|Journal of General Virology current issue|
Part of the Togaviridae family, alphaviruses are arthropod-borne viruses that are widely distributed throughout the globe. Alphaviruses are able to infect a variety of vertebrate hosts, but in humans, infection can result in extensive morbidity and mortality. Symptomatic infection can manifest as fever, an erythematous rash and/or significant inflammatory pathologies such as arthritis and encephalitis. Recent overwhelming outbreaks of alphaviral disease have highlighted the void in our understanding of alphavirus pathogenesis and the re-emergence of alphaviruses has given new impetus to anti-alphaviral drug design. In this review, the development of viable mouse models of Old Word and New World alphaviruses is examined. How mouse models that best replicate human disease have been used to elucidate the immunopathology of alphavirus pathogenesis and trial novel therapeutic discoveries is also discussed.
In human cytomegalovirus (HCMV)-infected cells, a dramatic remodelling of the nuclear architecture is linked to the creation, utilization and manipulation of subnuclear structures. This review outlines the involvement of several viral and cellular subnuclear structures in areas of HCMV replication and virusnndash;host interaction that include viral transcription, viral DNA synthesis and the production of DNA-filled viral capsids. The structures discussed include those that promote or impede HCMV replication (such as viral replication compartments and promyelocytic leukaemia nuclear bodies, respectively) and those whose role in the infected cell is unclear (for example, nucleoli and nuclear speckles). Viral and cellular proteins associated with subnuclear structures are also discussed. The data reviewed here highlight advances in our understanding of HCMV biology and emphasize the complexity of HCMV replication and virusnndash;host interactions in the nucleus.
We investigated the infectivity and transmissibility of the human seasonal H3N2, pandemic (pdm) H1N1 (2009) and B influenza viruses in dogs. Dogs inoculated with human seasonal H3N2 and pdm H1N1 influenza viruses exhibited nasal shedding and were seroconverted against the viruses; this did not occur in the influenza B virus-inoculated dogs. Transmission of human H3N2 virus between dogs was demonstrated by observing nasal shedding and seroconversion in naiiuml;ve dogs after contact with inoculated dogs. The seroprevalence study offered evidence of human H3N2 infection occurring in dogs since 2008. Furthermore, serological evidence of pdm H1N1 influenza virus infection alone and in combination with canine H3N2 virus was found in the serum samples collected from field dogs during 2010 and 2011. Our results suggest that dogs may be hosts for human seasonal H3N2 and pdm H1N1 influenza viruses.
H9N2 avian influenza viruses are enzootic around the world and can infect many different avian and mammalian hosts, including humans. Unlike the H9N2 viruses, which mainly originated in other countries and possess a non-structural protein 1 (NS1) of 230 aa, 98 % of the H9N2 viruses isolated in China lack the 13 aa at the C terminus of NS1 (217 aa in total). The biological significance of NS1 elongation remains elusive. To examine the effect of NS1 C-terminal elongation in the influenza virus, we used reverse genetics to generate a wt avian influenza H9N2 virus containing a 217 aa NS1 (H9N2NS1217) and two mutant viruses with elongated NS1s of 230 and 237 aa (H9N2NS1230 and H9N2NS1237). C-terminal elongation of NS1 did not have a significant impact on virus replication in Madinnndash;Darby canine kidney cells or DF-1 cells. The three variants exhibited similar replicability in mice; however, the H9N2NS1230 and H9N2NS1237 variants exhibited an upregulation in the level of inflammatory cytokines. In addition, both the H9N2NS1230 and H9N2NS1237 viruses increased replication and induced a high level of inflammatory cytokines and transmission in chickens, compared with the wt virus. These findings suggest that the NS1 extension conferred a gain of fitness to some extent.
Introductions of H7 influenza A virus (IAV) from wild birds into poultry have been documented worldwide, resulting in varying degrees of morbidity and mortality. H7 IAV infection in domestic poultry has served as a source of human infection and disease. We report the detection of H7N9 subtype IAVs in Minnesota (MN) turkey farms during 2009 and 2011. The full genome was sequenced from eight isolates as well as the haemagglutinin (HA) and neuraminidase (NA) gene segments of H7 and N9 virus subtypes for 108 isolates from North American wild birds between 1986 and 2012. Through maximum-likelihood and coalescent phylogenetic analyses, we identified the recent H7 and N9 IAV ancestors of the turkey-origin H7N9 IAVs, estimated the time and geographical origin of the ancestral viruses, and determined the relatedness between the 2009 and 2011 turkey-origin H7N9 IAVs. Analyses supported that the 2009 and 2011 viruses were distantly related genetically, suggesting that the two outbreaks arose from independent introduction events from wild birds. Our findings further supported that the 2011 MN turkey-origin H7N9 virus was closely related to H7N9 IAVs isolated in poultry in Nebraska during the same year. Although the precise origin of the wild-bird donor of the turkey-origin H7N9 IAVs could not be determined, our findings suggested that, for both the NA and HA gene segments, the MN turkey-origin H7N9 viruses were related to viruses circulating in wild birds between 2006 and 2011 in the Mississippi Flyway.
The retinoic acid-induced gene I (RIG-I) plays a crucial role in sensing viral RNA and IFN-bbeta; production. RIG-I varies in length and sequence between different species. We assessed the functional differences between RIG-I proteins derived from mammals and birds. The transfection of duck caspase recruitment domains (CARDs) and duck RIG-I (dCARDs and dRIG-I) and goose CARDs and goose RIG-I (gCARDs and gRIG-I) into chicken DF-1 cells increased the production of IFN-bbeta; mRNA and IFN-stimulated genes and decreased influenza A virus (IAV) replication; whereas human CARDs and RIG-I (hCARDs and hRIG-I) and mouse CARDs and RIG-I (mCARDs and mRIG-I) had no effect. In human 293T and A549 cells, hCARDs had the strongest IFN-inducing activity, followed by mCARDs, dCARDs and gCARDs. The IFN-inducing activity of hRIG-I was stronger than that of mRIG-I, dRIG-I and gRIG-I, in that order. The results showed that, although the ability of dCARDs to activate IFN was stronger than that of gCARDs in DF-1, 293T and A549 cells, dRIG-I had a weaker ability to activate IFN than gRIG-I in DF-1 cells with or without IAV infection. These data suggest that RIG-I proteins from different species have different amino acid sequences and functions. This genetic and functional diversity renders RIG-I flexible, adaptable and capable of recognizing many viruses in different species.
The dengue virus (DENV) envelope protein domain 3 (ED3) is the target of potent virus neutralizing antibodies. The DENV-2 ED3 contains adjacent type-specific and DENV complex-reactive antigenic sites that are composed of a small number of residues that were previously demonstrated to be critical for antibody binding. Site-directed mutagenesis of a DENV-2 16681 infectious clone was used to mutate critical residues in the DENV-2 type-specific (K305A and P384A) and DENV complex-reactive (K310A) antigenic sites. The K305A mutant virus multiplied like the parent virus in mosquito and mammalian cells, as did the P384A mutant virus, which required a compensatory mutation (G330D) for viability. However, the K310A mutant virus could not be recovered. The DENV-2 type-specific critical residue mutations K305A and P384A+G330D reduced the ability of DENV-2 type-specific, but not DENV complex-reactive, mAbs to neutralize virus infectivity and this was directly correlated with mAb binding affinity to the rED3 mutants.
Chikungunya virus (CHIKV) has recently affected millions of people in the Indian Ocean, with rare cases of encephalopathy and encephalitis occurring in neonates. In the study described herein, the capacity of mouse brain cells to control infection through innate immune antiviral responses was assessed. In vitro, CHIKV principally infected a subpopulation of mouse GFAP+ primary astrocytes. Oligodendrocytes and neurons could also be infected. An innate immune response was engaged by CHIKV-infected astrocytes with elevated expression of mRNAs for IFN-aalpha;nndash;bbeta;, inflammatory cytokines (e.g. IL-1bbeta;, IL-12, IL-10, IL-24) and proapoptotic factors (e.g. TNF-aalpha;, FasL, Lymphotoxin B). Programmed cell death through the intrinsic caspase-9 pathway was observed by immunofluorescence in infected astrocytes and neurons but not in oligodendrocytes. Interestingly, microglia did not replicate CHIKV but responded by elevated mitogen-activated protein kinase (MAPK) activity. Intracerebroventricular injection of CHIKV in neonate mice led to the infection of astrocytes. The astrogliosis response was accompanied by a dendritic CD206+ cell mobilization restricted to the site of infection. The results of this study support the paradigm that a multifaceted innate immune response can be mobilized by both professional immune and glial cells to control CHIKV neuroinfection events in neonates.
Core plays a critical role during hepatitis C virus (HCV) assembly, not only as a structural component of the virion, but also as a regulator of the formation of assembly sites. In this study, we observed that core is expressed later than other HCV proteins in a single viral cycle assay, resulting in a relative increase of core expression during a late step of the viral life cycle. This delayed core expression results from an increase of core half-life, indicating that core is initially degraded and is stabilized at a late step of the HCV life cycle. Stabilization-mediated delayed kinetics of core expression were also observed using heterologous expression systems. Core stabilization did not depend on its interaction with non-structural proteins or lipid droplets but was correlated with its expression levels and its oligomerization status. Therefore in the course of a HCV infection, core stabilization is likely to occur when the prior amplification of the viral genome during an initial replication step allows core to be synthesized at higher levels as a stable protein, during the assembly step of the viral life cycle.
Coxsackievirus B4 (CV-B4) belongs to the genus Enterovirus within the family Picornaviridae. To investigate target proteins recognized by T-cells in human enterovirus B infections, virus-encoded structural [VP0 (VP4 and VP2), VP1, VP3] and non-structural (2A, 2B, 2C, 3C and 3D) proteins were expressed and purified in Escherichia coli. Peripheral blood of 19 healthy adult donors was used to create enterovirus-specific T-cell lines by repeated stimulation with CV-B4 cell lysate antigen. T-cell lines responded in individual patterns, and responses to all purified proteins were observed. The most often recognized enteroviral protein was VP0, which is the fusion between the most conserved structural proteins, VP4 and VP2. T-cell responses to VP0 were detected in 15 of the 19 (79 %) donor lines. Non-structural 2C protein was recognized in 11 of the 19 (58 %) lines, and 11 of the 19 (58 %) lines also had a response to 3D protein. Furthermore, responses to other non-structural proteins (2A, 2B and 3C) were also detected. T-cell responses did not correlate clearly to the individual HLA-DR-DQ phenotype or the history of past coxsackie B virus infections of the donors.
Coronaviruses are enveloped RNA viruses that have evolved complex relationships with their host cells, and modulate their lipid composition, lipid synthesis and signalling. Lipid rafts, enriched in sphingolipids, cholesterol and associated proteins, are special plasma membrane microdomains involved in several processes in viral infections. The extraction of cholesterol leads to disorganization of lipid microdomains and to dissociation of proteins bound to lipid rafts. Because cholesterol-rich microdomains appear to be a general feature of the entry mechanism of non-eneveloped viruses and of several coronaviruses, the purpose of this study was to analyse the contribution of lipids to the infectivity of canine coronavirus (CCoV). The CCoV life cycle is closely connected to plasma membrane cholesterol, from cell entry to viral particle production. The methyl-bbeta;-cyclodextrin (Mbbeta;CD) was employed to remove cholesterol and to disrupt the lipid rafts. Cholesterol depletion from the cell membrane resulted in a dose-dependent reduction, but not abolishment, of virus infectivity, and at a concentration of 15 mM, the reduction in the infection rate was about 68 %. Mbbeta;CD treatment was used to verify if cholesterol in the envelope was required for CCoV infection. This resulted in a dose-dependent inhibitory effect, and at a concentration of 9 mM Mbbeta;CD, infectivity was reduced by about 73 %. Since viral entry would constitute a target for antiviral strategies, inhibitory molecules interacting with viral and/or cell membranes, or interfering with lipid metabolism, may have strong antiviral potential. It will be interesting in the future to analyse the membrane microdomains in the CCoV envelope.
Feline rotaviruses, members of the species Rotavirus A, are an infrequent source of zoonotic infections, and were previously shown by RNAnndash;RNA hybridization assays to possess two distinct genomic RNA constellations, represented by strains FRV-1 and FRV64. Due to the lack of whole genome sequence information for FRV-1, human rotavirus strain AU-1 has been used as a surrogate for the genotype constellation of feline rotaviruses. The aim of this study was to determine the whole genome sequence of FRV-1 and FRV64 to help understand the genetic relationships among existing feline rotaviruses from the evolutionary perspective. The genotype constellations of FRV-1 and FRV64 were G3-P-I3-R3-C3-M3-A3-N3-T3-E3-H3 and G3-P-I3-R3-C2-M3-A9-N2-T3-E3-H6, respectively. FRV-1 has a genotype constellation identical to that of the AU-1 strain. Although for individual genes they shared lineages, with the exception of genes encoding VP2, VP6 and VP7, the sequence identity between FRV-1 and AU-1 was considered to be sufficiently high for the AU-1 to be regarded as an example of the direct transmission of a feline rotavirus to a child. On the other hand, the FRV64 strain was not only similar in all the 11 genome segments to another feline rotavirus strain, Cat97, but also to canine rotavirus strains (K9 and CU-1) and feline/canine-like human rotavirus strains (Ro1845 and HCR3A). In conclusion, this study revealed intermingled sharing of genotypes and lineages among feline rotaviruses, suggesting the occurrence of frequent reassortment events over the course of evolution to emerge in four genotype constellations represented by FRV-1, FRV64/Cat97, Cat2 and BA222 strains.
