antisense sRNAs were predominantly 21 and 22 nt. HiPV sRNAs do not have the typical features of PIWI-interacting RNAs, but their 3' ends were preferentially cleaved at UA-rich sequences. Our data suggest that HiPV sRNAs may be derived both from activities of the RNA interference pathway and from cleavage of the viral genome by other host RNases.
Infection of the lepidopteran insect Trichoplusia ni with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) by the oral route stimulates activation of host matrix metalloproteases (MMP) and effector caspases, a process dependent on expression of the viral fibroblast growth factor (vFGF). This pathway leads to tracheal cell basal lamina remodelling, enabling virus escape from the primary site of infection, the midgut epithelium, and establishment of efficient systemic infection. In this study, we asked whether the MMPnndash;caspase pathway was also activated following infection by intrahaemocoelic injection. We found that intrahaemocoelic infection did not lead to any observable tracheal cell or midgut epithelium basal lamina remodelling. MMP and caspase activities were not significantly stimulated. We conclude that the main role of the AcMNPV vFGF is in facilitating virus midgut escape.
Phylogenomic evidence suggested that recombination is an important evolutionary force for potyviruses, one of the larger families of plant RNA viruses. However, mixed-genotype potyvirus infections are marked by low levels of cellular coinfection, precluding template switching and recombination events between virus genotypes during genomic RNA replication. To reconcile these conflicting observations, we evaluated the in vivo recombination rate (rg) of Tobacco etch virus (TEV; genus Potyvirus, family Potyviridae) by coinfecting plants with pairs of genotypes marked with engineered restriction sites as neutral markers. The recombination rate was then estimated using two different approaches: (i) a classical approach that assumed recombination between marked genotypes can occur in the whole virus population, rendering an estimate of rg = 7.762x10nndash;8 recombination events per nucleotide site per generation, and (ii) an alternative method that assumed recombination between marked genotypes can occur only in coinfected cells, rendering a much higher estimate of rg = 3.427x10nndash;5 recombination events per nucleotide site per generation. This last estimate is similar to the TEV mutation rate, suggesting that recombination should be at least as important as point mutation in creating variability. Finally, we compared our mutation and recombination rate estimates to those reported for animal RNA viruses. Our analysis suggested that high recombination rates may be an unavoidable consequence of selection for fast replication at the cost of low fidelity.
The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) inhibits host responses to jasmonic acid (JA), a chemical signal regulating resistance to insects. Previous experiments with a CMV subgroup IA strain and its 2b gene deletion mutant suggested that VSRs might neutralize aphid (Myzus persicae) resistance by inhibiting JA-regulated gene expression. To further investigate this, we examined JA-regulated gene expression and aphid performance in Nicotiana benthamiana infected with Potato virus X, Potato virus Y, Tobacco mosaic virus and a subgroup II CMV strain, as well as in transgenic plants expressing corresponding VSRs (p25, HC-Pro, 126 kDa and 2b). All the viruses or their VSRs inhibited JA-induced gene expression. However, this did not always correlate with enhanced aphid performance. Thus, VSRs are not the sole viral determinants of virus-induced changes in hostnndash;aphid interactions and interference with JA-regulated gene expression cannot completely explain enhanced aphid performance on virus-infected plants.
Rosellinia necatrix megabirnavirus 1 (RnMBV1) with a bipartite dsRNA genome (dsRNA1 and dsRNA2) confers hypovirulence to its natural host, the white root rot fungus, and is thus regarded as a potential virocontrol (biocontrol) agent. Each segment has two large ORFs: ORF1 and partially overlapping ORF2 on dsRNA1 encode the major capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), whilst ORF3 and ORF4 on dsRNA2 encode polypeptides with unknown functions. Here, we report the biological and molecular characterization of this virus in the chestnut blight fungus, Cryphonectria parasitica, a filamentous fungus that has been used as a model for mycovirus research. Transfection with purified RnMBV1 particles into an RNA-silencing-defective strain (dcl-2) of C. parasitica and subsequent anastomosis with the WT strain (EP155) resulted in stable persistent infection in both host strains. However, accumulation levels in the two strains were different, being ~20-fold higher in dcl-2 than in EP155. Intriguingly, whilst RnMBV1 reduced both virulence and growth rate in dcl-2, it attenuated virulence without affecting significantly other traits in EP155. Western blot analysis using antiserum against recombinant proteins encoded by either ORF1 or ORF2 demonstrated the presence of a 250 kDa protein in purified virion preparations, suggesting that RdRp is expressed as a CP fusion product via a nndash;1 frameshift. Antiserum against the ORF3-encoded protein allowed the detection of 150, 30 and 23 kDa polypeptides specifically in RnMBV1-infected mycelia. Some properties of an RnMBV1 mutant with genome rearrangements, which occurred after transfection of dcl-2 and EP155, were also presented. This study provides an additional example of C. parasitica serving as a versatile, heterologous fungus for exploring virusnndash;host interactions and virus gene expression strategies.
Bacteriophages have been found to be the most abundant and also potentially most diverse biological entities on Earth. In the present study, Bacillus phages were isolated rapidly and shown to have a high degree of diversity. The genomes of a newly isolated phage, phiCM3, and a prophage, proCM3, from the Bacillus thuringiensis strain YM-03 were sequenced and characterized. Comparative genome analysis showed that the phiCM3 genome is highly similar to the genomes of eight other Bacillus phages and seven of these phages were classified as the Wbbeta; group of phages. Analysis of the differential evolution of the genes in the Wbbeta;-group phages indicated that the genes encoding the antirepressor and tail fibre protein were more highly conserved than those encoding the major capsid protein, DNA replication protein, and RNA polymerase factor, which might have diverged to acquire mechanisms suitable for survival in different microbial hosts. Genome analysis of proCM3 revealed that proCM3 might be a defective phage because of mutations in the minor structural protein, and it was not inducible by mitomycin C treatment. The proCM3 genome was similar to those of two lytic Bacillus phages in sequence, but had a different genomic structure, composed of three regions in a different order. These data suggest that the three phages might have had a common ancestor and that genome rearrangement might have occurred during evolution. The findings of this study enrich our current knowledge of Bacillus phage diversity and evolution, especially for the Wbbeta;-group and TP21-L-like phages, and may help the development of practical applications of Bacillus phages.
|Journal of General Virology Publish Ahead of Print|
The Newcastle disease virus (NDV) matrix (M) protein is a highly basic and nucleocytoplasmic shuttling viral protein. Previous study has demonstrated that the N-terminal 100 amino acids of NDV M protein are somewhat acidic overall, but the remainder of the polypeptide is strongly basic. In this study, we investigated the role of the N-terminal basic residues in the subcellular localization of M protein and in the replication and pathogenicity of NDV. We found that mutation of the basic residue arginine (R) to alanine (A) at position 42 disrupted Mrrsquo;s nuclear localization. Moreover, a recombinant virus with R42A mutation in the M protein reduced viral replication in DF-1 cells and attenuated the virulence and pathogenicity of the virus in chickens. This is the first report to show that a basic residue mutation in the NDV M protein abrogates its nuclear localization and attenuates viral replication and pathogenicity.