Several members of the family Circoviridae have been shown to encode proteins with apoptotic activity. For example, both porcine circovirus type 2 (PCV2) and chicken anemia virus (CAV) encode a third viral protein (VP3) that has been shown to be cytotoxic. Interestingly, in the case of the CAV protein (designated apoptin), apoptosis is specific to transformed cell types. Similarities in genome structure and organization suggest that PCV type 1 (PCV1) may also contain a third ORF, which codes for a protein with homologous activity. To investigate this, ORF prediction followed by gene expression analyses were conducted on a gene found to be homologous to CAV and PCV2 VP3. Our data presented herein elucidate a putative ORF3 that codes for a viral protein with functional similarity to that of apoptin and PCV2 VP3. Unlike its homologues, sequence analysis revealed a highly hydrophobic, extended C-terminal domain in PCV1 VP3, which harbours a strong nuclear export signal. Subcellular localization analysis demonstrated divergent PCV1 VP3 localization patterns compared with that of CAV VP3. Interestingly, cytotoxicity studies revealed evidence that apoptosis may be selective to transformed cell types, similar to apoptin; however, PCV1 VP3 induced a dramatic G1 cell cycle arrest as opposed to the G2/M arrest observed with apoptin. These results indicate that nuclear localization of PCV1 VP3 is necessary neither for induction of apoptosis nor for transformed cell selectivity, and suggest a mechanism of action distinct from that of apoptin.
The comparative long-term kinetics of human cytomegalovirus (HCMV) load and HCMV-specific antibody responses in the immunocompetent and immunocompromised solid-organ transplanted host during primary HCMV infection was investigated. In total, 40 immunocompetent subjects and 17 transplanted patients were examined for viral load as well as for IgG antibody responses to HCMV glycoproteins gH/gL/pUL128L, gH/gL and gB, and neutralizing antibodies in ARPE-19 epithelial cells and human fibroblasts. In parallel, the CD4+ and CD8+ HCMV-specific T-cell responses were determined by cytokine flow cytometry. Transplanted patients reached significantly higher viral DNA peaks, which persisted longer than in immunocompetent subjects. The ELISA-IgG responses to the pentamer, gH/gL and gB were significantly higher in primary infections of the immunocompetent until six months after onset, with the two antibody levels then overlapping from six to 12 months. Antibody levels neutralizing infection of epithelial cells were significantly higher in transplanted patients after six months, persisting for up to a year after transplantation. This trend was not observed for antibodies neutralizing infection of human fibroblasts, which showed higher titres in the immunocompetent over the entire one-year follow-up. In conclusion, in immunocompromised patients the viral load peak was much higher, while the neutralizing antibody response exceeded that detected in the immunocompetent host starting six months after onset of follow-up, often concomitantly with a lack of specific CD4+ T cells. In this setting, the elevated antibody response occurred in the presence of differentiated follicular helper T cells in the blood, which decreased in number as did antibody titres upon reappearance of HCMV-specific CD4+ T cells.
Human herpesvirus-6A (HHV-6A) is rarer than HHV-6B in many infant populations. However, they are similarly prevalent as germline, chromosomally integrated genomes (ciHHV-6A/B). This integrated form affects 0.1nndash;1 % of the human population, where potentially virus gene expression could be in every cell, although virus relationships and health effects are not clear. In a Czech/German patient cohort ciHHV-6A was more common and diverse than ciHHV-6B. Quantitative PCR, nucleotide sequencing and telomeric integration site amplification characterized ciHHV-6 in 44 German myocarditis/cardiomyopathy and Czech malignancy/inflammatory disease (MI) patients plus donors. Comparisons were made to sequences from global virus reference strains, and blood DNA from childhood-infections from Zambia (HHV-6A mainly) and Japan (HHV-6B). The MI cohort were 86 % (18/21) ciHHV-6A, the cardiac cohort 65 % (13/20) ciHHV-6B, suggesting different disease links. Reactivation was supported by findings of 1) recombination between ciHHV-6A and HHV-6B genes in 20 % (4/21) of the MI cohort; 2) expression in a patient subset, of early/late transcripts from the inflammatory mediator genes chemokine receptor U51 and chemokine U83, both identical to ciHHV-6A DNA sequences; and 3) superinfection shown by deep sequencing identifying minor virus-variants only in ciHHV-6A, which expressed transcripts, indicating virus infection reactivates latent ciHHV-6A. Half the MI cohort had more than two copies per cell, median 5.2, indicative of reactivation. Remarkably, the integrated genomes encoded the secreted-active form of virus chemokines, rare in virus from childhood-infections. This shows integrated virus genomes can contribute new human genes with links to inflammatory pathology and supports ciHHV-6A reactivation as a source for emergent infection.
Human monkeypox is a viral zoonosis caused by monkeypox virus, an orthopoxvirus (OPXV). The majority of human monkeypox cases have been reported in moist forested regions in West and Central Africa, particularly in the Democratic Republic of the Congo (DRC). In this study we investigated zoonotic OPXV infection among wild animals in Zambia, which shares a border with DRC, to assess the geographical distribution of OPXV. We screened for OPXV antibodies in sera from non-human primates (NHPs), rodents and shrews by ELISA, and performed real-time PCR to detect OPXV DNA in spleen samples. Serological analysis indicated that 38 of 259 (14.7 %) rodents, 14 of 42 (33.3 %) shrews and 4 of 188 (2.1 %) NHPs had antibodies against OPXV. The OPXV DNA could not be detected in spleens from any animals tested. Our results indicated that wild animals living in rural human habitation areas of Zambia have been infected with OPXV.
Vaccinia virus (VACV) is a large DNA virus that replicates in the cytoplasm and encodes about 200 proteins of which approximately 50 % may be non-essential for viral replication. These proteins enable VACV to suppress transcription and translation of cellular genes, to inhibit the innate immune response, to exploit microtubule- and actin-based transport for virus entry and spread, and to subvert cellular metabolism for the benefit of the virus. VACV strain WR protein C16 induces stabilization of the hypoxia-inducible transcription factor (HIF)-1aalpha; by binding to the cellular oxygen sensor prolylhydroxylase domain-containing protein (PHD)2. Stabilization of HIF-1aalpha; is induced by several virus groups, but the purpose and consequences are unclear. Here, 1H-NMR spectroscopy and liquid chromatography-mass spectrometry are used to investigate the metabolic alterations during VACV infection in HeLa and 2FTGH cells. The role of C16 in such alterations was examined by comparing infection to WT VACV (strain WR) and a derivative virus lacking gene C16L (vC16). Compared with uninfected cells, VACV infection caused increased nucleotide and glutamine metabolism. In addition, there were increased concentrations of glutamine derivatives in cells infected with WT VACV compared with vC16. This indicates that C16 contributes to enhanced glutamine metabolism and this may help preserve tricarboxylic acid cycle activity. These data show that VACV infection reprogrammes cellular energy metabolism towards increased synthesis of the metabolic precursors utilized during viral replication, and that C16 contributes to this anabolic reprogramming of the cell, probably via the stabilization of HIF-1aalpha;.
Two strains of African swine fever virus (ASFV), the high-virulence Lisboa60 (L60) and the low-virulence NH/P68 (NHV), which have previously been used in effective immunization/protection studies, were sequenced. Both were isolated in Portugal during the 11-year period after the introduction of ASFV to the European Continent in 1957. The predicted proteins coded by both strains were compared, and where differences were found these were also compared to other strains of known virulence. This highlighted several genes with significant alterations in low-virulence strains of ASFV that may constitute virulence factors, several of which are still uncharacterized regarding their function. Phylogenetic analysis grouped L60 and NHV closest to other P72 genotype I ASFV strains from Europe and West Africa, consistent with the assumed West African origin of all European strains. Interestingly, a relatively lower genomic identity exists between L60 and NHV, both isolated in a similar geographical location 8 years apart, than with other European and west African strains isolated subsequently and in more distant locations. This may reflect the intensive passage in tissue culture, during the early 1960s, of a Portuguese isolate to obtain an attenuated vaccine, which may have led to NHV. This study contributes to a better understanding of the evolution of ASFV, and defines additional potential virulence genes for future studies of pathogenesis towards the development of effective vaccines.
The pattern of flavivirus infection in mosquitoes belonging to the genera Aedes and Culex collected in two regions of north-eastern Italy (Trentino and Veneto) was assessed. Mosquitoes were collected during 2012 and screened for flaviviruses using a generic reverse transcription-nested-PCR targeted on a region of the non-structural NS5 gene. The phylogenetic analysis was performed on a fragment of ~1000 bp. Virus isolation was attempted in C6/36 insect cell lines and the infected cell cultures were studied by electron microscopy. We detected a wide distribution of Aedes flavivirus (AeFV) in Aedes albopictus, with higher infection prevalence in Trentino than in Veneto. In Culex pipiens collected in Veneto, we detected a new sequence of an insect-specific flavivirus and one of Usutu virus. Interestingly, we detected AeFV in C. pipiens, for the first time to our knowledge, in both regions. Viral isolation in cell culture was successful for AeFV. AeFV sequences found in Veneto showed a high percentage of similarity to those detected in Trentino and to those previously reported in other areas of northern Italy. Co-infections with different flaviviruses were not detected.
In this study, we identified a novel virus from gentian (Gentiana triflora) that causes ring-spots on ovaries. Furthermore, the virus causes unusual symptoms, ring-spots that appear specifically on the outer surface of the ovarian wall after pollination. Pollen grains carrying the virus were used to infect host plants by hand-pollination. RNA extracted from purified virions indicated that the virus had two segments, RNA1 and RNA2. The full-length cDNA sequence indicated that RNA1 had two ORFs: ORF1 had methyltransferase and helicase motifs, and ORF2 had an RNA-dependent RNA polymerase motif. RNA2 had five ORFs encoding a coat protein, triple gene block proteins 1nndash;3 and a cysteine-rich protein. The length of RNA1 was 5519 bases and that of RNA2 was 3810 bases not including a polyU/polyA region between the first and second ORFs. Viral RNA does not have a polyA tail at the 3' end. Sequence similarity and phylogenetic analysis suggested that the virus is closely related to members of the genera Pecluvirus and Hordeivirus but distinct from them. These combined results suggest that the causal agent inducing ring-spot symptoms on gentian ovaries is a new virus belonging to the family Virgaviridae but not to any presently known genus. We tentatively name the virus gentian ovary ring-spot virus.
Shrews are small insectivorous mammals that are distributed worldwide. Similar to rodents, shrews live on the ground and are commonly found near human residences. In this study, we investigated the enteric virome of wild shrews in the genus Crocidura using a sequence-independent viral metagenomics approach. A large portion of the shrew enteric virome was composed of insect viruses, whilst novel viruses including cyclovirus, picornavirus and picorna-like virus were also identified. Several cycloviruses, including variants of human cycloviruses detected in cerebrospinal fluid and stools, were detected in wild shrews at a high prevalence rate. The identified picornavirus was distantly related to human parechovirus, inferring the presence of a new genus in this family. The identified picorna-like viruses were characterized as different species of calhevirus 1, which was discovered previously in human stools. Complete or nearly complete genome sequences of these novel viruses were determined in this study and then were subjected to further genetic characterization. Our study provides an initial view of the diversity and distinctiveness of the shrew enteric virome and highlights unique novel viruses related to human stool-associated viruses.
PRD1 is a Gram-negative bacteria infecting complex tailless icosahedral virus with an inner membrane. This type virus of the family Tectiviridae contains at least 18 structural protein species, of which several are membrane associated. Vertices of the PRD1 virion consist of complexes recognizing the host cell, except for one special vertex through which the genome is packaged. Despite extensive knowledge of the overall structure of the PRD1 virion and several individual proteins at the atomic level, the locations and interactions of various integral membrane proteins and membrane-associated proteins still remain a mystery. Here, we demonstrated that blue native PAGE can be used to probe proteinnndash;protein interactions in complex membrane-containing viruses. Using this technique and PRD1 as a model, we identified the known PRD1 multiprotein vertex structure composed of penton protein P31, spike protein P5, receptor-binding protein P2 and stabilizing protein P16 linking the vertex to the internal membrane. Our results also indicated that two transmembrane proteins, P7 and P14, involved in viral nucleic acid delivery, make a complex. In addition, we performed a zymogram analysis using mutant particles devoid of the special vertex that indicated that the lytic enzyme P15 of PRD1 was not part of the packaging vertex, thus contradicting previously published results.
Bacteriophages and their derivatives are continuously gaining impetus as viable alternative therapeutic agents to control harmful multidrug-resistant bacterial pathogens, particularly in the food industry. The reduced efficacy of conventional antibiotics has resulted in a quest to find novel alternatives in the war against infectious disease. This study describes the full-genome sequence of Cronobacter phage vB_CsaP_Ss1, with subsequent cloning and expression of its endolysin, capable of hydrolysing Gram-negative peptidoglycan. Cronobacter phage vB_CsaP_Ss1 is composed of 42 205 bp of dsDNA with a G+C content of 46.1 mol%. A total of 57 ORFs were identified of which 18 could be assigned a putative function based on similarity to characterized proteins. The genome of Cronobacter phage vB_CsaP_Ss1 showed little similarity to any other bacteriophage genomes available in the database and thus was considered unique. In addition, functional analysis of the predicted endolysin (LysSs1) was also investigated. Zymographic experiments demonstrated the hydrolytic activity of LysSs1 against Gram-negative peptidoglycan, and this endolysin thus represents a novel candidate with potential for use against Gram-negative pathogens.
|Journal of General Virology Publish Ahead of Print|
Rabbit hemorrhagic disease virus (RHDV), a Lagovirus of the family Caliciviridae, causes rabbit hemorrhagic disease (RHD) in the European rabbit (Oryctolagus cuniculus). The disease was first documented in 1984 in China and rapidly spread worldwide. In 2010 a new RHDV variant emerged, tentatively classified as "RHDVb". RHDVb is characterized by affecting vaccinated rabbits and those llt;2 months old, and is genetically distinct (~20%) from older strains. To determine the evolution of RHDV, including the new variant, we generated 28 full-genome sequences from samples collected between 1994 and 2014. Phylogenetic analysis of the gene encoding the major capsid protein, VP60, indicated that all viruses sampled from 2012 to 2014 were RHDVb. Multiple recombination events were detected in the more recent RHDVb genomes, with a single major breakpoint located in the 5' region of VP60. This breakpoint divides the genome into two regions: one that encodes the non-structural proteins, and another that encodes the major and minor structural proteins, VP60 and VP10, respectively. Additional phylogenetic analysis of each region revealed two types of recombinants with distinct genomic backgrounds. Recombinants always include the structural proteins of RHDVb, with non-structural proteins from non-pathogenic lagoviruses or from pathogenic genogroup 1 strains. Our results show that in contrast to the evolutionary history of older RHDV strains, recombination plays an important role in generating diversity in the newly emerged RHDVb.