During three seasons of mumps outbreaks in the Netherlands (September 2009 nndash; August 2012), 822 mumps cases were laboratory confirmed at the national laboratory (RIVM). Most patients were vaccinated young adults. Given the protracted endemic circulation, we studied the genetic diversity and changes of mumps virus over a period of three years. Phylogenetic analysis of the SH gene (316 bp) was performed on a representative set of 808 PCR positive specimens. Additionally, the HN gene (1749 bp) and F gene (1617 bp) were sequenced for a subset of samples (n=17). Correlations between different sequence types and epidemiological and clinical data were investigated. The outbreaks in the Netherlands were dominated by two SH gene sequence types within genotype G, termed MuVs/Delft.NLD/03.10 (variant 1) and MuVs/Scheemda.NLD/12.10 (variant 2). Sequence analysis of the HN and F genes indicated that the outbreaks were initiated by separately introduced genetic lineages. The predominance of variant 2 by the end of the first outbreak season could not be explained by any of the epidemiological factors investigated. Orchitis was more frequently reported in males infected with variant 2, irrespective of age and vaccination status. These findings illustrate genetic heterogeneity of an emerging mumps genotype and raise questions about the mechanisms driving mumps epidemiology and immunity, in relation to vaccination.
Typical avian influenza A viruses are restricted from replicating efficiently and causing disease in humans. However, an avian virus can become adapted to humans by mutating or recombining with currently circulating human viruses. These viruses have the potential to cause pandemics in an immunologically naiiuml;ve human population. It is critical that we understand the molecular basis of host range restriction and how this can be overcome. Here we review our current understanding of the mechanisms by which influenza viruses adapt to replicate efficiently in a new host. We predominantly focus on the influenza polymerase which remains one of the least understood host range barriers.
Five neutralizing antigenic sites have been described for serotype O foot-and-mouth disease viruses (FMDV) based on monoclonal antibody (mAb) escape mutant studies. However, a mutant virus selected to escape neutralization of mAb binding at all five sites was previously shown to confer complete cross-protection with the parental virus in guinea pig challenge studies, suggesting that amino acid residues outside of the mAb binding sites contribute to antibody-mediated in vivo neutralization of FMDV. Comparison of the ability of bovine antisera to neutralize a panel of serotype O FMDV identified three novel putative sites at VP2-74, VP2-191 and VP3-85, where amino acid substitutions correlated with changes in sero-reactivity. The impact of these positions was tested using site-directed mutagenesis to effect substitutions at critical amino acid residues within an infectious copy of FMDV O1 Kaufbeuren (O1K). Recovered viruses containing additional mutations at VP2-74 and VP2-191 exhibited greater resistance to neutralization with both O1K guinea pig and O BFS bovine antisera than a virus that was engineered to include only mutations at the five known antigenic sites. The changes at VP2-74 and VP3-85 are adjacent to critical amino acids that define antigenic sites 2 and 4, respectively. However VP2-191 (17AAring; away from VP2-72), located at the 3-fold axis and more distant from previously identified antigenic sites exhibited the most profound effect. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and will improve our strategies for vaccine strain selection and rational vaccine design.
The full-length genome sequence of a porcine picobirnavirus detected in Italy in 2004 was determined. The S segment was 1730 nucleotide (nt) in length, coding for a putative RNA-dependent RNA polymerase. Two distinct sub-populations of L segment (LA and LB) were identified in the sample, with the sizes ranging from 2351 to 2666 nt. The ORF1 coding for a protein of unknown function, contained repetitions of the ExxRxNxxxE motif in variable number. The capsid protein coding ORF2 spanned nt 810-2447 in the LB variants and started at nt 734 in the LA variants. However, a termination codon was present only in one of all LA segment variants. Three dimensional modelling of the porcine PBV capsids suggested structural differences in the protruding domain, tentatively involved as antigens in humoral immune response. Altogether, these findings suggest simultaneous presence of two different picobirnavirus strains sharing the same S segment but displaying genetically diverse L segments. In addition, the sample probably contained a mixture of PBVs with aberrant RNA replication products. Altered structure in the L segments could be tolerated and retained in the presence of functionally integer cognate genes and represent a mechanism of virus diversification.
Respiratory syncytial virus (RSV) is one of the primary causative agents of upper and lower respiratory tract infections in young children, in particular infants. Recently, we reported the protective efficacy of a RSV vaccine formulation consisting of a truncated version of the fusion (F) protein formulated with a TLR agonist and an immunostimulatory peptide in a carrier system (F/TriAdj). To evaluate the duration of immunity induced by this vaccine candidate, we carried out long-term trials. The F was formulated with TriAdj containing either poly(I:C) or CpG ODN and administered intranasally (IN) to mice. One year after the second vaccination all mice were challenged with RSV. Both F/TriAdj formulations mediated the induction of high levels of IgG1, IgG2a and virus neutralizing antibodies, and IgA in the lungs. Based on the numbers of IFN- and IL-5 secreting cells in the spleens, the immune response was slightly Th1-biased. This was confirmed by the presence of F85nndash;93-specific CD8+ effector T cells in the lungs of both F/TriAdj(polyI:C)- and F/TriAdj(CpG)-immunized mice. Both F/TriAdj formulations induced RSV-specific CD8 T cells. However, F/TriAdj(polyI:C) generated significantly higher IgG affinity maturation and higher numbers of RSV-specific CD8+ effector memory T cells in lungs and CD8+ central memory T cells in spleens and lymph nodes than F/TriAdj(CpG). After RSV challenge, no virus replication and no evidence of vaccine-induced pathology were detected in mice immunized with either of the F/TriAdj formulations, which demonstrates that the duration of immunity induced with these vaccines is at least one year.
Significant loss of RNA followed by severely reduced cellular protein pool, a phenomenon termed host shutoff, is associated with a number of lytic virus infections and is a critical player in virus pathogenesis. Until recently, viral DNA exonucleases were associated only with processing of viral genomic DNA and encapsidation. However, recent observations have identified a combined host shutoff and exonuclease function for the highly conserved viral exonucleases in - herpesviruses which include Kaposirrsquo;s sarcoma-associated herpesvirus, Epstein-Barr virus and the murine model murine gammaherpesvirus-68. In this study, we show that although ablation of the MHV-68 exonuclease ORF37 caused a restrictive phenotype in interferon-aalpha;/bbeta; receptor-positive cells, lack of ORF37 was tolerated in cells lacking the interferon- aalpha; /bbeta; receptor: the ORF37 deletant virus was capable of forming infectious particles and caused loss of RNA in interferon-aalpha;/bbeta; receptor knockout cells. Moreover, ORF37 deletant virus was able to establish lytic infection in lungs of mice lacking the interferon-aalpha;/bbeta; receptor. These observations provide further evidence that this family of viral nucleases play a key role in providing a window of opportunity for virus growth by overcoming type I IFN-dependent responses by host shutoff of cellular transcripts.
Morphogenesis of member viruses of genus Enterovirus of Picornaviridae is still poorly understood despite decades-long investigations. However, we recently provided evidence that 2CATPase gives specificity to poliovirus encapsidation through an interaction with capsid protein VP3. Polypeptide 2CATPase is a highly conserved nonstructural protein of enteroviruses with important roles in RNA replication, encapsidation and uncoating. We have identified a site (K279/R280) near the C-terminus of the polypeptide that is required for morphogenesis. The aim of our current project was to search for additional functional sites near the C-terminus of the 2CATPase polypeptide, with particular interest in those that are required for encapsidation. We selected for analysis a cysteine-rich site of the polypeptide and constructed four mutants in which cysteines or a histidine was changed to an alanine. The RNA transcripts were transfected into HeLa cells yielding 2 lethal, one temperature-sensitive and one quasi-infectious mutant. All four mutants exhibited normal protein translation in vitro and 3 of them possessed severe RNA replication defects. The quasi-infectious mutant (C286A) yielded variants with a pseudoreversion at the original site (A286D) but some also contained one additional mutation: A138Vor M293V. The temperature-sensitive mutant (C272A/H273A) exhibited an encapsidation, and possibly also an uncoating defect, at 37ddeg;C. Variants of this mutant revealed suppressor mutations at 3 different sites in the 2CATPase polypeptide: A138V, M293V, or K295R. We conclude that the cysteine-rich site near the C-terminus of 2CATPase is involved in encapsidation, possibly through an interaction with an upstream segment located between boxes A and B of the nucleotide-binding domain.