Polyomaviruses infect a diverse range of mammalian and avian hosts and are associated with a variety of symptoms. However, it is unknown whether the viruses are found in all mammalian families and the evolutionary history of the polyomaviruses is still unclear. Here we report the discovery of a novel polyomavirus in the European badger (Meles meles), which to our knowledge represents the first polyomavirus to be characterised in the Mustelidae family, and within a European carnivoran. Although the virus was discovered serendipitously in the supernatant of a cell culture inoculated with badger material, we subsequently confirmed its presence in wild badgers. The European badger polyomavirus was tentatively named Meles meles polyomavirus 1 (MmelPyV1). The genome is 5187bp long and encodes proteins typical of polyomaviruses. Phylogenetic analyses including all known polyomavirus genomes consistently group MmelPyV1 with California sea lion polyomavirus 1 across all regions of the genome. Further evolutionary analyses revealed phylogenetic discordance among polyomavirus genome regions, possibly arising from evolutionary rate heterogeneity, and a complex association between polyomavirus phylogeny and host taxonomic groups.
The route of pathogen entry can have a major effect on the ability of the virus to induce a prolific infection, but it can also affect the ability of the host organism to induce an immune response to fight the infection. Transmission of arboviruses that cause serious diseases in humans often begin by an insect ingesting a virus, which then disseminates through the internal organs and tissues and ultimately culminates in viral transmission to a human host. Understanding the effect of a natural route of infection on the host-pathogen interaction may facilitate development of approaches to prevent viral dissemination. Drosophila has been a useful model organism for understanding host-virus interactions, however most studies have achieved infection by artificially injecting the virus into the host. Here we develop a single-stranded quantitative PCR able to detect only actively replicating Drosophila C virus (DCV) to study the effect of viral feeding at the early stages of larval development. Exposure of newly hatched larvae to DCV leads to 20 % of larvae becoming infected within 12 hours post-contamination, and causes a 14 % egg to adult mortality. This is the first time to our knowledge that it has been shown experimentally that DCV is able to establish a prolific infection following larval feeding. Using the newly developed tools, the results suggest that the larvae that become infected die before adult eclosion.
CTLA-4 is a negative regulator of TCR-mediated CD4+ T-cell activation and function. Up-regulation of CTLA-4 during HIV-1 infection on activated T-cells, particularly on HIV-specific CD4+ T-cells, correlates with immune dysfunction and disease progression. As HIV-1 infects and replicates in activated CD4+ T cells, we investigated mechanisms by which HIV-1 modulates CTLA-4 expression to establish productive viral infection in these cells. Here we demonstrate that HIV-1 infection in activated CD4+ T cells was followed by Nef-mediated down-regulation of CTLA-4. This was associated with a decreased T-cell activation threshold and significant resistance to CTLA-4 triggering. In line with these in vitro results, quantification of proviral HIV-DNA from treatment-naive HIV-infected subjects demonstrated a preferential infection of memory CD4+CTLA-4+ T-cells, thus identifying CTLA-4 as a biomarker for HIV-infected cells in vivo. As transcriptionally active HIV-1 and Nef expression in vivo were previously shown to take place mainly in the CD3+CD4negCD8neg (double negative, DN) cells, we further quantified HIV DNA in the CTLA-4+ and CTLA-4neg sub-populations of these cells. Our results showed that DN T-cells lacking CTLA-4 expression were enriched in HIV-DNA compared to DN CTLA-4+ cells. Together, these results suggest that HIV-1 preferential infection of CD4+CTLA-4+ T-cells in vivo is followed by Nef-mediated concomitant down-regulation of both CD4 and CTLA-4 upon transition to productive infection. This also highlights the propensity of HIV-1 to evade restriction of the key negative immune regulator CTLA-4 on cell activation and viral replication and therefore contributes to the overall HIV-1 pathogenesis.
A variant Australian West Nile virus (WNV) strain, WNVNSW2011, emerged in 2011 causing an unprecedented outbreak of encephalitis in horses in south-eastern Australia. However no human cases associated with this strain have yet been reported. Studies using mouse models for WNV pathogenesis showed that WNVNSW2011 was less virulent than pathogenic in humans American strain of WNV, New York 99 (WNVNY99). To identify viral genes and mutations responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, we constructed chimeric viruses with substitution of large genomic regions coding for the structural genes, non-structural genes, and the untranslated regions, as well as seven individual non-structural gene chimeras using modified Circular Polymerase Extension Cloning method. Our results showed that complete non-structural region of WNVNSW2011, when substituted with that of WNVNY99, significantly enhanced viral replication and the ability to suppress type I interferon (IFN) response in cells resulting in higher virulence in mice. Analysis of the individual non-structural gene chimeras showed predominant contribution of WNVNY99 NS3 to increased virus replication and evasion of IFN response in cells and virulence in mice. From other WNVNY99 NS proteins, NS2A, NS4B and NS5, were shown to contribute in various degrees to the enhanced replication, modulation of IFN response, and virus-induced disease. Thus combination of non-structural proteins, likely NS2A, NS3, NS4B and NS5, is primarily responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains and accumulative mutations within these proteins would likely to be required for the Australian WNVNSW2011 strain to become significantly more virulent.
Positive-stranded RNA viruses include important human, animal and plant pathogens. Their genomes are able to fold into complex structures stabilised by base pairing between individual nucleotides, many of which are highly conserved and have essential functions during virus replication. With new studies and technological advances the diversity of roles, mechanisms and interactions in which such structured viral RNA functions is becoming increasingly clear. It is also evident that many RNA structures do not function as discrete elements but through mechanisms involving multiple, long-range and often dynamic RNA-RNA interactions. Through a range of examples and recent advances, this review illustrates the diverse roles and mechanisms of structured viral RNA during the replication of positive-stranded RNA viruses infecting humans and animals.
Thermostable Newcastle disease virus (NDV) vaccines have been used widely to control Newcastle disease (ND) for village flocks, due to their independence of cold chains for delivery and storage. To explore the potential use of the thermostable NDV as a vaccine vector, an infectious clone of thermostable avirulent NDV strain TS09-C was developed using reverse genetics technology. The green fluorescence protein (GFP) gene, along with the self-cleaving 2A gene of foot-and-mouth disease virus and Ubiquitin monomer (2AUbi), were inserted immediately upstream of the NP, M, or L gene translation start codon in the TS09-C infectious clone. Detection of GFP expression in the recombinant virus-infected cells showed that the recombinant virus, rTS-GFP/M, with the GFP inserted into the M gene expressed the highest level of GFP. The rTS-GFP/M virus retained the same thermostability, growth ability, and pathogenicity as its parental rTS09-C virus. Vaccination of specific pathogen free (SPF) chickens with the rTS-GFP/M virus conferred complete protection against virulent NDV challenge. Taken together, the data suggested that the rTS09-C virus could be used as a vaccine vector to develop bivalent thermostable vaccines against ND and the target avian diseases for village chickens, especially in the developing and least-developed countries.
We have previously reported that betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole exhibited a modified SJNNV capsid amino acid sequence, with two amino acid changes at positions 247 and 270. In the current study, we have investigated the possible role of both residues as putative virulence determinants. Three recombinant viruses harbouring site-specific mutations in the capsid protein sequence, rSs160.03247 [S247A], rSs160.03270 [S270N], and rSs160.03247+270 [S247A/S270N], have been generated using a reverse genetics system. These recombinant viruses were studied in cell culture and in vivo in the natural fish host. The three mutant viruses were shown to be infectious and able to replicate in E-11 cells, reaching final titers similar to the wild-type virus, although with a somewhat slower kinetics of replication. When the effect of the amino acid substitutions on virus pathogenicity was evaluated in Senegalese sole, typical clinical signs of betanodavirus infection were observed in all groups. However, fish mortality induced by all three mutant viruses was clearly affected. Roughly 40% of the fish survived in these 3 groups in contrast to the wild-type virus which killed 100% of the fish. These data demonstrate that residues 247 and 270 play a major role in the betanodavirus virulence although when both mutated amino acids 247 and 270 are present, corresponding recombinant virus was not further attenuated.
Influenza A viruses circulate in a wide range of animals. H3N8 equine influenza virus (EIV) is an avian-origin virus that has established in dogs as canine influenza virus (CIV) and has also been isolated from camels and pigs. Previous work suggests that adaptive mutations acquired during EIV evolution might have played a role in CIV emergence. Given the potential role of pigs as a source of human infections, we determined the ability of H3N8 EIVs to replicate in pig cell lines and in respiratory explants. We show that evolutionary distinct EIVs display different infection phenotypes along the pig respiratory tract, but not in cell lines. Our results suggest that EIV displays a dynamic host range along its evolutionary history, supporting the view that evolutionary processes play important roles on host range and tropism, and also underscore the utility of using explants cultures to study influenza pathogenesis.
Coxsackievirus A6 (CAV6) is an enterically transmitted enterovirus. Until recently, CAV6 infections have been considered as being of minor clinical significance and only rarely aetiologically linked with hand, foot and mouth disease (HFMD) associated with other species A enteroviruses (particularly enterovirus 71 and CAV16). From 2008 onwards, however, CAV6 infections have been associated with several outbreaks worldwide of atypical HFMD (aHFMD) accompanied by a varicelliform rash. We recently reported CAV6-associated eczema herpeticum (EH) occurring predominantly in children and young adults in Edinburgh in January - February, 2014. To investigate genetic determinants of novel clinical phenotypes of CAV6, we genetically characterised and analysed CAV6 variants associated with EH in Edinburgh, 2014 and those with aHFMD in CAV isolates collected from 2008. A total of eight recombinant forms have circulated worldwide over the past 10 years, with particularly recent appearance of recombinant form H (RF-H) associated with EH cases in Edinburgh in 2014. Comparison of phylogenies and divergence of complete genome sequences of CAV6 identified recombination breakpoints in 2A-2C, within VP3 and between VP1 and the 5'untranslated region. A Bayesian temporal reconstruction of CAV6 evolution since 2004 provided estimates of dates and the actual recombination events that generated more recently appearing RFs (-E, -F, -G and -H). Associations were observed between recombination groups and clinical presentations of herpangina, aHFMD and EH, but not with VP1 or other structural genes. These observations provide evidence that NS gene regions may potentially contribute to clinical phenotypes and outcomes of CAV6 infection.
Cystoviridae is a family of bacteriophages with a tri-segmented dsRNA genome enclosed in a tri-layered virion structure. Here, we present a new putative member of the Cystoviridae family, bacteriophage NN. NN was isolated from a Finnish lake in contrast to the previously identified cystoviruses, which originate from various legume samples collected in the U.S.A. Nucleotide sequence of the virus reveals a strong genetic similarity to Pseudomonas phage 6 (~ 80% for the L-segments, ~ 55% for the M-segments and ~ 84% for the S-segments), the type member of the virus family. However, the relationship between NN and other cystoviruses is more distant. In general, proteins located in the internal parts of the virion were more conserved than those exposed on the virion surface, a phenomenon previously reported among eukaryotic dsRNA viruses. Structural models of several putative NN proteins propose that cystoviral structures are highly conserved.
The neonatal Fc receptor (FcRn) is the only receptor known to be able to transport IgG across cell barriers and may therefore modulate viral infection. FcRn is efficiently expressed in hepatocytes. We therefore investigated the possible involvement of an FcRn-dependent mechanism in hepatitis C virus (HCV) neutralization. Our study, in both HCV pseudoparticles and HCV in cell culture models, showed that FcRn was not involved in the intracellular neutralization of HCV, by contrast to the situation observed for influenza A virus.
Porcine reproductive and respiratory syndrome virus (PRRSV) usually establishes a prolonged infection and causes an immunosuppressive state. It has been proposed that interleukin-10 (IL-10) plays an important role in PRRSV-induced immunosuppression. However, this mechanism has not been completely elucidated. In this study, we found that transfection of 3D4/2 macrophages with the N protein gene of type 2 PRRSV significantly upregulated IL-10 expression at the transcriptional level. Moreover, alanine substitution mutation analysis revealed that the N protein residues 33-37, 65-68, and 112-123 were related to the upregulation of IL-10 promoter activity. Recombinant PRRSV with mutations at residues 33-37 in the N protein (rQ33-5A and rS36A), recovered from corresponding infectious cDNA clones, induced significantly lower levels of IL-10 production in infected monocyte-derived dendritic cells, as compared to their revertants rQ33-5A(R) and rS36A(R), and the wild-type recombinant PRRSV strain rNT/wt. These data indicate that type 2 PRRSV N protein plays an important role in IL-10 induction and the N-N non-covalent domain is associated with this activity.
Hepatitis E virus (HEV) infection causes high mortality in pregnant women. However, the pathogenic mechanisms of HEV infection in pregnant women remain unknown. In this study, the roles of pregnancy serum in HEV infection were investigated using an efficient cell culture system. HEV infection was exacerbated by supplementing with pregnancy serum, especially the serum in third trimester of pregnancy. Estrogen receptors (ER-aalpha; and ER-bbeta;) were activate in cells supplemented with pregnancy serum, and significantly inhibited when HEV infection. Type I interferon, especially IFN-bbeta;, showed a delayed up-regulation in HEV infected cells supplemented with the serum in third trimester of pregnancy, which indicated that the delayed IFN-bbeta; expression may facilitate viral replication. Results suggested that pregnancy serum accelerate HEV replication by suppressing estrogen receptors and type I interferon in the early stage of infection.
Rapid biosynthesis is key to the success of bacteria and viruses. Highly expressed genes in bacteria exhibit strong codon bias corresponding to differential availability of tRNAs. However, a large clade of lambdoid coliphages exhibit relatively poor codon adaptation to the host translation machinery, in contrast to other coliphages that exhibit strong codon adaptation to the host. Three possible explanations were previously proposed but dismissed: 1) the phage-borne tRNA genes that reduce the dependence of phage translation on host tRNAs, 2) lack of time needed for evolving codon adaptation due to recent host switching, and 3) strong strand asymmetry with biased mutation disrupting codon adaptation. Here we examine the possibility that phages with relatively poor codon adaptation have poor translation initiation which would weaken the selection on codon adaptation. We measure translation initiation by: 1) the strength and position of the Shine-Dalgarno (SD) sequence and (2) stability of secondary structure of sequences flanking SD and start codon known to affect accessibility of SD and start codon. Phage genes with strong codon adaptation have significantly stronger SD sequences than those with poor codon adaptation. The former also have significantly weaker secondary structure in sequences flanking SD and start codon than the latter. Thus, lambdoid phages do not exhibit strong codon adaptation because they have relatively inefficient translation initiation and would benefit little from increased elongation efficiency. We also provide evidence suggesting that phage lifestyle (virulent versus temperate) affects selection intensity on the efficiency of translation initiation and elongation.
Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor which blocks cytosolic entry of numerous viruses that utilise acidic endosomal entry pathways. In humans and mice, IFITM3 limits influenza-induced morbidity and mortality. Although many IFITM3-sensitive viruses are zoonotic, whether IFITMs function as antiviral restriction factors in mammalian species other than humans and mice is unknown. Here, IFITM3 orthologues in the microbat Myotis myotis and the pig (Sus scrofa domesticus) were identified using rapid amplification of cDNA ends. Amino acid residues known to be important for IFITM3 function were conserved in the pig and bat orthologues. Ectopically-expressed pig and microbat IFITM3 co-localised with transferrin (early endosomes) and CD63 (late endosomes/multivesicular bodies) and trafficked from the plasma membrane into endosomes following live cell staining. Pig and microbat IFITM3 restricted cell entry mediated by multiple influenza HA subtypes and lyssavirus G proteins. Expression of pig or microbat IFITM3 in A549 cells reduced influenza virus yields and nucleoprotein expression. Conversely siRNA knockdown of IFITM3 in pig NPTr cells and primary microbat cells enhanced virus replication, demonstrating that these genes are functional in their species of origin at endogenous levels. In sum, we show that IFITMs function as potent broad-spectrum antiviral effectors in two mammals - pigs and bats - identified as major reservoirs for emerging viruses.
Epitopes on the surface of the foot-and-mouth disease virus (FMDV) capsid have been identified by monoclonal antibody (mAb) escape mutant studies leading to the designation of four antigenic sites in serotype A FMDV. Previous work focused on viruses isolated mainly from Asia, Europe and Latin America. In this study we report prediction of epitopes in African serotype A FMDVs and tested selected epitopes using reverse genetics. Twenty-four capsid amino acid residues were predicted to be of antigenic significance by analyzing the capsid sequences (n=56) using in-silico methods and six residues by correlating capsid sequence with serum-virus neutralization data. The predicted residues were distributed on the surface-exposed capsid regions, VP1-VP3. The significance of residue changes at eight of the predicted epitopes was tested by site directed mutagenesis using a cDNA clone resulting in the generation of 12 mutant viruses involving seven sites. The effect of the amino acid substitutions on the antigenic nature of the virus was assessed by virus neutralisation (VN) test. Mutations at four different positions, namely VP1-43, VP1-45, VP2-191, and VP3-132 led to significant reduction in VN titre (P-value = 0.05, 0.05, 0.001 and 0.05, respectively). This is the first time that the antigenic region encompassing amino acids VP1-43 to 45 (equivalent to antigenic site 3 in serotype O), VP2-191 and VP3-132 were predicted as epitopes and serologically evaluated for serotype A FMD viruses. This identifies novel capsid epitopes of recently circulating serotype A FMD viruses in East Africa.
The group A rotavirus (RVA) P genotype has been sporadically detected in humans and is thought to be acquired through zoonotic transmission. The present study describes the full-length genome analysis of two G8P and one G10P human RVAs detected in Italy. The strains possessed the typical bovine-like I2-R2-C2-M2-A3/A11-N2-T6-E2-H3 genotype constellation. All the segments of the two G8P RVAs were most closely related to bovine (-like) strains but were relatively distant to each other suggesting two independent interspecies transmission events. Likewise, the G10P RVA gene segments were most similar to bovine (-like) RVAs, but distinct from the G8 strains. The natural history of these strains probably involved the interspecies transmission of these viruses to humans from a yet unidentified animal host, without evidence of reassortment events involving human RVAs. These results reinforce the potential of animal viruses to cross the host-species barrier causing disease and increase viral genetic diversity in humans.
West Nile Virus (WNV), a mosquito-borne Flavivirus, is the major cause of arboviral encephalitis in the United States. As other members of the Japanese encephalitis virus serogroup, WNV produces an additional nonstructural protein, NS1', a carboxy-terminal extended product of NS1 generated as the result of a -1 programmed ribosomal frameshift (PRF). We have previously shown that mutations abolishing the PRF and consequently NS1', resulted in reduced neuroinvasiveness. However whether this was caused by the PRF event itself or by the lack of a PRF product, NS1', or a combination of both, remains undetermined. Here we show that WNV NS1' forms a unique sub-population of heat- and low pH- stable dimers. C-terminal truncations and mutational analysis employing an NS1'-expressing plasmid showed that stability of NS1' dimers is linked to the penultimate ten amino acids. To examine the role of NS1' heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS1' to create a WNV producing a truncated version of NS1' lacking the last 20 amino acids but not affecting the PRF. NS1' protein produced by this mutant virus was secreted more efficiently than wild type NS1', indicating that the sequence of the last 20 amino acids of NS1' is responsible for its cellular retention. Further analysis of this mutant showed similar to the wild type WNVKUN growth kinetics in cells and virulence in weanling mice after peripheral infection suggesting that full length NS1' is not essential for virus replication in vitro and for virulence in mice.
In spite of the success of the mumps vaccination, recent mumps outbreaks were reported even among individuals with a history of mumps vaccination. For better understanding why the vaccination failed in cases of vaccinees which fell ill during recent mumps outbreaks, the immunological events during infection and/or vaccination should be better defined. In the work presented here we sought for new neutralization sites on the mumps virus surface glycoproteins. By using anti-mumps monoclonal antibodies (mAbs), three amino acid positions at residues 221, 323 and 373 in the F protein of mumps virus were shown to be located in at least two conformational neutralization epitopes. MAbs which specifically target these sites effectively neutralized mumps virus in vitro. The newly acquired glycosylation site at position 373 or loss of the existing one at position 323 were identified as the mechanism behind the escape from the specific mAbs. Based on the findings of this study we suggest that the influence of the antigenic structure of the F protein should not be ignored in a thorough investigation of the underlying mechanism of the mumps vaccine failure or when making a strategy for development of a new vaccine.
Aquareoviruses (AqRV) have a close relationship with orthoreoviruses. However, they contain an additional genome segment S11, which encodes nonstructural protein NS26. We previously showed that NS26 can enhance the fusogenic activity of the fusion-associated small transmembrane (FAST) protein NS16 from AqRV. In this study, a TLPK motif in NS26 was identified to be important for the enhancement. When the TLPK motif was deleted from NS26, the enhanced efficiency on the NS16 mediated cell-cell fusion was significantly impaired. Further mutational analysis showed that the lysine (K) residue in the TLPK motif was critical for the enhancement. Additionally, deletion of the TLPK motif prevented NS26 from interacting with the lysosomes. These findings suggested that the TLPK motif is important for NS26 to enhance the fusogenic activity of NS16, and NS26 may utilize the lysosome to benefit the fusion process.
Eight avian influenza A(H5N6) viruses were isolated from live poultry markets (LPMs) in Sichuan and Jiangxi Provinces in 2014, including those close to the country where the human H5N6 infection occurred. Genetic and phylogenetic analyses revealed that these H5N6 viruses are novel reassortants between H5N1 clade 2.3.4 and H6N6 viruses, and have evolved into two distinct lineages (Sichuan and Jiangxi). Moreover, the human-H5N6 virus was closely related to the avian-source viruses of Sichuan lineage, Notably, H5N6 viruses contained a T160A substitution in the HA protein and an 11-amino acid-deletion in the NA stalk, which may aid in enhancing viral affinity for human-like receptors, and virulence in mammals. As the H5N1 virus infects humans through direct contact, infection with the novel H5N6 virus raises significant concerns that the H5 subtype is a likely candidate for a pandemic. Therefore, extensive and long-term surveillance of avian influenza viruses (AIVs) in LPMs is essential.
The prM glycoprotein is thought to be a chaperon for the proper folding, membrane association and assembly of the envelope protein (E) of flaviviruses. The prM-E and E proteins of the Japanese encephalitis virus (JEV) were expressed in insect cells by using both the baculovirus-expression system and the transient expression method. Protein expression was analysed by Western blots, and the cytopathic effect was observed by microscopy. In the baculovirus-expression system, the E protein, with or without the prM protein induced syncytial formation in Sf9 cells. Transient expression of prM-E also induced syncytia in Sf9 cells. Immno-fluorescence revealed that in presence of prM, E proteins were ER-like in distribution, while in the absence of prM, E proteins were located on the cell surface. Sucrose gradient sedimentation and Western blot analysis indicated that the E protein expressed with or without the prM protein was secreted into the culture medium in particulate form. And the formation of virus-like particles (VLPs) in the medium was confirmed by electron microscopy (EM) and immuno-electron microscopy (IEM). The results suggest that E protein of JEV in the absence of prM retained its fusion ability, by either cell surface expression or formation of VLPs. Moreover, based on the observation that co-expression of prM-E in Sf9 cells induce considerable syncytial formation, a novel, safe and simple antiviral screening approach is proposed when studying inhibitory antibodies, peptides or small molecules targeting to JEV E protein.
A full-length infectious cDNA clone of the genotype 1 Korean avian hepatitis E virus (avian HEV) (pT11-aHEV-K) was constructed and its infectivity and pathogenicity were investigated in LMH chicken cells and broiler breeders. We demonstrated that capped RNA transcripts from the pT11-aHEV-K clone were translation-competent when transfected into LMH cells and infectious when intrahepatically injected into the livers of chickens. Gross and microscopic pathological lesions underpinned the avian HEV infection and help characterize its pathogenicity in broiler breeder chickens. The avian HEV genome contains a hypervariable region (HVR) in the ORF1. To demonstrate the utility of the avian HEV infectious clone, several mutants with various deletions in and beyond the known HVR were derived from the pT11-aHEV-K clone. The HVR-deletion mutants were replication competent in LMH cells, although the deletion mutants extending beyond the known HVR were non-viable. By using the pT11-aHEV-K infectious clone as the backbone, an avian HEV luciferase reporter replicon and HVR-deletion mutant replicons were also generated. The luciferase assay results of the reporter replicon and its mutants support the data obtained from the infectious clone and its derived mutants. To further determine the effect of HVR deletion on virus replication, the capped RNA transcripts from the wild-type pT11-aHEV-K clone and its mutants were intrahepatically injected in the chickens. The HVR-deletion mutants that were translation competent in LMH cells displayed in chickens an attenuation phenotype of avian HEV infectivity, suggesting that the avian HEV HVR is important in modulating the virus infectivity and pathogenicity.
Amphibian populations suffer massive mortalities from infection with Frog virus 3 (FV3, Ranavirus, Iridoviridae), a pathogen also involved in mortalities of fish and reptiles. Experimental oral infection with FV3 in captive-raised adult wood frogs, Rana sylvatica [Lithobates sylvaticus], was performed as the first step in establishing a native North American animal model of ranaviral disease to study pathogenesis and host-response. Oral dosing was successful; LD50 was 10^2.93 (2.42-3.44) pfu for frogs averaging 35 mm in length. Onset of clinical signs occurred 6-14 days post-infection (dpi) (median 11 dpi) and time-to-death 10-14 dpi (median 12 dpi). Each ten-fold increase in virus dose increased the odds of dying by 23-fold and accelerated onset of clinical signs and death by approximately 15%. Ranavirus DNA was demonstrated in skin and liver of all frogs that died or were euthanized because of severe clinical signs. Shedding of virus occurred in feces (7-10 dpi; 3-4.5 d before death) and skin sheds (10 dpi; 0-1.5 d before death) of some frogs dead from infection. Most common lesions were dermal erosion and hemorrhages, hematopoietic necrosis in bone marrow, kidney, spleen and liver, necrosis in renal glomeruli and in tongue, gastrointestinal tract, and urinary bladder mucosa. Presence of ranavirus in lesions was confirmed by immunohistochemistry. Intracytoplasmic inclusion bodies (probably viral) were present in the bone marrow and the epithelia of the oral cavity, gastrointestinal tract, renal tubules and urinary bladder. Our work describes a Ranavirus-wood frog model and provides estimates that can be incorporated into ranavirus disease ecology models.
Annexin A2 (ANXA2) is an important host factor regulating several key processes in many viruses. To evaluate the potential involvement of ANXA2 in the life cycle of classical swine fever virus (CSFV), RNAi approach was utilized. Knockdown of ANXA2 didn't impair CSFV RNA replication but significantly reduced CSFV production. A comparable reduction of extracellular and intracellular infectivity levels was detected, indicating that ANXA2 might play a role in CSFV assembly rather than in genome replication and virion release. Furthermore, ANXA2 was found to bind CSFV NS5A, an essential replicase component. Amino acids R338, N359, G378 of the NS5A were revealed to be pivotal for ANXA2-NS5A interaction. Substitutions of these amino acids had no effect on viral RNA replication but substantially reduced CSFV production, which might partly be due to these mutations destroying ANXA2-NS5A interaction. These results suggested that ANXA2 might participate in CSFV production process by binding NS5A.
Flavivirus NS4A and NS4B are important membrane proteins for viral replication that are assumed to serve as the scaffold for the formation of replication complexes (RCs). We previously demonstrated that a single Lys-to-Arg mutation at position 79 in NS4A protein (NS4A-K79R) significantly impaired Japanese encephalitis virus (JEV) replication. In this study, the mutant virus was subject to genetic selection to search for the potential interaction between NS4A and other viral components. Sequencing of the recovered viruses revealed that, besides a A97E change in NS4A protein itself, a Y3N compensatory mutation located in NS4B protein had emerged from independent selections. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that both adaptive mutations restored greatly the replication defect caused by NS4A-K79R. Our results, for the first time, clearly showed the genetic interaction between NS4A and NS4B although the mechanism underlying their interaction is unknown.
Infection with multiple genetically distinct strains of pathogen is common and can lead to positive (complementation) or negative (competitive) within-host interactions. These interactions can alter aspects of the disease process and help shape pathogen evolution. Infection of the host with multiple strains of cytomegalovirus (CMV) infection occurs frequently in humans and mice. Profound, NK cell mediated (apparent) competition, has been identified in C57BL/6 mice and prevented the replication and shedding of certain co-infecting CMV strains. However the frequency of such strong competition has not been established. Other within-host interactions such as complementation or alternative forms of competition remain possible. Moreover high rates of recombination in both human CMV (HCMV) and murine CMV (MCMV) suggest prolonged periods of viral co-replication, rather than strong competitive suppression. An established model was employed to investigate the different possible outcomes of multi-strain infection in other mouse strains. In this study, co-replication of up to four strains of MCMV in the spleens, livers and salivary glands was observed in both MCMV susceptible and MCMV resistant mice. In the absence of apparent competition, no other forms of competition were unmasked. In addition, no evidence of complementation between viral strains was observed. Importantly, co-replication of MCMV strains was apparent for up to 90 days in the salivary glands. These data indicate that competition is not the default outcome of multi-strain CMV infection. Prolonged, essentially neutral, co-replication may be the norm, allowing for multi-strain transmission and prolonged opportunities for recombination.
Porcine circovirus type 2 (PCV2) is the main etiological agent of postweaning multisystemic wasting syndrome (PMWS). The mechanism of pathogenicity associated with PCV2 infection is still not fully understood. Nevertheless, the fact that large amounts of proinflammatory cytokines within lymphoid tissues are released during the early stage of PCV2 infection may induce chronic inflammatory responses followed by the destruction of lymphoid tissues. However, how PCV2 infection causes an excessive inflammatory response in the host immune system during the early stage of PCV2 infection is still not elucidated. In this study, we show that direct interaction between the PCV2 ORF3 and regulator of G protein signaling 16 (RGS16) within the cytoplasm of host cells leads to ubiquitin-mediated proteasomal degradation of RGS16. Facilitated degradation of the RGS16 by PCV2 ORF3 further enhances NFB translocation into the nucleus through the ERK 1/2 signaling pathway and increased IL-6 and IL-8 mRNA transcripts. Consequently, more severe inflammatory responses and leukocyte infiltration occur around host cells. This evidence may be the first clue explaining the molecular basis of how excessive amounts of proinflammatory cytokines within lymphoid tissues are released during the early stage of PCV2 infection.
The serpin family of serine proteinase inhibitors plays key roles in a variety of biochemical pathways. In insects, one of the important functions carried out by serpins is regulation of the phenoloxidase cascade, a pathway that produces melanin and other compounds that are important in insect humoral immunity. Recent sequencing of the baculovirus Hemileuca sp. nucleopolyhedrovirus (HespNPV) genome revealed the presence of a gene, hesp018, with homology to insect serpins. To our knowledge hesp018 is the first viral serpin homolog to be characterized outside of the chordopoxviruses. The Hesp018 protein was found to be a functional serpin with inhibitory activity against a subset of serine proteinases. Hesp018 also inhibited phenoloxidase activation when mixed with lepidopteran hemolymph. The Hesp018 protein was secreted when expressed in lepidopteran cells, and a baculovirus expressing Hesp018 exhibited accelerated production of viral progeny during in vitro infection. Expression of Hesp018 also reduced caspase activity induced by baculovirus infection, but caused increased cathepsin activity. In infected insect larvae, expression of Hesp018 resulted in faster larval melanization, consistent with increased activity of viral cathepsin. Finally, expression of Hesp018 increased the virulence of a prototype baculovirus by 4-fold in orally-infected neonate Trichoplusia ni larvae. Based on our observations, we hypothesize that the hesp018 may have been retained in HespNPV due to its ability to inhibit the activity of select host proteinases, possibly including proteinases involved in the phenoloxidase response, during infection of host insects.
Three (MoCAV/F2, MoCAV/F8, MoCAV/F11) of 4 mouse monoclonal antibodies (mAbs) established against the A2/76 strain of chicken anemia virus (CAV) showed neutralization activity. Immunoprecipitation showed a band at approximately 50 kDa in A2/76-infected cell lysates by neutralizing mAbs, corresponding to the 50-kDa capsid protein (VP1) of CAV, and the mAbs reacted with recombinant VP1 proteins expressed in Cos7 cells. MoCAV/F2 and MoCAV/F8 neutralized the 14 CAV strains tested, whereas MoCAV/F11 did not neutralize 5 of the strains, indicating distinct antigenic variation among the strains. In blocking immunofluorescence tests with the A2/76-infected cells, binding of MoCAV/F11 was not inhibited by the other mAbs. MoCAV/F2 inhibited the binding of MoCAV/F8 to the antigens and vice versa, suggesting that the 2 mAbs recognized the same epitope. However, mutations were found in different parts of VP1 of the escape mutants of each mAb: EsCAV/F2 (deletion of T89+A90), EsCAV/F8 (I261T), and EsCAV/F11 (E144G). Thus, the epitopes recognized by MoCAV/F2 and MoCAV/F8 seemed to be topographically close in the VP1 structure, suggesting that VP1 has at least 2 different neutralizing epitopes. However, MoCAV/F8 did not react to EsCAV/F2 or to EsCAV/F8, suggesting that binding of MoCAV/F8 to the epitope requires coexistence of the epitope recognized by MoCAV/F2. In addition, MoCAV/F2, with a titer of 1:12,800 to the parent strain, neutralized EsCAV/F2 and EsCAV/F8 with low titers of 32 and 152, respectively. The similarity of the reactivity of MoCAV/F2 and MoCAV/F8 to VP1 may also suggest the existence of a single epitope recognized by these mAbs.
Epidemiological studies have suggested that consumption of beef may correlate with an increased risk of colorectal cancer. One hypothesis to explain this proposed link might be the presence of a carcinogenic infectious agent capable of withstanding cooking. Polyomaviruses are a ubiquitous family of thermostable non-enveloped DNA viruses that are known to be carcinogenic. Using virion enrichment, rolling circle amplification (RCA), and next-generation sequencing, we searched for polyomaviruses in meat samples purchased from several supermarkets. Ground beef samples were found to contain three polyomavirus species. One species, bovine polyomavirus 1 (BoPyV1), was originally discovered as a contaminant in laboratory fetal calf serum. A previously unknown species, BoPyV2, occupies the same clade as human Merkel cell polyomavirus and raccoon polyomavirus 1, both of which are carcinogenic in their native hosts. A third species, BoPyV3, is related to human polyomaviruses 6 and 7. Examples of additional DNA virus families, including herpesviruses, adenoviruses, circoviruses, and gyroviruses were also detected either in ground beef samples or in comparison samples of ground pork and ground chicken. The results suggest that the virion enrichment/RCA approach is suitable for random detection of essentially any DNA virus with a detergent-stable capsid. It will be important for future studies to address the possibility that animal viruses commonly found in food might be associated with disease.
The human cytomegalovirus (HCMV) UL112-113 gene is implicated in lytic viral replication. The UL112-113 proteins p34, p43, p50, and p84 are expressed via alternative splicing. However, the mechanism for the generation of three additional virus-associated proteins (p20, p26, and p28), which share the UL112 reading frame, remains unknown. Bioinformatic analyses indicated that p34, p43, p50, and p84 contain potential PEST-like degradation motifs. In this study, inhibitors of calpains, lysosomes, and proteasomes reduce p20, p26, and p28 levels in virus-infected cells, suggesting the involvement of proteolytic modification. Moreover, maitotoxin, which increases intracellular calcium levels and activates calpain activity, induces the intracellular proteolysis of p34 into p20, p26, and p28 and the cleavage of p43, p50, and p84 into p38 and a novel protein, p34c. Proteolytic assays further indicated that p34, p43, p50, and p84 were substrates of calpain-1 and calpain-2 and that they generated proteolytic products that corresponded to those detected during the HCMV infectious period. Furthermore, substitution mutations in the putative calpain cleavage sites of p34 reduced accumulation of proteolytic products. The knockdown of endogenous calpain-1 and calpain-2 by RNA interference reduced accumulation of p20, p26, and p28 and concurrently increased levels of nascent p43, p50, and p84 during the infectious cycle. Intriguingly, the calpain depletion enhanced viral genome synthesis. Moreover, HCMV-permissive cells that stably expressed p20, p26 or p28 exhibited reduced viral genome synthesis and mature virus production. Our findings suggest that cognate UL112-113 proteins derived from calpain-catalysed proteolysis are involved in the HCMV replication process.
The members of Vesiculovirus which belongs to the family of Rhabdoviridae can cause great economic loss in fish culture. In the present report, a vesiculovirus (named as SHVV) was isolated from diseased hybrid snakehead fish. The SHVV shared 94% nucleotide sequence identity at the genomic level with Siniperca chuatsi rhabdovirus (SCRV) which infected Mandarin fish (Siniperca chuatsi). We showed that SHVV was able to replicate and proliferate very well in SSN-1 cells which were original from striped snakehead fish (Channa striatus). Furthermore, Mandarin fish was susceptible to SHVV by bath exposure as well as by intraperitoneal injection. The infected fish showed typical clinical signs of rhabdovirus infection, including hemorrhage and edema. Histopathological analysis revealed that extensive inflammation and necrosis were observed in the spleen, kidney, liver, heart and brain of the moribund Mandarin fish. The present results will shed new lights on the epidemic of vesiculoviruses among fish.
Little is known about viruses associated with Antarctic animals, though they are likely widespread. We recovered a novel polyomavirus from Adeeacute;lie penguin (Pygoscelis adeliae) faecal matter sampled in a sub-colony at Cape Royds, Ross Island, Antarctica. The 4988 nt Adeeacute;lie penguin polyomavirus (AdPyV) has a typical polyomavirus genome organisation with three open reading frames (ORFs) that encode capsid proteins on the one strand and two non-structural protein coding ORFs on the complementary strand. The genome of AdPyV shares ~60% pairwise identity with all avipolyomaviruses. Maximum likelihood phylogenetic analysis of the large T-antigen (T-Ag) amino acid sequences shows that the T-Ag of AdPyV clusters with those of avipolyomaviruses sharing between 48-52% identities. Only three viruses associated with Adeeacute;lie penguins have been identified at a genomic level, Avian influenza virus subtype H11N2 from the Antarctic Peninsula and, respectively, Pygoscelis adeliae papillomavirus and AdPyV from capes Crozier and Royds on Ross Island.
There is no large-scale therapy available against Human Respiratory Syncytial Virus (hRSV), a major pathogen responsible for acute respiratory diseases. Macaques represent an interesting animal model to evaluate potential treatments because of their genetic, anatomical and immunological proximity with human. However, the parameters that influence hRSV growth and control in this model are still poorly understood. We have documented in the following the influence of age as well as repeated infections on the virological, clinical and immunological parameters of this animal model. Following intranasal inoculation, hRSV replicated in the upper respiratory tract for less than 15 days with no clinical signs regardless of age. Interestingly, we observed the induction of a local immune response at the nasal mucosa as assessed by expression profiles of inflammatory and interferon-stimulated genes. Animals also developed specific antibodies, and were immune to re-infection. Thus, we showed that even in infant macaques, intranasal hRSV infection induced both local and systemic immune responses to efficiently control the virus.
Infection with hepatitis C virus (HCV) is characterized by systemic oxidative stress that is caused by either viral Core or chronic inflammation. It is well recognized that reactive oxygen species (ROS) like H2O2 can induce apoptotic cell death and can therefore function as anti-tumorigenic species. However, the detailed mechanisms by which ROS induces apoptotic cell death and HCV copes with the oxidative condition are largely unknown. In the present study, we found that H2O2 induced apoptotic cell death in the p53-positive human hepatocytes but not in the p53-negative ones. For this effect, H2O2 up-regulated levels of p14, increased ubiquitin-dependent degradation of mouse double minute 2 (MDM2), and reduced the interaction between MDM2 and p53 to prevent p53 degradation, resulting in accumulation of p53 and subsequent activation of the p53-dependent apoptotic pathways. Interestingly, HCV Core repressed p14 expression via promoter hypermethylation to abolish the potential of H2O2 to activate the p14-MDM2-p53 pathway. As a consequence, the HCV Core-expressing cells could overcome the p53-mediated apoptosis provoked by H2O2. Taken together, HCV Core can contribute to hepatocellular carcinoma formation by removing deleterious roles of ROS inducing cell death.
During the last decades, metagenomic studies expanded the numbers of newly described, often unclassified, viruses within the Circoviridae family. Using broad-spectrum circo-/cyclovirus PCRs, we characterized a novel circo-like virus in Aedes vexans mosquitoes from Germany whose main putative open reading frames (ORFs) shared very low amino acid identity with those of previously characterized circo-/cycloviruses. Phylogenetic and genetic distance analysis revealed that this new virus species defines, with previously described mosquito- and bat feces-derived circo-like viruses, a different genus, tentatively called "krikovirus", within Circoviridae. We further demonstrated that viruses of the putative krikovirus genus all share a genomic organization which is unique among Circoviridae. Further investigations are needed to determine the host range, tissue tropism and transmission route(s). This report increases the current knowledge of the genetic diversity and evolution of the members of the Circoviridae family.
Analysing the evolution of FIV on the intra-host level is important, in order to address whether the diversity and composition of viral quasispecies affects disease progression.
We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at the rate of 1.16 x 10-3 substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3-V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3-V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation.
Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximal evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared to HIV might explain why many naturally infected cats do not progress to AIDS rapidly.