A thorough characterization of the genetic diversity of viruses present in vector and vertebrate host populations is essential for the early detection of and response to emerging pathogenic viruses, yet genetic characterization of many important viral groups remains incomplete. The Simbu serogroup of the genus Orthobunyavirus, family Bunyaviridae is an example. The Simbu serogroup currently consists of a highly diverse group of related arboviruses that infect both humans and economically important livestock species. Here, we report complete genome sequences for 11 viruses within this group, with a focus on the large and poorly characterized Manzanilla and Oropouche species complexes. Phylogenetic and pairwise divergence analyses indicate the presence of high-levels of genetic diversity within these two species complexes, on par with that seen among the five other species complexes in the Simbu serogroup. Based on previously reported divergence thresholds between species, the data suggest that these two complexes should actually be divided into at least five species. Together these five species form a distinct phylogenetic clade apart from the rest of the Simbu serogroup. Pairwise sequence divergences among viruses of this clade and viruses in other Simbu serogroup species complexes are similar to levels of divergence among the other orthobunyavirus serogroups. The genetic data also suggest relatively high levels of natural reassortment, with three potential reassortment events present, including two well-supported events involving viruses known to infect humans.
In Europe and Asia, Ixodid ticks transmit tick-borne encephalitis virus (TBEV), a flavivirus that causes severe encephalitis in humans but appears to show no virulence for livestock and wildlife. In the British Isles, where TBEV is absent, a closely related tick-borne flavivirus, named Louping ill virus (LIV), is present. However, unlike TBEV, LIV causes a febrile illness in sheep, cattle, grouse and some other species, that can progress to fatal encephalitis. The disease is detected predominantly in animals from upland areas of the United Kingdom and Ireland. This distribution is closely associated with the presence of its arthropod vector, the hard tick Ixodes ricinus. The virus is a positive-strand RNA virus belonging to the Flavivirus genus, exhibiting a high degree of genetic homology to TBEV and other mammalian tick-borne viruses. In addition to causing acute encephalomyelitis in sheep, other mammals and some avian species, the virus is recognised as a zoonotic agent with occasional reports of seropositive individuals, particularly those whose occupation involves contact with sheep. Preventative vaccination in sheep is effective although there is no treatment for disease. Surveillance for LIV in Great Britain (GB) is limited despite an increased awareness of emerging arthropod-borne diseases and potential changes in distribution and epidemiology. This review provides an overview of LIV and highlights areas where further effort is needed to control this disease.
Summary Semaphorins are a large, phylogenetically conserved family of proteins that are involved in a wide range of biological processes including axonal steering, organogenesis, neoplastic transformation, as well as immune responses. In this study, a novel semaphorin homolog gene belonging to the Singapore grouper iridovirus (SGIV), ORF155R (termed as SGIV-sema), was cloned and characterized. The coding region of SGIV-sema is 1728 bp in length, encoding a predicted protein with 575 amino acids (aa). SGIV-sema contains a ~370-aa N-terminal Sema domain, a conserved PSI domain, and an Ig-like domain near the C terminus. SGIV-sema is an early gene product during viral infection and predominantly distributed in the cytoplasm with a speckled and clubbed pattern of appearance. Functionally, SGIV-sema could promote viral replication during SGIV infection in vitro, with no effect on the proliferation of host cells. Intriguingly, ectopically expressed SGIV-sema could alter the cytoskeletal structure of fish cells, characterized by a circumferential ring of microtubules near the nucleus and a disrupted microfilament organization. Furthermore, SGIV-sema was able to attenuate the cellular immune response, as demonstrated by decreased expression of inflammation/immune-related genes such as interleukin-8 (IL-8), IL-15, tumor necrosis factor alpha (TNF-aalpha;), and mediator of IRF3 activation (MITA), in SGIV-sema-expressing cells before and after SGIV infection. Ultimately, our study identified a novel, functional SGIV gene that could regulate cytoskeletal structure, immune responses and facilitate viral replication. Keywords: semaphorin, Singapore grouper iridovirus, viral replication, cytoskeletal structure, immune response
Murine hybridomas producing neutralizing monoclonal antibodies (MAbs) specific of the pandemic influenza virus A/Cal/07/09 hemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extr/6530/10) lacked reactivity with the monoclonals and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to ablate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus hemagglutinin during virus evolution (Shih et al., Proc. Nal. Acad. Sci. USA, 104:6283-6288, 2007). The changes found in the A/Extr/6530/10 HA were not found in escape mutants selected in vitro with one of the monoclonals, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may alter similarly epitopes of the new antigenic site identified in this work on the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of MAbs and human postinfection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.
The multifunctional potyviral Helper component Protease (HcPro) contains variable regions with some functionally conserved domains, such as the FRNK box. Natural variants occur at FRNK box, a conserved central domain, known for its role in RNA binding and RNAi suppression activities, although no dominant natural variant for N182 residue are known to occur. Here, a mutant at HcProN182L was developed to ascertain its indispensible nature in natural populations. Using in vitro studies, we found an increase in the small RNA (sRNA) binding potentiality of HcProN182Lwithout affecting the protein-protein interaction property, suggesting that the presence of N182 is critical to maintain threshold levels of sRNAs, but does not interfere in self-interaction of HcPro. Furthermore, we found that expression of HcProN182L in Nicotiana benthamiana affected plant growth. Transient expression of HcProN182L supported reporter gene expression in 16c GFP transgenic plants more than HcPro, suggesting that replacement of asparagine in FRNK box favors RNA silencing suppression. HcPro was found to be distributed in the nucleus and cytoplasm, whereas HcProN182L was observed only in cytoplasmic inclusion bodies in N. benthamiana leaves, when fused to GFP tag and expressed by agro-infiltration, suggesting mutation favours oligomerisation of HcPro. These findings suggest that amino acid N182 of the conserved FRNK box may impose stringent regulation on the RNA silencing mechanisms and is required to maintain subcellular localisation of the protein for its multi-functionality. Hence, the N182 residue of the FRNK box seems to be indispensable for potyvirus infection during evolution.
From Jan. 1, 2009, to May 31, 2013, 15, 287 respiratory specimens submitted to the Clinical Virology laboratory at Childrenrrsquo;s Hospital Colorado were tested for human coronavirus RNA by RT-PCR. Human coronaviruses HKU1, OC43, 229E, and NL63 co-circulated during each of the respiratory seasons but with significant year to year variability, and cumulatively accounted for 7.4 nndash; 15.6% of all samples tested during months of peak activity. A total of 79 (0.5% prevalence) specimens were positive for human betacoronavirus HKU1 RNA. Genotypes HKU1 A and B were both isolated from clinical specimens and propagated on primary human tracheal-bronchial epithelial cells cultured at the air-liquid interface and were neutralized in vitro by human intravenous immunoglobulin and by polyclonal rabbit antibodies to the spike glycoprotein of HKU1. Phylogenetic analysis of deduced amino acid sequences of 7 full length genomes of Colorado HKU1 viruses and the spike glycoproteins from 4 additional HKU1 viruses from Colorado and 3 from Brazil, demonstrated remarkable conservation of these sequences with genotypes circulating in Hong Kong and France. Within genotype A, all but one of the Colorado HKU1 sequences formed a unique subclade defined by 3 amino acid residue substitutions (W197F, F613Y, S752F) in the spike glycoprotein and exhibited a unique signature in the acidic tandem repeat in the N terminal region of the nsp3 subdomain. Elucidating the function of and mechanisms responsible for the formation of theses varying tandem will increase our understanding of the replication process and pathogenicity of HKU1 and potentially other coronaviruses.