Adeno-associated virus (AAV) type 5 represents the genetically most distant AAV serotype and the only one isolated directly from human tissue. Seroepidemiological evidence suggests HSV as helper virus for human AAV5 infections underlining the in vivo relevance of the AAV-herpesvirus relationship. In this study we analyzed for the first time herpes simplex virus (HSV) helper functions for productive AAV5 replication and compared these to AAV2. Using a combination of HSV strains and plasmids for individual genes, the previously defined HSV helper functions for AAV2 replication were shown to induce AAV5 gene expression, DNA replication and production of infectious progeny. The helper functions comprise the replication genes for ICP8 (UL29), helicase-primase (UL5/8/52), and DNA polymerase (UL30/42). HSV immediate-early genes for ICP0 and ICP4 further enhanced AAV5 replication, mainly by induction of rep gene expression. In the presence of HSV helper functions AAV5 Rep colocalized with ICP8 in nuclear replication compartments and HSV alkaline exonuclease (UL12) enhanced AAV5 replication, similar to AAV2. UL12 in combination with ICP8 was shown to induce DNA strand exchange on partially double-stranded templates to resolve and repair concatemeric HSV replication intermediates. Similarly, concatemeric AAV replication intermediates appeared to be processed to yield AAV unit-length molecules, ready for AAV packaging. Taken together, our findings show that productive AAV5 replication is promoted by the same combination of HSV helper functions as AAV2.
Various herpesviruses have been discovered in marine mammals, associated with a wide spectrum of disease. In the present study we describe the detection and phylogenetic analysis of a novel gammaherpesvirus, tentatively called phocine herpesvirus 7 (PhHV-7), that was detected in samples collected during an outbreak of ulcerative gingivitis and glossitis from juvenile harbor seals (Phoca vitulina) at the Seal Rehabilitation and Research Centre, the Netherlands. The presence of this novel gammaherpesvirus was confirmed by viral metagenomics, while no other viruses except four novel anelloviruses were detected. However, PhHV-7 DNA was also detected in harbor and grey seals (Halichoerus grypus) without gingivitis or glossitis. Genetic analysis of the partial polymerase gene of PhHV-7 detected in both species revealed limited sequence variation. Additional studies are needed to elucidate whether the discovered viruses played a role in the observed disease.
A granulovirus (GV) producing occlusion bodies (OBs) with an unusual appearance was isolated from Adoxophyes spp. larvae in a field. Ultrastructural observations revealed that its OBs were significantly larger and cuboidal in shape, rather than the standard ovo-cylindrical shape typical of GVs. N-terminal amino acid sequence analysis of the OB matrix protein from this virus suggested that it was a variant of Adoxophyes orana granulovirus (AdorGV). Bioassays of this GV (termed AdorGV-M) and an English isolate of AdorGV (termed AdorGV-E) indicated that the two isolates were equally pathogenic against larvae of A. honmai. However, AdorGV-M retained more infectivity towards larvae after irradiation with UV light than AdorGV-E. Sequencing and analysis of the AdorGV-M genome revealed little sequence divergence between this isolate and AdorGV-E. Comparison of selected genes among the two AdorGVs isolates and other Japanese AdorGV isolates revealed differences that may account for the unusual OB morphology of the AdorGV-M.
African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens responsible for protection in the pig. Available data from vaccination/challenge experiments in pigs indicate ASF protective immunity is hemadsorption inhibition (HAI) serotype-specific. A better understanding of ASFV HAI serologic groups and their diversity in nature, as well as improved methods to serotype ASFV isolates, is needed. Here, we demonstrate that the genetic locus encoding ASFV CD2v and C-type lectin proteins mediates HAI serologic specificity and that CD2v/C-type lectin genotyping provides a simple method to group ASF viruses by serotype, thus facilitating study of ASFV strain diversity in nature and providing information necessary for eventual vaccine design, development and efficacious use.
Hepatitis B virus X protein (HBx) is involved in the development of hepatocellular carcinoma (HCC). The HBx sequence is a preferential site of integration into the human genome, leading to the formation of C-terminal truncated HBx proteins (Ct-HBx). We previously reported that Ct-HBx proteins were able to potentiate cell transformation in vitro. Our present goal was to compare the ability of Ct-HBx and full-length HBx (FL-HBx) proteins to develop or enhance HCC in transgenic mice. In the absence of treatment, neither Ct-HBx nor FL-HBx transgenic mice developed HCC. In young mice treated with diethylnitrosamine (DEN), at 8 months of age, a significantly higher incidence and number of liver lesions were observed in Ct-HBx mice than in FL-HBx and control mice. The earlier development of tumours in Ct-HBx transgenic mice was associated with increased liver inflammation. At 10 months, macroscopic and microscopic analyses showed that statistically, FL-HBx mice developed more liver lesions with a larger surface area compared to control mice. Following the DEN-induced initiation of HCC, Ct-HBx and FL-HBx transgenic mice displayed an increase in the inflammatory response, evidenced by IL-6, TNF-aalpha; and IL-1bbeta; expression and the activation of STAT3 ERK and JNK. This was associated with an increase in cell apoptosis following DEN treatment. In conclusion, in DEN-treated transgenic mice, the expression of Ct-HBx protein causes a more rapid onset of HCC than that of FL-HBx protein. HBV genome integration leading to the expression of a truncated form of HBx protein may therefore facilitate HCC development in chronically infected patients.
The HMPV fusion protein (F) is the most immunodominant protein, yet subunit vaccines containing only this protein do not confer complete protection. The HMPV matrix protein (M) induces the maturation of antigen presenting cells in vitro. The inclusion of the M protein into an F protein subunit vaccine might therefore provide an adjuvant effect. We administered the F protein twice intramuscularly, adjuvanted with alum, the M protein or both, to BALB/c mice at 3-week intervals. Three weeks after the boost, mice were infected with HMPV and monitored for 14 days. At day 5 post challenge, pulmonary viral titers, histopathology and cytokine levels were analyzed. Mice immunized with F+alum and F+M+alum generated significantly more neutralizing antibodies than mice immunized with F only (titers of 47pplusmn;7 (pllt;0.01) and 147pplusmn;13 (pllt;0.001) vs 17pplusmn;2). Unlike F only (1.6pplusmn;0.5x103 TCID50/g lung), pulmonary viral titers in mice immunized with F+M and F+M+alum were undetectable. Mice immunized with F+M presented the most important reduction in pulmonary inflammation and the lowest Th2/Th1 cytokine ratio. In conclusion, addition of the HMPV M protein to an F protein-based vaccine modulates both humoral and cellular immune responses to subsequent infection, thereby increasing the protection conferred by the vaccine.
The segment-specific non-coding regions (NCRs) of influenza A virus RNA genome play important roles in controlling viral RNA transcription, replication and genome packaging. In this report, we present, for the first time to our knowledge, a full view of the segment-specific (NCRs) of all influenza A viruses by bioinformatics analysis. Our systematic functional analysis reveal that the eight segment-specific NCRs could differently regulate viral RNA synthesis and protein expression at both transcription and translation levels. Interestingly, a highly conserved suboptimal nucleotide at -3 position of Kozak sequence, that could down-regulate protein expression at translation level, is only present in the segment-specific NCR of PB1. By reverse genetics, we demonstrate that recombinant viruses with an optimized Kozak sequence at -3 position in PB1 results in a significant multiple-cycle replication reduction that is independent of PB1-F2 expression. Our detailed dynamic analysis of the virus infection reveals that, the mutant virus displays slightly altered dynamics from the wild type virus on both viral RNA synthesis and protein production. Furthermore, we demonstrate that the level of PB1 expression is involved in regulating type I interferon production. Together, these data reveal a novel strategy exploited by influenza A virus to fine-tune virus replication dynamics and host anti-viral response through regulating PB1 protein expression.
Bats are important reservoirs of many viruses with zoonotic potential worldwide, including Europe. Among bat viruses, members of the Picornaviridae family remain a neglected group. We performed viral metagenomic analyses on Miniopterus schreibersii bat fecal samples, collected in Hungary in 2013. In the present study we report the first molecular data and genomic characterization of a novel picornavirus from the bat species M. schreibersii in Europe. Based on phylogenetic analyses, the novel bat picornaviruses unambiguously belong to the Mischivirus genus and were highly divergent from other bat-derived picornaviruses of the Sapelovirus genus. Although the Hungarian viruses were most closely related to Mischivirus A, they formed a separate monophyletic branch within the genus.
Yellow fever virus (YFV)-17D is an empirically developed, highly effective live-attenuated vaccine that has been administered to human beings for almost a century. YFV-17D has stood as a paradigm for a successful viral vaccine, and has been exploited as a potential virus vector for the development of recombinant vaccines against other diseases. In this study, a DNA-launched YFV-17D construct (pBeloBAC-FLYF) was explored as a new modality to the standard vaccine to combine the commendable features of both DNA vaccine and live attenuated viral vaccine. The DNA-launched YFV-17D construct was characterized extensively both in cell culture and in mice. High titers of YFV-17D were generated upon transfection of the DNA into cells, whereas a mutant with deletion in the capsid coding region (pBeloBAC-YF/C) was restricted to a single round of infection, with no release of progeny virus. Homologous prime-boost immunization of mice with both pBeloBAC-FLYF and pBeloBAC-YF/C elicited specific dose-dependent cellular immune response against YFV-17D. Vaccination of A129 mice with pBeloBAC-FLYF resulted in induction YFV-specific neutralizing antibodies in all vaccinated subjects. These promising results underlined the potential of the DNA-launched YFV both as an alternative to standard YFV-17D vaccination but also as a vaccine platform for the development of DNA-based recombinant YFV vaccines.
Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cellular DNA deaminases that restrict a broad spectrum of lentiviruses. This process is counteracted by the viral infectivity factor (Vif) of lentiviruses, which binds APOBEC3s and promotes their degradation. Core binding factor subunit bbeta; (CBF-bbeta;) is an essential co-factor for the function of human immunodeficiency virus type 1 Vif to degrade human APOBEC3s. However, the requirement for CBF-bbeta; in Vif-mediated degradation of other mammalian APOBEC3 proteins is less clear. Here we determine the sequence of feline CBFB and perform phylogenetic analyses. These analyses reveal that mammalian CBFB is under purifying selection. Moreover, we demonstrate that CBF-bbeta; is dispensable for feline immunodeficiency virus Vif-mediated degradation of APOBEC3s of its host. These findings suggest that primate lentiviruses have adapted to use CBF-bbeta;, an evolutionary stable protein, to counteract APOBEC3 proteins of their hosts after diverging from other lentiviruses.
Many insect-transmissible pathogens are transmitted by specific insect species and not by others, even if the insect species are closely related. The molecular mechanisms underlying such strict pathogen-insect specificity are poorly understood. Rice dwarf virus (RDV), a plant reovirus, is mainly transmitted by the leafhopper species Nephotettix cincticeps, but is ineffectively transmitted by the leafhopper Recilia dorsalis. Here, we demonstrated that virus-containing tubules composed of viral nonstructural protein Pns10 of RDV, associated with the intestinal microvilli of N. cincticeps, but not with those of R. dorsalis. Furthermore, Pns10 of RDV specifically interacted with cytoplasmic actin, the main component of microvilli of N. cincticeps, but not with that of R. dorsalis, suggesting that the interaction of Pns10 with insect cytoplasmic actin was consistent with the transmissibility of RDV by leafhoppers. All these results suggested that the interaction of Pns10 of RDV with insect cytoplasmic actin may determine pathogen-vector specificity.
We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here we show that the same mutations improve the growth kinetics of a virus containing the A/VietNam/1203/2004 (H5N1) HA and NA. Total protein yield and neuraminidase activity were increased when a chimeric NA was included. These increases indicate that the synergistic effect is due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.
During productive infection with Epstein-Barr virus (EBV), a dramatic suppression of cellular protein expression is caused by the viral alkaline exonuclease BGLF5. Among the proteins downregulated by BGLF5 are multiple immune components. Here, we show that shutoff reduces expression of the innate EBV-sensing Toll-like receptor-2 and the lipid antigen-presenting CD1d molecule, thereby identifying these proteins as novel targets of BGLF5.
To silence BGLF5 expression in B cells undergoing productive EBV infection, we employed an shRNA approach. Viral replication still occurred in these cells, albeit with reduced late gene expression. Surface levels of a group of proteins, including immunologically relevant molecules such as CD1d and HLA class I and class II, were only partly rescued by depletion of BGLF5, suggesting that additional viral gene products interfere with their expression. Our combined approach thus provides a means to unmask novel EBV (innate) immune evasion strategies that may operate in productively infected B cells.
Human noroviruses are one of the major causes of acute gastroenteritis worldwide. Due to the lack of an efficient human norovirus cell culture system coupled with an animal model, human norovirus research mainly relies on human volunteer studies and surrogate models. Current models either utilize human norovirus infected animals including the gnotobiotic pig or calf and the chimpanzee models, or employ other members of the Caliciviridae family including cell culture propagable surrogate caliciviruses such as the feline calicivirus, murine norovirus and most recently the Tulane virus. One of the major features of human noroviruses is their extreme biological diversity, including genetic, antigenic and histo-blood group antigen binding diversity, and possible differences of virulence and environmental stability. This extreme biological diversity and its effect on intervention/prevention strategies cannot be modeled by uniform groups of surrogates much less by single isolates. Tulane virus, the prototype recovirus strain, was discovered in 2008. Since then, several other novel recoviruses have been described and cell culture adapted. Recent studies indicate that the epidemiology, the biological features and diversity of recoviruses and the course of infection and clinical disease in recovirus infected macaques more closely reflect those properties of human noroviruses than any of the current surrogates. This review aims to summarize what is currently known about recoviruses, highlight their biological similarities to human noroviruses and discuss applications of the model in addressing questions relevant for human norovirus research.
Marek's disease virus (MDV) is an important oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts. The Meq-clustered miRNAs encoded by MDV have been suggested to play potentially critical roles in the induction of lymphomas. Using the technique of bacterial artificial chromosome mutagenesis, we have presently constructed a series of specific miRNA-deleted mutants and demonstrate that these miRNAs are not essential for replication of MDV and have no effects on the early cytolytic or latent phases of the developing disease. However, compared to the parental GX0101, mortality of birds infected with the mutants GXmiR-M2, GXmiR-M3, GXmiR-M5, GXmiR-M9 and GXmiR-M12 was reduced from 100% to 18%, 30%, 48%, 24% and 14%, coupled with the gross tumour incidence reduction from 28% to 8%, 4%, 12%, 8% and 0%, respectively. Our data confirms that except for mdv1-miR-M4, the other Meq-clustered miRNAs also play critical roles in MDV oncogenesis. Further work will be needed to elucidate the miRNA-mediated regulatory mechanisms that trigger the development of MD lymphomas.