The recent identification of a new nanovirus, pea necrotic yellow dwarf virus (PNYDV), from pea in Germany prompted us to survey wild and cultivated legumes for nanovirus infections in several European countries. This led to the identification of two new nanoviruses: black medic leaf roll virus (BMLRV) and pea yellow stunt virus (PYSV), each considered a putative new species. The complete genomes of a PYSV isolate from Austria and three BMLRV isolates from Austria, Azerbaijan and Sweden were sequenced. In addition, the genomes of five isolates of faba bean necrotic yellows virus (FBNYV) from Azerbaijan and Spain and those of four faba bean necrotic stunt virus (FBNSV) isolates from Azerbaijan were completely sequenced, leading to the first identification of FBNSV occurring in Europe. Sequence analyses uncovered evolutionary relationships, extensive reassortment, potential remnants of mixed nanovirus infections as well as intra- and intercomponent recombination events within the nanovirus genomes. In some virus isolates, diverse types of the same genome component (paralogs) were observed, a type of genome complexity not previously described for any member of the Nanoviridae family. Moreover, infectious and aphid-transmissible nanoviruses from cloned genomic DNAs of FBNYV and BMLRV were reconstituted that, for the first time, allow experimental reassortments for studying the genome functions and evolution of these nanoviruses.
The present study was aimed at evaluating an underlying mechanism of the antiviral activity of the sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri (G. fisheri) against white spot syndrome virus (WSSV) infection in haemocytes of the black tiger shrimp Penaeus monodon. Primary culture of haemocytes from P. monodon was performed and inoculated with WSSV, after which the cytopathic effect (CPE), cell viability, and viral load were determined. Haemocytes treated with WSSV-SG premix showed decreased CPE, viral load, and cell mortality from the viral infection. Solid-phase virus-binding assay revealed that SG bound to WSSV in a dose-related manner. Far Western blotting analysis indicated that SG bound to VP 26, and VP 28 proteins of WSSV. In contrast to the native SG, desulfated SG did not reduce CPE and cell mortality, and showed low binding activity with WSSV. The current study suggests that SG from G. fisheri elicits its anti-WSSV activity by binding to viral proteins which are important for the process of viral attachment to the host cells. It is anticipated that the sulfate groups of SG are important for the viral binding.
We previously reported that Pin1 facilitates the human immunodeficiency virus type 1 (HIV-1) uncoating by interacting with the capsid (CA) core through the phosphorylated Ser16-Pro17 motif. However, the specific kinase responsible for Ser16 phosphorylation has remained unknown. Here, we show that virion-associated extracellular signal-regulated kinase 2 (ERK2) phosphorylates Ser16. The characterization of immature virions produced by exposing of CEM/LAV-1 cells to 10 mmicro;M saquinavir indicated that Ser16 is phosphorylated after the initiation of Pr55gag processing. Furthermore, a mass spectrometry-based in vitro kinase assay demonstrated that ERK2 specifically phosphorylated the Ser16 residue in the Ser16-Pro17-motif-containing substrate. The treatment of CEM/LAV-1 cells with the ERK2 inhibitor sc-222229 decreased the Ser16 phosphorylation level inside virions, and the virus partially defective in Ser16 phosphorylation showed an impaired reverse transcription and an attenuated replication owing to an attenuated Pin1-dependent uncoating. Furthermore, the suppression of ERK2 expression by RNA interference in CEM/LAV-1 cells resulted in suppressed ERK2 packaging inside virions and decreased the Ser16 phosphorylation level inside virions. Interestingly, the ERK2-packaging-defective virus showed an impaired reverse transcription and an attenuated HIV-1 replication. Taken together, these findings provide insights into the as yet obscure processes in Pin1-dependent HIV-1 uncoating.
Circulating coxsackievirus A16 (CA16) is a major cause of hand, foot, and mouth disease (HFMD) in Southeast Asia. At present, there is no vaccine against CA16 viruses. Pathogenic animal models that are sensitive to diverse circulating CA16 viruses would be desirable for vaccine development and evaluation. In this study, we isolated and characterized several circulating CA16 viruses from recent HFMD patients. These CA16 viruses currently circulating in humans were highly pathogenic in a newly developed neonatal mouse model; we also observed and analyzed the pathogenesis of representative circulating recombinant form (CRF) CA16 viruses. An inactivated CA16 vaccine candidate, formulated with alum adjuvant and containing sub-microgram quantities of viral proteins, protected neonatal mice born to immunized female mice from lethal-dose challenge with a series of CA16 viruses. Further analysis of humoral immunity showed that antibody elicited from both the immunized dams and their pups can neutralize various lethal viruses by cytopathic effect (CPE) in vitro. Moreover, viral titers and loads in the tissues of challenged pups in the vaccine group were far lower than those in the control group, and some were undetectable. This lethal challenge model using pathogenic CA16 viruses and the vaccine candidates that mediated protection in this model could be useful tools for the future development and evaluation of CA16 vaccine.
In 2009, a novel lyssavirus (subsequently named Ikoma lyssavirus, IKOV) was detected in the brain of an African civet (Civettictis civetta) with clinical rabies in the Serengeti National Park of Tanzania. The degree of nucleotide divergence between genomes of IKOV and other lyssaviruses predicted antigenic distinction from, and lack of protection provided by, available rabies vaccines. In addition, the index case was considered likely to be an incidental spill over event, and therefore the true reservoir of IKOV remained to be identified. The advent of sensitive molecular techniques has led to a rapid increase in the discovery of novel viruses. Detecting viral sequence alone however, only allows for prediction of phenotypic characteristics and not their measurement. In the present study we describe the in-vitro and in-vivo characterisation of IKOV, demonstrating that it is (1) pathogenic by peripheral inoculation in an animal model, (2) antigenically distinct from current rabies vaccine strains, and (3) is poorly neutralised by sera from humans and animals immunised against rabies. In a laboratory mouse model, no protection was elicited by a licensed rabies vaccine. We also investigated the role of bats as reservoirs of IKOV. We found no evidence for infection among 483 individuals of at least 13 bat species sampled across sites in the Serengeti and Southern Kenya.