Cassava brown streak disease (CBSD) has emerged as the most important viral disease of cassava (Manihot esculenta) in Africa and is a major threat to food security. CBSD is caused by two distinct species of ipomoviruses, Cassava brown streak virus and Ugandan cassava brown streak virus, belonging to the family Potyviridae. Previously CBSD was reported only from the coastal lowlands of East Africa, but recently it has begun to spread as an epidemic throughout the Great Lakes region of East Africa and Central Africa. This new spread represents a major threat to the cassava-growing regions of West Africa. CBSD resistant cassava cultivars are being developed through breeding and transgenic RNAi-derived field resistance to CBSD has also been demonstrated. This review aims to provide a summary of the most important studies on aetiology, epidemiology and control of CBSD and highlight key research areas that need prioritization.
CRISPR/Cas9 system is a highly efficient and powerful tool for RNA-guided editing of cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here we reported on CRISPR/Cas9-mediated editing of EBV genome in human cells. Two guide RNAs were used to direct a targeted deletion of 558 bp in the promoter region of BART transcript which encodes viral microRNAs. Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and guide RNAs. No off-target cleavage was found by deep sequencing. The loss of BART microRNA expression and activity was verified, supporting that the BART promoter is the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses can be recovered and introduced into other cells at low multiplicity of infection. Recombinant viruses with an edited genome can be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provides not only the first genetic evidence that the BART promoter drives the expression of BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.
As a member of the newly established Betaflexiviridae family, Grapevine rupestris stem pitting-associated virus (GRSPaV) has a RNA genome containing five open reading frames (ORFs). ORF1 encodes a putative replicase polyprotein typical of the Alphavirus superfamily of positive-strand, ssRNA viruses. Several viruses of this superfamily have been demonstrated to replicate in structures designated as viral replication complexes associated with intracellular membranes. However, structure and cellular localization of the replicase complex have not been studied for members of Betaflexiviridae, a family of mostly woody plant viruses. As a first step toward the elucidation of the replication complex of GRSPaV, we investigated the subcellular localization of full-length and truncated versions of its replicase polyprotein via fluorescent tagging, followed by fluorescence microscopy. We found that the replicase polyprotein formed distinctive punctate bodies in both Nicotiana benthamiana leaf cells and tobacco protoplasts. We further mapped a region of 76 amino acid in the methyl-transferase domain responsible for the formation of these punctate structures. These punctate structures are distributed in close proximity to the endoplasmic reticulum network. Membrane flotation and biochemical analyses demonstrate that the N-terminal region responsible for punctate structure formation associated with cellular membrane likely through an amphipathic a helix serving as an in-plane anchor. The identity of this membrane is yet to be determined. This is, to our knowledge, the first report on the localization and membrane association of the replicase proteins of a member of the family Betaflexiviridae.
Interferon-induced restriction factors can significantly affect the replicative capacity of retroviruses in mammals. Tripartite motif protein 5, isoform aalpha; (TRIM5aalpha;) is a restriction factor that acts at early stages of the virus life cycle by intercepting and destabilizing incoming retroviral cores. Sensitivity to TRIM5aalpha; maps to the N-terminal domain of the retroviral capsid (CA) proteins. In several New World and Old World monkey species, independent events of retrotransposon-mediated insertion of the cyclophilin A (CypA) coding sequence in the trim5 gene have given rise to TRIMCyp (also called TRIM5-CypA), a hybrid protein that is active against some lentiviruses in a species-specific fashion. In particular, TRIMCyp from the owl monkey (omkTRIMCyp) very efficiently inhibits HIV-1. Previously, we showed that disrupting the integrity of microtubules (MTs) and of cytoplasmic dynein complexes partially rescued replication of retroviruses, including HIV-1, from restriction mediated by TRIM5aalpha;. Here we show that efficient restriction of HIV-1 by omkTRIMCyp is similarly dependent on the MT network and on dynein complexes, but in a context-dependent fashion. When omkTRIMCyp was expressed in human HeLa cells, restriction was partially counteracted by pharmacological agents targeting MTs or by siRNA-mediated inhibition of dynein. The same drugs (nocodazole and paclitaxel) also rescued HIV-1 from restriction in cat CRFK cells, although to a lesser extent. Strikingly, neither nocodazole, paclitaxel nor depletion of the dynein heavy chain (DHC) had a significant effect on the restriction of HIV-1 in an owl monkey cell line. These results suggest the existence of cell-specific functional interactions between MTs/dynein and TRIMCyp.
The division of viruses into orders, families, genera and species provides a classification framework that seeks to organise and make sense of the diversity of viruses infecting animals, plants and bacteria. Classifications are based on similarities in genome structure and organisation, the presence of homologous genes and sequence motifs, and, at lower levels such as species, host range, nucleotide and antigenic relatedness and epidemiology. Classification below the level of family must also be consistent with phylogeny and virus evolutionary histories. Recently developed methods such as PASC, DEMaRC and NVR offer alternative strategies for genus and species assignments that are based purely on degrees of divergence between genome sequences. They offer the possibility of automating classification of the vast number of novel virus sequences being generated by next generation metagenomic sequencing. However, distance-based methods struggle to deal with the complex evolutionary history of virus genomes which are shuffled by recombination and re-assortment and where taxonomic lineages evolve at different rates. In biological terms, classifications based on sequences distances alone are also arbitrary whereas the current system of virus taxonomy is of utility precisely because it is primarily based upon phenotypic characteristics. However, a separate system is clearly needed by which virus variants that lack biological information might be incorporated in to the ICTV classification even if based solely on sequence relationships to existing taxa. For these, simplified taxonomic proposals and naming conventions represent a practical way to expand the existing virus classification and catalogue our rapidly increasing knowledge of virus diversity.
The human genome is comprised of 8% endogenous retroviruses (ERVs), the majority of which are defective due to deleterious mutations. Nonetheless, transcripts of ERVs are found in most tissues and these transcripts could either be reverse transcribed to generate single-stranded DNA or expressed to generate proteins. Thus, the expression of ERVs could produce nucleic acids or proteins with viral signatures, much like the pathogen-associated molecular patterns (PAMPs) of exogenous viruses, which would enable them to be detected by the innate immune system. The activation of some pattern recognition receptors (PRRs) in response to ERVs has been described in mice and in the context of human autoimmune diseases. Here, we review the evidence for detection of ERVs by PRRs and the resultant activation of innate immune signalling. This is an emerging area of research within the field of innate antiviral immunity, showing how ERVs could possibly initiate immune signalling pathways and might have implications for numerous inflammatory diseases.
Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally transmitted by mosquitoes. During the infection, JEV generally enters host cells via receptor-mediated clathrin-dependent endocytosis that requires the involvement of the 70 kDa heat shock protein (Hsp70). Heat shock cognate protein 70 (Hsc70) is one member of the Hsp70 family and is mainly constitutive; thus, it may be expressed at physiological condition. In C6/36 cells, Hsc70 is up-regulated in response to JEV infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface, it probably does not serve as the receptor according to our results in the present study. In contrast, Hsc70 is evidently associated with virus penetration into the cell and resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly involved in clathrin-mediated endocytosis, particularly at a late stage of viral entry into host cells. Furthermore, we found that Hsc70 is composed of at least three isoforms, including B, C, and D. Of them, the isoform D is the one that helps JEV to penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant evidence that sheds light on the regulatory mechanisms of JEV infection in host cells, especially on the process of clathrin-mediated endocytosis.
Chronic hepatitis B (CHB) is treated with nucleos(t)ide analogs (NA). The reverse transcriptase (RT) region in the Hepatitis B virus (HBV) genome mutates to resist NA treatment, yet the RT mutations have not been well characterized. Further, the HBV genotype might influence RT sequence evolution, NA resistance (NAr) mutation patterns, and drug resistance development. We examined 42 NAr mutation sites in 169 untreated and 131 NA-treated CHB patient samples. Patients were identified with genotype B (HBV-B) and HBV-C infections, with a higher prevalence and mutation frequency of HBV-C than HBV-B. Seventeen reported NAr mutation sites and 13 novel mutations were detected. NAr-related mutation prevalence was significantly higher in NA-treated versus untreated patients. Primary antiviral-resistant mutants only existed in NA-treated patients. Sequencing data revealed 7 HBV-C-specific mutations and 3 HBV-B-specific mutations. In conclusion, NA treatment and HBV genotype might constitute the selection basis and promote NA-resistant HBV strain evolution under antiviral therapy.
Pathogenesis of viral hemorrhagic fevers (VHF) is associated with alteration of vascular barrier function and hemorrhage. To date, the specific mechanism behind this is unknown. Programmed cell death and regulation of apoptosis in response to viral infection is an important factor for host or virus survival but this has not been well-studied in the case of Crimean-Congo hemorrhagic fever virus (CCHFV).
In this study, we demonstrated that CCHFV infection suppresses cleavage of poly (ADP-ribose) polymerase (PARP), triggered by staurosporine at early post infection. We also demonstrated that CCHFV infection suppresses activation of caspase-3 and caspase-9. Most interestingly, we found that CCHFV N can suppress induction of apoptosis by Bax and inhibit the release of cytochrome-c from the inner membrane of mitochondria to cytosol. However, CCHFV infection induces activation of Bid at late post infection, suggesting the activation of extrinsic apoptotic signaling. Consistently, supernatant from late post-infected cells stimulated was found to induce PARP cleavage, most probably through the TNF-aalpha; dead receptor pathway. In summary, we found that CCHFV has strategies to interplay with apoptosis pathways and thereby regulate caspase cascade. We suggest that CCHFV suppresses caspase activation at early stages of the CCHFV replication cycle, which perhaps benefits the establishment of infection. Furthermore, we suggest that the host cellular response at late post infection induces host cellular pro-apoptotic molecules through the death receptor pathway. External host-derived stimuli most probably initiate the apoptotic process, and the route continues either by crosstalk between the death receptor and mitochondria routes, or separately.
Baculoviruses are arthropod-specific pathogens and find extensive applications in pest control strategies and recombinant protein expression. Spodoptera litura nucleopolyhedrovirus infects the Tropical armyworm, Spodoptera litura which is an important polyphagous crop pest widely distributed in regions of Asia and Oceania. Using next generation sequencing, we report stage specific profiling of SpltNPV encoded miRNAs at different time intervals post infection of Sf21 cell lines. Sequence length distribution analysis of the small RNA libraries revealed a significant increase in 20nt reads and reduction of other size fractions during late phases of infection. In-silico miRNA prediction tools identified 48 novel SpltNPV encoded miRNAs, out of which ten were experimentally validated in Sf21 cell lines using northern blot analysis and Taqman qPCR. The viral miRNAs were found to express in fat body and mid gut tissues of infected fifth instar Spodoptera litura larva as well. Real time PCR analysis confirmed that expression of most viral miRNAs was triggered post 12 hours of infection and continued thereafter. Gene Ontology and KEGG pathway annotation of computationally predicted targets of the reported miRNAs suggests a major impact of these miRNAs on cell signalling, protein translation and metabolic processes.
Porcine reproductive and respiratory syndrome virus (PRRSV) is now prevalent throughout the world and has caused major economic losses to the pig industry. Arterivirus nonstructural protein 10 (nsp10) is a superfamily 1 helicase participating in multiple processes of virus replication. Nsp10 of PRRSV, however, has not yet been well characterized. In this study, a series of nsp10 mutants were constructed and analyzed for functional sites of different enzymatic activities. We found that nsp10 could bind both ssDNA and dsDNA, and this binding activity could be inactivated by mutations at Cys25 and His32. These two mutations also abolished unwinding activity without affecting ATPase activity. In addition, substitution of Ala227 by serine eliminated helicase activity, while substitution by valine enhanced unwinding activity. Taken together, our results showed that Cys25 and His32 in PRRSV nsp10 were critical for nucleic acid binding and unwinding, and that Ala227 played an important role in helicase activity.
Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for epidemics of debilitating arthritic disease. The recent outbreak (2004-2014) resulted in an estimated 1.4-6.5 million cases, with imported cases reported in nearly 40 countries. The development of CHIKV-specific diagnostics and research tools is thus highly desirable. Herein we describe the generation and characterization of the first monoclonal antibodies (mAbs) specific for the capsid protein (CP) of CHIKV. The antibodies recognized isolates representing the major genotypes of CHIKV, as well as several other alphaviruses, and were reactive in a range of assays including ELISA, Western blot, immunofluorescence (IFA) and immunohistochemistry (IHC). We have also used the anti-CP mAb, 5.5G9, in IHC studies to show that capsid antigen is persistently expressed 30 days post-infection in cells with macrophage morphology in a mouse model of chronic CHIKV disease. These antibodies may thus represent useful tools for further research including investigations on the structure and function of CHIKV CP, and as valuable reagents for CHIKV detection in a range of settings.
Since one of us co-authored a review on NS5A a decade ago the hepatitis C virus (HCV) field has changed dramatically, primarily due to the advent of the JFH-1 cell culture infectious clone which allowed the study of all aspects of the virus lifecycle from entry to exit. This review will describe advances in our understanding of NS5A biology over the past decade, highlighting how the JFH-1 system has allowed us to determine that NS5A is essential not only in genome replication but also in the assembly of infectious virions. We will review the recent structural insights - NS5A is predicted to comprise 3 domains, X-ray crystallography has revealed the structure of domain I but there is a lack of detailed structural information about the other two domains which are predicted to be largely unstructured. Recent insights into the phosphorylation of NS5A will be discussed, and we will highlight a few pertinent examples from the ever expanding list of NS5A binding partners identified over the past decade. Lastly, we will review the literature showing that NS5A is a potential target for a new class of highly potent small molecules that function to inhibit virus replication. These direct acting antivirals (DAAs) are now either licenced, or in the late stages of approval, for clinical use both in the USA and UK/Europe. In combination with other DAAs targeting the viral protease (NS3) and polymerase (NS5B), they are revolutionising treatment for HCV infection.