A novel picornavirus from commercial broiler chickens (Gallus gallus domesticus) has been identified and genetically characterised. The viral genome consists of a single-stranded, positive-sense RNA genome of ggt;9243 nucleotides (nt) excluding the poly (A) tail and as such represents one of the largest picornavirus genomes reported to date. The virus genome is GC-rich with a G+C content of 54.5 %. The genomic organisation is similar to other picornaviruses: 5rrsquo; UTR-L-VP0-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3rrsquo; UTR. The partially characterised 5rrsquo; UTR of ggt;373 nt appears to possess a type II internal ribosomal entry site (IRES), found in Aphthovirus and Cardiovirus. This IRES exhibits significant sequence homology to turkey "Gallivirus A". The 3rrsquo; UTR of 278 nt contains the conserved 48 nt llsquo;barbell-likerrsquo; structure identified in "Passerivirus", "Gallivirus", Avihepatovirus and some Kobuvirus genus members. A predicted large open reading frame (ORF) of 8592 nt encodes a potential polyprotein precursor of 2864 amino acids. In addition, the virus contains a predicted large L protein of 462 amino acids. Pairwise sequence comparisons, along with phylogenetic analysis revealed the highest percentage identity to "passerivirus A" (formerly called turdivirus 1), forming a monophyletic group across the P1, P2 and P3 regions, llt;40, llt;40 and llt;50 % amino acid identity respectively. Reduced identity was observed against "Gallivirus A" and members of the Kobuvirus genus. Quantitative PCR analysis estimated a range of 4 x 105 to 5 x 108 viral genome copies/gram in 22 (73 %) of 30 PCR-positive faeces. Based on sequence and phylogenetic analysis, we propose that this virus is the first member of a potential novel genus within the family Picornaviridae. Further studies are required to investigate the pathogenic potential of this virus within the avian host.
Chronic infections by the hepatotropic viruses hepatitis B virus (HBV) and hepatitis C virus (HCV) are major risk factors for the development of hepatocellular carcinoma (HCC). It is estimated that more than 700,000 individuals per year die from hepatocellular carcinoma, and around 80% of HCC is attributable to HBV or HCV infection. Despite the clear clinical importance of virus-associated HCC, the underlying molecular mechanisms remain largely elusive. Oxidative stress, in particular DNA lesions associated with oxidative damage, play a major contributory role in carcinogenesis, and are strongly linked to the development of many cancers, including HCC. A large body of evidence demonstrates that both HBV and HCV induce hepatic oxidative stress, with increased oxidative DNA damage being observed both in infected individuals and in murine models of infection. Here, we review the impact of HBV and HCV on the incidence and repair of oxidative DNA damage. We begin by giving a brief overview of oxidative stress and the repair of DNA lesions induced by oxidative stress. We then review in detail the evidence surrounding the mechanisms by which both viruses stimulate oxidative stress, before focusing on how the viral proteins themselves may perturb the cellular response to oxidative DNA damage, impacting upon genome stability and thus hepatocarcinogenesis.
The 5rrsquo; end of eukaryotic mRNA contains the type type-1 (m7GpppNm) or type-2(m7GpppNmNm) cap structure. Many viruses have evolved various mechanisms to develop their 22 own capping enzymes (e.g., flavivirus and coronavirus) or to "steal" caps from host mRNAs 23 (e.g., influenza virus). Other viruses have developed "cap-mimicking" mechanisms by attaching a peptide to the 5rrsquo; end of viral RNA (e.g., picornavirus and calicivirus) or by having a complex 5rrsquo; RNA structure (IRES) for translation initiation (e.g., piconovirus, pestivirus, and hepacivirus). Here we review the diverse viral RNA capping mechanisms. Using flavivirus as a model, we summarize how a single methyltransferase catalyzes two distinct N-7 and 2'-O methylations of viral RNA cap in a sequential manner. For antiviral development, a structural feature unique to the flavivirus methyltransferase was successfully used to design selective inhibitors that block viral methyltransferase without affecting host methyltransferases. Functionally, capping is essential for prevention of triphosphate-triggered innate immune activation; N-7 methylation is critical for enhancement of viral translation; 2'-O methylation is important for subversion of innate immune response during viral infection. Flaviviruses defective in 2'-O methyltransferase are replicative, but their viral RNAs lack 2'-O methylation and are recognized and eliminated by the host immune response. Such mutant viruses could be rationally designed as live attenuated vaccines. This concept has recently been proved with Japanese encephalitis virus and dengue virus. The findings obtained with flavivirus should be applicable to other RNA viruses.
An epidemic of diarrhea in adult cows occurred at a total of 105 dairy farms in Yamagata Prefecture, Japan between 2003 and 2010. Reverse transcription-polymerase chain reaction (RT-PCR) diagnostic tests revealed the presence of bovine rotavirus species C (RVCs) in samples from each of 6 farms (5.7%). In this study, we determined the full-length nucleotide sequences of 11 RNA segments from six bovine RVC strains and investigated genetic diversity among them, including two bovine RVC strains identified in our previous study. Comparisons of all segmental nucleotide and the deduced amino acid sequences among bovine RVCs indicated high identities across all genes except for the VP4 gene. Phylogenetic analysis of each gene revealed that the 6 bovine RVCs belonged to a bovine cluster distinct from human and porcine RVCs. Bovine RVC strains could be clearly divided into two lineages of the VP4 genes. The nucleotide sequence identity for VP4 genes between lineage I and II was 83.7-84.8 %. Moreover, bovine RVC strains belonging to lineage I exhibited one amino acid deletion and three amino acid insertions, which differed for those strains belonging to lineage II. Our data suggest that multiple bovine RVCs originated from a common ancestor, but had different genetic backgrounds not only in Yamagata Prefecture but also in the rest of Japan.
Since the discovery of a non-virulent infectious salmon anaemia virus (ISAV) HPR0 variant, many studies have speculated on the functional role of deletions within the highly polymorphic region (HPR) of genomic segment 6, which codes for the haemagglutinin-esterase (HE) protein. To address this issue, mutant HE proteins with deletions in their HPR were generated from the Scottish HPR0 template (NWM10) and fusion inducing activity measured using lipid (octadecyl Rhodamine B) and content mixing assays (Firefly luciferase). Segment 6 HPR was found to have a strong influence on ISA viral fusion, and deletions in this near membrane region predominantly increased the fusion inducing ability of the resulting HE proteins. The position and length of the HPR deletions were not significant factors, suggesting that they may affect fusion non-specifically. In comparison, the amino acid composition of the associated fusion (F) protein was a more crucial criterion. Antibody co-patching and confocal fluorescence demonstrated that HE and F protein were highly co-localised forming defined clusters on the cell surface, post-transfection. The binding of erythrocyte ghosts on the attachment protein caused a reduction in the percentage of co-localisation suggesting that ISAV fusion might be triggered through physical separation of the F and HE protein. In this process HPR deletion appeared to modulate and reduce the strength of interaction between the two glycoproteins, causing more F protein to be released and activated. This work provides a first insight into the mechanism of virulence acquisition through HPR deletion, with fusion enhancement acting as a major contributing factor.
Bagaza virus (BAGV) and Israel turkey meningoencephalomyelitis virus (ITV) are classified in the Flavivirus genus of the Flaviviridae family. Serologically, they are closely related, belonging to the Ntaya serocomplex. Nucleotide sequences available to date consist of several complete sequences of BAGV isolates, but only partial sequences of ITV isolates. Sequence comparisons of partial envelope (E) and NS5 regions reveal a close genetic relationship between these viruses. Despite this, BAGV and ITV are considered as separate virus species in the database of the International Committee on Taxonomy of Viruses (ICTV). In this work, complete nucleotide sequences for five ITV isolates are provided, thereby permitting a phylogenetic comparison with other complete sequences of flaviviruses in the Ntaya serogroup. We conclude that BAGV and ITV are the same virus species and propose that both viruses be designated by a new unified name: Avian meningoencephalomyelitis virus (AMEV).
The human zinc finger antiviral protein (hZAP) gene is spliced to yield a short (hZAP-S) and a long (hZAP-L) isoform. The long isoform possesses a poly(ADP-ribose) polymerase (PARP)-like domain in its C-terminus predicted to be inactive due to alterations in its triad motif compared to bona fide PARPs. Using Sindbis virus as prototype member of alphaviruses we confirmed that hZAP-L is a more potent inhibitor of alphaviruses than hZAP-S. Specific siRNA knockdown of hZAP-L but not hZAP-S demonstrated a role of endogenous hZAP-L in restriction of alphavirus replication. While single amino acid substitutions in the triad motif of hZAP-Lrrsquo;s PARP-like domain reduced the antiviral activity, exchange of all three triad motif residues to alanine or to the amino acids of active PARPs virtually abolished the antiviral effect. Contrary to previous assumptions these results indicate an essential function of the PARP-like domain in hZAP-L's antiviral activity.
Baculoviruses infect insects, producing two distinct phenotypes during the viral life cycle: the budded virus (BV) and, the occlusion-derived virus (ODV) for intra and host spread, respectively. Since the 80's several countries have been using the multiple Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) as a biological control agent against the velvet bean caterpillar (A. gemmatalis). The genome of the AgMNPV-2D carries at least 152 potential genes, with twenty-four that possibly code for structural proteins. Proteomic studies were done on a few baculoviruses, with six ODV and two BV proteomes finished so far. Moreover, there are limited data on virion proteins carried by the AgMNPV-2D. Therefore, structural proteins of AgMNPV-2D were analyzed by MALDI-Q-TOF and LC-MS/MS. A total of 44 proteins were associated to the ODV and 33 to the BV of AgMNPV-2D. Although 38 structural proteins were already known, we found six new proteins in ODV and seven new proteins carried by the AgMNPV-2D BV. Eleven cellular proteins that were found on several other enveloped viruses were also identified, which are possibly carried with the virion. These findings may provide novel insights into baculovirus biology and its host interaction. Moreover, our data may be helpful in subsequent applied studies aiming towards the improvement of the AgMNPV as biopesticide and as biotechnology tool for gene expression or delivery.
HIV 1 transcription depends on cellular transcription factors that bind to sequences in the Long Terminal Repeat (LTR) promoter. Each HIV 1 subtype has a specific LTR promoter configuration and minor sequence changes in transcription factor binding sites (TFBS) or their arrangement can influence transcriptional activity, virus replication and latency properties. Previously, we investigated the proviral latency properties of different HIV 1 subtypes in the SupT1 T cell line. Here, subtype-specific latency and replication properties were studied in primary PHA-activated T lymphocytes. No major differences in latency and replication capacity were measured among the HIV 1 subtypes. The subtype B and AE LTRs were studied in more detail with regard to a putative AP 1 binding site using luciferase reporter constructs. C Jun, a member of the AP 1 transcription factor family, can activate both subtype B and AE LTRs, but the latter showed a stronger response, reflecting a closer match with the consensus AP 1 binding site. C Jun over-expression enhanced Tat-mediated transcription of the viral LTR but in the absence of Tat inhibited basal promoter activity. Thus, c Jun can exert a positive or negative effect via the AP 1 binding site in the HIV-1 LTR promoter, depending on the presence or absence of Tat.
A novel reovirus, designated Cimodo virus (CMDV), was isolated from mosquitoes collected in a rainforest region in Coocirc;te dIvoire. The entire genome comprised 24.835 bp divided into 12 segments ranging from 585 to 4,080 bp. The icosahedral non-enveloped virions were 80 nm in diameter. Eight major viral proteins of about 150, 135, 120, 80, 66, 59, 42, and 30 kDa were identified and seven proteins were mapped to the corresponding genome segments by liquid chromatography mass spectrometry (LC-MS). Predicted protein genes diverged by ggt;77% encoded amino acids from their closest reovirus relatives. The deep phylogenetic branching suggests that CMDV defines an as yet unidentified genus within the subfamily Spinareovirinae.
The recently emerged Middle East respiratory syndrome coronavirus or MERS-CoV, a beta-coronavirus, is associated with severe pneumonia and renal failure. The environmental origin of MERS-CoV is yet unknown, however, its genome sequence is closely related to those of two bat coronaviruses, named BtCoV-HKU4 and BtCoV-HKU5, which were derived from Chinese bat samples. A hallmark of highly pathogenic respiratory viruses is their ability to evade the innate immune response of the host. Coronavirus accessory proteins, for example those from severe acute respiratory syndrome coronavirus (SARS-CoV), have been shown to block innate anti-viral signaling pathways. MERS-CoV, similar to SARS-CoV, has been shown to inhibit type I interferon induction in a variety of cell types in vitro. We therefore hypothesized that MERS-CoV and the phylogenetically related coronaviruses BtCoV-HKU4 and BtCoV-HKU5 may encode proteins with similar capabilities. In this study, we demonstrate that the ORF4b-encoded accessory proteins (p4b) of MERS-CoV, BtCoV-HKU4, and BtCoV-HKU5 may indeed facilitate innate immune evasion by inhibiting the type I IFN and NF-kB signaling pathways. We also analyzed the subcellular localization of p4b from MERS-CoV, BtCoV-HKU4, and BtCoV-HKU5 and demonstrate that all are localized to the nucleus
Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. The emergence of very virulent IBDV (vvIBDV) has brought more challenges for effective prevention of this disease. The molecular basis for the virulence of vvIBDV is not fully understood. In this study, twenty IBDV strains were phylogenically analyzed and clustered in three branches based on their full-length B-segments. The amino acid triplet located at 145/146/147 of VP1 was found highly conserved in branch I non-vvIBDVs as asparagine/glutamic acid/glycine (NEG), in branch II vvIBDVs as threonine/glutamic acid/glycine (TEG) while in branch III vvIBDVs as threonine/aspartic acid/asparagine (TDN). The further studies showed that the three amino acids play a critical role in the replication and pathogenicity of vvIBDV. Substitution of the TDN triplet with TEG or NEG reduced viral replication and pathogenicity of the vvIBDV HuB-1 strain in chickens. On the other hand, the replication of the attenuated IBDV Gt strain was reduced in CEF cells while was enhanced in the bursa by substituting NEG with TEG or TDN. The exchange of the three amino acids was also found to be capable of affecting the polymerase activity of VP1. The important role of segment B in the pathogenicity of IBDV was confirmed in this study. These results also provide new insights into the mechanism of the virulence of vvIBDVs and may offer new targets for their attenuation to develop potential vaccines using reverse genetics.
Pigs can be severely harmed by flu, and represent important reservoir hosts, in which new human pathogens like the recent pandemic swine-origin H1N1 influenza A virus can arise by mutation and reassortment of genome segments. To obtain novel, safe influenza vaccines for pigs, and to investigate the antigen-specific immune response, we modified an established live-virus vaccine against Aujeszkys disease of swine, pseudorabies virus (PrV) strain Bartha, to serve as vector for the expression of hemagglutinin (HA) of swine-origin H1N1 virus. To facilitate transgene insertion, the genome of PrV-Ba was cloned as a bacterial artificial chromosome. HA expression occurred under control of the human or murine cytomegalovirus immediate early promoters (P-HCMV, P-MCMV), but could be substantially enhanced by synthetic introns and adaptation of the codon usage to that of PrV. However, despite abundant expression, the heterologous glycoprotein was not detectably incorporated into mature PrV particles. Replication of HA-expressing PrV in cell culture was only slightly affected compared to that of the parental virus strain. A single immunization of pigs with the PrV vector expressing the codon-optimized HA gene under control of P-MCMV induced high levels of HA-specific antibodies. The vaccinated animals were protected from clinical signs after challenge with a related swine-origin H1N1 influenza A virus, and challenge virus shedding was significantly reduced.
We analyzed natural recombination in 79 HEV-A strains representing 13 serotypes by sequencing of VP1, 2C, and 3D genome regions. The half-life of a non-recombinant tree node in coxsackieviruses 2, 4 and 10 was only 3.5 years, and never more than 9 years. All coxsackieviruses that differed by more than 7% of the nucleotide sequence in any genome region were recombinants relative to each other. Enterovirus 71, on the contrary, displayed remarkable genetic stability. Three major EV71 clades were stable for 19-29 years, with a half-life of non-recombinant viruses between 13 and 18.5 years in different clades. Only five EV71 strains out of over 150 recently acquired non-structural genome regions from coxsackieviruses, while none of 80 contemporary coxsackieviruses had non-structural genes transferred from the three EV71 clades. In contrast to earlier observations, recombination between VP1 and 2C genome regions was not more frequent than between 2C and 3D regions.
Arboretum (ABTV) and Puerto Almendras (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11,482 nt (ABTV) and 11,876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family.
Recently, several novel human polyomaviruses (HPyV) have been detected. HPyV6, 7, 9 and 10 are not associated with any disease so far. Trichodysplasia spinulosa (TS)-associated polyomavirus (TSPyV) can cause the rare skin disease TS. We have evaluated cutaneous DNA prevalence and viral loads of five HPyV in HIV-infected men compared to healthy male controls. 449 forehead swabs were analysed by HPyV-specific real-time PCRs. HPyV6, HPyV7, TSPyV, and HPyV10 were found significantly more frequent on the skin of 210 HIV-infected compared to 239 HIV-negative men (HPyV6: 39.0 vs. 27.6%, HPyV7: 21.0 vs. 13.4%, TSPyV: 3.8 vs. 0.8%, HPyV10: 9.3 vs. 3.4%; p llt; 0.05, respectively). HPyV9 was not detected. Multiple infections were more frequent in HIV-positive men, but HPyV-DNA loads did not differ significantly in both groups. In contrast to HPyV6, 7, and 10, TSPyV and HPyV9 do not seem to be a regular part of the human skin microbiome.
In the present work we investigated the importance of Raf/MEK/ERK signaling pathway in the multiplication of the arenavirus Junin (JUNV) in monkey and human cell cultures. We established that JUNV induces a biphasic activation of ERK and we proved that a specific inhibitor of ERK pathway, U0126, impairs viral replication. Furthermore, U0126 exerted inhibitory action against the arenaviruses Tacaribe and Pichindeeacute;. Moreover, treatment with known ERK activators such as phorbol 12-myristate 13-acetate and serum increased viral yields whereas ERK silencing by small interfering RNAs caused the inhibition of viral multiplication. Therefore, activation of Raf/MEK/ERK signaling pathway is required to ensure JUNV efficient replication and may constitute a host target for the development of novel effective therapeutic strategies to deal with arenavirus infections.
Some respiratory tract infections remain unexplained despite extensive testing for common pathogens. Nasopharyngeal aspirates (NPA) from 120 Chilean infants from Santiago with acute lower respiratory tract infections were analyzed by viral metagenomics revealing the presence of nucleic acids from anelloviruses, adenovirus associated virus, as well as 12 known respiratory viral pathogens. A single sequence read showed translated protein similarity to cycloviruses. We used inverse PCR to amplify the complete circular ssDNA genome of a novel cyclovirus we named CyCV-ChileNPA1. Closely related variants were detected using PCR in the NPA of three other affected children that also contained anelloviruses. This report increases the current knowledge of the genetic diversity of cycloviruses whose detection in multiple NPAs may reflect tropism for human respiratory tissues.
Orthoreoviruses have been associated with a variety of diseases in domesticated poultry and wild-living birds. In 2002 a reovirus strain, named Tvaauml;rminne avian virus (TVAV), was identified in Finland in a crow showing neurological disorders. The objective of this study was the molecular characterization of this novel reovirus strain. Genome sequencing was performed by combining semiconductor sequencing and traditional capillary sequencing. Sequence and phylogenetic analyses showed that TVAV shares low nucleotide sequence identity with other reoviruses (range for each gene, 31-72%) including strains belonging to the Avian orthoreovirus species. The most closely related reovirus strain was an isolate identified in Steller sea lion. Our data indicate that TVAV is a divergent avian origin reovirus that may be the first representative of a distinct virus species within the Orthoreovirus genus.
The threat of highly pathogenic avian influenza (HPAI) H5N1 viruses to cause the next pandemic remains a major concern. Here, we evaluated the cross-protection induced by natural infection of human seasonal influenza strains or immunization with trivalent inactivated influenza vaccine (TIV) against HPAI H5N1 (A/Vietnam/1203/2004) virus in ferrets. Groups were treated with phosphate buffered saline (group A), infected with H1N1 (group B) or H3N2 (group C) virus, or immunized with TIV (group D). Twelve weeks after the last treatment, serological assays revealed that groups B and C sustained moderate immunogenicity against homologous viruses but not D; cross-reactivity against the H5N1 virus was not detected in any group. Following challenge with A/Vietnam/1203/2004(H5N1) virus, only groups B and C exhibited attenuated viral loads leading to 100% survival. Our data suggest that natural infection with human seasonal strains could potentially provide better heterosubtypic protection against HPAI H5N1 virus infection compared to TIV immunization.
The catalytic subunit of the herpes simplex virus 1 DNA polymerase (HSV-1 Pol) is essential for viral DNA synthesis and production of infectious virus in cell culture. While mutations that affect 5-3 polymerase activity have been evaluated in animal models of HSV-1 infection, mutations that affect other functions of HSV-1 Pol have not. In a previous report, we utilized bacterial artificial chromosome technology to generate defined HSV-1 pol mutants with lesions in the previously uncharacterized pre-NH2-terminal domain. We found that the extreme N-terminal 42 residues (deletion mutant polN43) were dispensable for replication in cell culture, while residues 44-49 (alanine-substitution mutant polA6) were required for efficient viral DNA synthesis and production of infectious virus. In this study, we sought to address the importance of these conserved elements in viral replication in a mouse corneal infection model. Mutant virus polN43 exhibited no meaningful defect in acute or latent infection despite strong conservation of residues 1-42 with HSV-2 Pol. The polA6 mutation caused a modest defect in replication at the site of inoculation, and was severely impaired for ganglionic replication, even at high inocula that permitted efficient corneal replication. Additionally, the polA6 mutation resulted in reduced latency establishment and subsequent reactivation. Moreover, we found that the polA6 replication defect in cultured cells was exacerbated in resting cells as compared to dividing cells. These results reveal an important role for the conserved motif at residues 44-49 of HSV-1 Pol for ganglionic viral replication.
The neuroinflammatory response to West Nile virus infection can be either protective or pathological depending on the context. Although several studies have examined chemokine profiles within brains of WNV-infected mice, little is known about how various cell types within the central nervous system (CNS) contribute to chemokine expression. Here, we assessed chemokine expression in brain microvascular endothelial cells and astrocytes, which comprise the major components of the blood-brain barrier (BBB), in response to a nonpathogenic (WNV-MAD78) and a highly pathogenic (WNV-NY) strain of WNV. Higher levels of the chemokine CCL5 were detected in WNV-MAD78-infected brain endothelial monolayers compared to WNV-NY-infected cells. However, the opposite profile was observed in WNV-infected astrocytes, indicating that pathogenic and nonpathogenic strains of WNV provoke different CCL5 profiles at the BBB. Thus, cells comprising the BBB may contribute to a dynamic proinflammatory response within the CNS that evolves as WNV infection progresses.
Human infection by H7N9 influenza virus was first identified in China in March 2013. As of August 12, 2013, a total of 135 documented cases with 44 fatalities had been reported. Genetic and laboratory analyses of the novel H7N9 viruses isolated from patients indicate that these viruses possess several polymerase gene mutations previously associated with human adaptation and potential pandemic capabilities. However, the function of these mutations in the emergence and pathogenicity of the viruses is not well known. In this study, we demonstrate that the PB2 E627K mutation, which occurs in over 70% of the H7N9 patient isolates, promotes the replication of H7N9 virus by enhancing PB2 polymerase activity and enhances virulence in mice. Our results show the PB2 E627K mutation has played an important role in this H7N9 influenza outbreak and in the pathogenicity of the H7N9 virus.
Neurological diseases caused by encephalitic flaviviruses are severe and associated with high level of mortality. However, detailed mechanisms of viral replication in the brain and features of viral pathogenesis remain poorly understood. We carried out the comparative analysis of replication of neurotropic flaviviruses, West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus (TBEV), in primary cultures of mouse brain neurons. All flaviviruses multiplied well in the primary neuronal cultures from hippocampus, cerebral cortex, or cerebellum. Distribution of viral-specific antigen in the neuron varied; TBEV infection induced the accumulations of viral antigen in the neuronal dendrites to greater extent than did infection with other viruses. Viral structural, non-structural proteins, and double-stranded RNA were detected in the regions of which viral antigens accumulated in dendrites after TBEV replication. Replication of TBEV replicon after the infection of TBEV virus-like particles also induced the antigen accumulation, indicating that accumulated viral antigens were the results of the viral RNA replication. Further, electron microscopic observation confirmed that TBEV replication induced the characteristic ultrastructural membrane alterations in the neurites; newly formed laminal membrane structure containing virion-like structures. This is the first report describing viral replication in and ultrastructural alterations of the neuronal dendrites, possibly causing the neuronal dysfunction. These findings encourage further study to understand the molecular mechanisms of viral replication in brains and the pathogenicity of neurotropic flaviviruses.
Tick-borne encephalitis virus (TBEV) is a major arbovirus that causes thousands of cases of severe neurological illness in humans annually. However, virulence factors and pathological mechanisms of TBEV remain largely unknown. To identify the virulence factors, we constructed chimeric viruses between two TBEV strains of the Far-Eastern subtype, Sofjin-HO (highly pathogenic) and Oshima 5-10 (low pathogenic).The replacement of the coding region for the structural and non-structural proteins from Sofjin into Oshima showed a partial increase of the viral pathogenicity in a mouse model. Oshima-based chimeric viruses with the variable region of the 3' untranslated region (3'-UTR) of Sofjin, which had a deletion of 207 nucleotides, killed 100% of mice and showed almost same virulence with Sofjin. Replacement of the variable region of 3'-UTR from Sofjin into Oshima did not increase viral multiplication in cultured cell and a mouse model at the early phase of viral entry into the brain. At the terminal phase of viral infection in mice, the virus titer of the Oshima-based chimeric virus with the variable region of the 3'-UTR of Sofjin reached a level identical to that of Sofjin, and showed a similar histopathological change in the brain tissue. This is the first report to show that the variable region of the 3'-UTR is a critical virulence factor in mice. These findings encourage further study to understand the mechanisms of the pathogenicity of TBEV and develop preventative and therapeutic strategies for TBE.
Human cytomegalovirus (HCMV) is known to evade extrinsic pro-apoptotic pathways not only by downregulating cells surface expression of the Death Receptors (DRs) TNFR1, TRAIL receptor 1 (TNFRSF10A) and TRAIL receptor 2 (TNFRSF10B), but also by impeding downstream signalling events. Fas (CD95/APO-1/TNFRSF6) also plays a prominent role in apoptotic clearance of virus-infected cells so its fate in HCMV infected cells needed to be addressed. Here, we show that cell surface expression of Fas was suppressed in HCMV-infected fibroblasts from 24 h onwards through the late phase of productive infection, and was dependent on de novo virus-encoded gene expression but not virus DNA replication. Significant levels of the fully glycosylated (endoglycosidase H-resistant) Fas were retained within HCMV infected cells throughout the infection within intracellular membranous structures. HCMV infection provided cells with a high level of protection from Fas-mediated apoptosis. Downregulation of Fas was observed with HCMV strains AD169, FIX, Merlin and TB40.
Full-length genome infectious clone is a powerful tool for functional assay in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank No. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered viruses from the cDNA clone replicated poorly in either BHK-21 cells or in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within NS2B and NS4A were recovered in the passaged viruses leading to viruses with large plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were on the order of NS2B-T102Mllt;NS4A-R79Kllt;NS2B-T102M+ NS4A-R79K. In vivo virulence assay in mice showed that the recombinant virus containing double mutations was similar virulent as wild type SA14 (GenBank No. M55506). This study reports the first chemical synthesized JEV, and reverse genetic assay demonstrated the substitutions of NS2B-T102M and NS4A-R79K altered JEV replication.
Porcine parvovirus (PPV) is a small DNA virus with restricted coding capacity. The 5kb genome can expresses 3 major nonstructural proteins (NS1, NS2, SAT), and 2 structural proteins (VP1, VP2). These few viral proteins are pleiotropic and interact with cellular components throughout viral replication. In this regard, very few cell lines were shown to efficiently replicate the virus. Cell lines were established from a primary culture of bovine cells that allowed distinguishing allotropic variants of PPV. Three cell lines were differentially sensitive to infection by two prototype PPV strains, NADL-2 and Kresse. In the first cell line (D10), infection was restricted early in the infectious cycle and was not productive. Infection of the second cell line (G11) was 1000 times less efficient with the NADL-2 strain compared to porcine cells while production of infectious virus of the Kresse strain was barely detectable. Restriction points in those cells were the initial generation of DNA replication intermediate and NS1 production. Infection with chimeras between NADL-2 and Kresse showed that residues outside the previously described allotropic determinant are also partially responsible for the restriction to Kresse replication in G11 cells. F4 cells were permissive to both strains although genome replication and infectious virus production were lower than in the porcine cells used for comparison. These results highlighted the dependent nature of parvovirus tropism on host factors and suggested that cells from a non-host origin can fully support a productive infection by both strains.
Trim 5aalpha; was the first member of the tripartite motif (TRIM) family of proteins that was identified to potently restrict human immunodeficiency virus type 1 (HIV-1) replication. The breadth of antiretroviral activity of TRIM family members is an active area of investigation. In this study, we demonstrate human Trim 37 possesses anti-HIV-1 activity. This antiretroviral activity and the manner in which it was displayed was implicated by i) decreased viral replication upon Trim 37 transient overexpression in virus-producing cells, ii) correlation of the reduction of viral infectivity with Trim 37 virion incorporation, iii) increased HIV-1 replication during siRNA depletion of Trim 37 expression, and iv) reduction in viral DNA synthesis upon Trim 37 transient overexpression. Our findings provide the first demonstration of the potent antiviral activity of human Trim 37, and implicate an antiviral mechanism whereby Trim 37 interferes with viral DNA synthesis.