Turnip mosaic virus (TuMV) is a potyvirus that is transmitted by aphids and infects a wide range of plant species. We investigated the evolution of this pathogen by collecting 32 isolates of TuMV, mostly from Brassicaceae plants, in Australia and in New Zealand. We performed a variety of sequence-based phylogenetic and population genetic analyses of the complete genomic sequences and of three non-recombinogenic regions of those sequences. The substitution rates, divergence times, and phylogeographic patterns of the virus populations were estimated. Six inter- and five intralineage recombination type patterns were found in the genomes of the Australian and New Zealand isolates, and all were novel. Only one recombination type pattern has been found in both countries. Australian and New Zealand populations were genetically different, and were different from the European and Asian populations. Our Bayesian coalescent analyses, based on a combination of novel and published sequence data from three non-recombinogenic protein-encoding regions, showed that TuMV probably started to migrate from Europe to Australia and New Zealand more than 80 years ago, and that distinct populations arose as a result of evolutionary drivers such as recombination. The basal-B2 subpopulation in Australia and New Zealand seems to be older than those of the world-B2 and B3 populations. To our knowledge, our study presents the first population genetic analysis of TuMV in Australia and New Zealand. We have shown that the time of migration of TuMV correlates well with the establishment of agriculture and migration of Europeans to these countries.
Mouse mammary tumour virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse tranferrin receptor 1 for cell entry. Several MMTV strains have been shown to productively infect, in addition to murine cells, various heterologous cell lines including those of human origin, albeit less efficiently than murine cells. Here, we analysed whether MMTV(C3H), previously reported to be incapable of infecting human cells, can productively infect human cells. Using a recently described high titer MMTV-based vector carrying the MMTV(C3H)Env we successfully transduced cells of human origin. Furthermore, wild-type MMTV(C3H) was able to infect human cells, albeit less efficiently than mouse cells. The established infection was, however, sufficient to enable virus spread to every cell in culture. Infectivity of wild-type MMTV(C3H) and MMTV-based vectors carrying MMTV(C3H)Env was blocked by heat-inactivation, an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine and pre-incubation with neutralizing anti-MMTV antibodies that did not neutralize vectors pseudotyped with amphotropic MLV Env, providing evidence for an authentic, receptor-mediated and reverse transcriptase-dependent infection process. Persistently infected human Hs578T cells produced infectious virions capable of infecting naive human breast cells in culture, the infectivity of which could also be blocked by neutralizing anti-MMTV antibodies, demonstrating that virus particles released by the persistently infected Hs578T cells are antigenically related to the virus produced from murine cells. Taken together, our results show that MMTV(C3H), like MMTV(GR) and MMTV(RIII), is able to not only infect, but also to replicate in cultured human breast cells.
Oropouche virus (OROV) is a medically important orthobunyavirus, which causes frequent outbreaks of a febrile illness in the Northern parts of Brazil. However, despite being the cause for an estimated half a million human infections since its first isolation in Trinidad, 1955, details of the molecular biology of this tripartite, negative-sense RNA virus remain limited. We have determined the complete nucleotide sequence of the Brazilian prototype strain of OROV, BeAn 19991, and found a number of differences compared to sequences in the database. Most notable were that the S segment contains an additional 204 nucleotides at the 3' end and that there is a critical nucleotide mismatch at position 9 within the base-paired terminal panhandle structure of each genome segment. In addition, we obtained the complete sequence of the Trinidadian prototype strain TRVL 9760 that showed similar characteristics to the BeAn 19991 strain. By using a T7 RNA polymerase-driven minigenome system, we demonstrated that cDNA clones of the BeAn 19991 L and S segments expressed functional proteins and also that the newly determined terminal untranslated sequences acted as functional promoters in the minigenome assay. By co-transfecting a cDNA to the viral glycoproteins, virus-like particles (VLP) were generated that packaged a minigenome and were capable of infecting naive cells.
Analysis of virus-derived small RNAs with high-throughput sequencing has been successful for detecting novel viruses in plants and invertebrates. However, the applicability of this method has not been demonstrated in fungi, although fungi were among the first organisms reported to utilize RNA silencing. Here, we used virus-infected isolates of the fungal species complex Heterobasidion annosum sensu lato as a model system to test whether mycovirus genome segments can be detected with small RNA deep sequencing. Species of Heterobasidion are some of the most devastating forest pathogens in boreal forests. These fungi cause wood decay and are commonly infected with species of Partitiviridae and the yet unassigned virus species Heterobasidion RNA virus 6 (HetRV6). Small RNA deep sequencing allowed the simultaneous detection of all eight double-stranded RNA virus strains known to be present in the tested samples and one putative mitovirus species (family Narnaviridae) with a single-stranded RNA genome, designated here as Heterobasidion mitovirus 1. Prior to this study, no members of the family Narnaviridae had been described as infecting species of Heterobasidion. Quantification of viral double- and single-stranded RNA with quantitative PCR indicated that co-infecting viral species and viruses with segmented genomes can be detected with small RNA deep sequencing despite vast differences in the amount of RNA. This is the first study demonstrating the usefulness of this method for detecting fungal viruses. Moreover, the results suggest that viral genomes are processed into small RNAs by different species of Heterobasidion.
Rotavirus A is a very common cause of acute diarrhea in infants and young children worldwide. Most of human strains are classified into 2 major Wa-like and DS-1-like genotype constellations, while a minor third strain, AU-1, was described in 1989 among human rotavirus isolates from Japan. AU-1 demonstrates a high degree of homology to a feline rotavirus, FRV-1, which suggests interspecies transmission of feline rotavirus. However, there has been no subsequent report of RVs possessing AU-1 genotype throughout all the 11 genes. Between March 1997 and December 1999, 157 rotavirus positive stool samples were collected from Brazilian children, and 16 of 157 (10.2%) were P genotype. We analyzed 8 strains by nearly full genome sequencing. These 8 strains were divided into 2 groups, that is, 5 AU-1-like and 3 Wa-like strains. Four of 5 AU-1-like strains had AU-1-like genotype constellation throughout the 11 genes. The remaining AU-1-like strain was considered to be a reassortant strain comprised of 9, 2 and 1 genes from AU-1-like, Wa-like and G9 strains, respectively. Three Wa-like strains were considered to be reassortant comprised of 7 to 8 genes and 3 to 4 genes from Wa-like and non-Wa-like strains, respectively. This report of human G3P rotavirus strains possessing AU-1 genotype constellation throughout all genes demonstrates stability and infectivity of AU-1-like strain with its original genotype beyond space and time.
A fundamental aspect of herpesviruses is their ability to establish a life long latent infection within the host. Current research would suggest that small regulatory RNAs called microRNAs play a central role in all three aspects of virus latency and persistence. In this review we will discuss the role of miRNAs in virus persistence and latency, specifically focusing on herpesviruses. We will cover the current knowledge on the role of miRNAs in establishing and maintaining virus latency and promoting survival of infected cells through targeting of both viral and cellular transcripts, highlighting key publications in the field. We will also discuss potential areas of future research and how novel technologies may aid in determining how miRNAs shape virus latency in the context of herpesvirus infections.
The stability and conservation of the sequences of RNA viruses in the field and the high error rates measured in vitro are paradoxical. The field stability indicates that there are very strong selective constraints on sequence diversity. The nature of these constraints is discussed. Apart from constraints on variation in cis-acting RNA and the amino acid sequences of viral proteins, there are other ones relating to the presence of specific dinucleotides such CpG and UpA as well as the importance of RNA secondary structures and RNA degradation rates. Recent other constraints identified in other RNA viruses such as effects of secondary RNA structure on protein folding or modification of cellular tRNA complements are also discussed. Using the family Paramyxoviridae I show that the codon usage pattern (CUP) is (i) specific for each virus species and (ii) that it is markedly different from the host - it does not vary even in vaccine viruses that have been derived by passage in a number of inappropriate host cells. The CUP might thus be an additional constraint on variation and I propose the concept of codon constellation to indicate the informational content of the sequences of RNA molecules relating not only to stability and structure but also to the efficiency of translation of a viral mRNA resulting from the CUP and the numbers and position of rare codons.
Human Papillomaviruses (HPVs) have been detected in urban wastewaters, demonstrating that epitheliotropic viruses can find their way into sewage through the washing of skin and mucous membranes. Papillomavirus shedding through feces is still an unexplored issue. The objective of the present study was to investigate the presence of HPVs in stool samples. We analyzed 103 fecal specimens collected from hospitalized patients with diarrhea using validated primers able to detect aalpha;, bbeta; and HPVs. PCR products underwent sequencing analysis and sequences were aligned to reference genomes from the Papillomavirus Episteme database. A total of 15 sequences were characterized from the fecal samples. Thirteen samples (12.6%) were positive for 9 genotypes belonging to aalpha; and bbeta; genus: HPV32 (LR, aalpha;1), HPV39 (HR, aalpha;7), HPV44 (LR, aalpha;10), HPV8 (bbeta;1), HPV9, HPV23, HPV37, HPV38, and HPV120 (bbeta;2). Two putative novel genotypes of the bbeta; genus, species 1 and 2 were also detected. The tissue (s) of origin is unknown, since feces can collect HPVs originating from or passing through the entire digestive system. To our knowledge, this is the first investigation on the occurrence and diversity of HPVs in fecal samples. Results from this study demonstrate that HPVs can find their way into sewage as a result of shedding in the feces. This highlights the need of further studies aimed at understanding the prevalence of HPV in different water environments and the potential for waterborne transmission.
The unfolded protein response (UPR) is a cellular defense mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1aalpha; become activated. PERK phosphorylates eIF2aalpha; leading to a general inhibition of cellular translation, while the expression of transcription factor ATF4 is upregulated. Active IRE1aalpha; splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Herein we demonstrate that transient expression of chikungunya virus (CHIKV) (Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2aalpha; and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIK infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV infected cells. Individual expression of CHIKV non-structural proteins (nsP) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that render nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER, but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.
T-lymphotropic feline leukemia virus (FeLV-T), a highly pathogenic variant of FeLV, induces severe immunosuppression in cats. FeLV-T is fusion-defective because in its PHQ motif, a gammaretroviral consensus motif in the N-terminal of an envelope protein, histidine is replaced with aspartate. Infection by FeLV-T requires FeLIX, a truncated envelope protein encoded by an endogenous FeLV, for transactivation of infectivity and Pit1 for binding FeLIX. Although Pit1 is present in most tissues in cats, the expression of FeLIX is limited to certain cells in lymphoid organs. Therefore, the host cell range of FeLV-T was thought to be restricted to cells expressing FeLIX. However, because FeLIX is a soluble factor and expressed constitutively in lymphoid organs, we presumed it to be present in blood and evaluated its activities in sera of various mammalian species using a pseudotype assay. We demonstrated that cat serum has FeLIX activity at a functional level, suggesting that FeLIX is present in cats and FeLV-T may be able to infect cells expressing Pit1 regardless of the expression of FeLIX in vivo. In addition, FeLIX activities in sera were detected only in domestic cats but not in other feline species tested. To our knowledge, this is the first report to prove that a large amount of truncated envelope protein of endogenous retrovirus is circulating in the blood to facilitate the infection of a pathogenic exogenous retrovirus.
Neutralising antibodies (NAbs) are believed to comprise an essential component of the protective immune response induced by vaccines against FIV and HIV infections. However, relatively little is known about the role of NAbs in controlling FIV infection and subsequent disease progression. Here we present studies examining the neutralisation of HIV-luciferase pseudotypes bearing homologous and heterologous FIV Envs (n=278) by sequential plasma samples collected at 6 month intervals from naturally infected cats (n=38) over a period of 18 months. We evaluated the breadth of the NAb response against non-recombinant homologous and heterologous clade A and clade B viral variants as well as recombinants and assessed the results, testing for evidence of an association between the potency of the NAb response and the duration of infection, CD4 T lymphocyte numbers, health status and survival times of the infected cats. Neutralisation profiles varied significantly between FIV infected cats and strong autologous neutralisation, assessed using luciferase based in vitro assays, did not correlate with the clinical outcome. No association was observed between strong NAb responses and either improved health status or increased survival time of infected animals, implying that other protective mechanisms are likely to be involved. Similarly, no correlation was observed between the development of autologous NAbs and the duration of infection. Furthermore, cross-neutralising antibodies were evident in only a small proportion (13%) of cats.
Pleconaril is a capsid-inhibitor previously used to treat enterovirus infections. A pleconaril resistant Echovirus (E) 11 strain was identified before pleconaril treatment was given in an immune-compromised patient. The patient was also treated with immune-globulins (IVIG) for a long period, but remained unresponsive. The pleconaril resistant strains could not be neutralized in vitro confirming IVIG treatment failure. To identify the basis of pleconaril resistance genetic and structural analyses were conducted. Analysis of a modelled viral capsid indicated conformational changes in the hydrophobic pocket that could prevent pleconaril docking. Substitutions (V117I, V119M, and I188L) in the pleconaril resistant viruses were found in the pocket region of VP1. Modelling suggests V119M could confer resistance, most probably due to the protruding sulphate side chain of methionine. Although pleconaril resistance induced in vitro in a susceptible E11 clinical isolate was characterised by a different substitution (I183M), resistance is suggested to also results by a similar mechanism i.e. due to a protruding sulphate side chain of methionine. Our results show resistant strains that arise in vivo display different markers than those identified in vitro and suggest that multiple factors may play a role in pleconaril resistance in patient strains. Based on IVIG treatment failure, we predict one of these factors could be immune-related. Thus both IVIG and capsid-inhibitors target the viral capsid and can induce mutations that can be cross-reactive enabling escape from both IVIG and drug. This could limit treatment options and should be investigated further.
Salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared to terrestrial alphavirus replicons (Olsen et al., 2013a). In this study, we have compared several modifications to a SAV replicon construct and analyzed their influence on foreign gene expression. We found that with an insertion of a translational enhancer consisting of the N-terminal 102 nucleotides of the capsid gene together with a nucleotide sequence encoding the FMDV 2A peptide caused a significant increase of the EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 5rrsquo; end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at 3rrsquo; end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production which can be applied for immunization of salmonid fish in the future.
Crimean-Congo hemorrhagic fever virus (CCHFV) is a Nairovirus of the family Bunyaviridae, which can cause severe hemorrhagic fever in humans, with mortality rates above 30%. CCHFV is the most widespread of the tick-borne human viruses and it is endemic in areas of central Asia, the Middle East, Africa and southern Europe. Its viral genome consists of three negative sense RNA segments. The large segment (L) encodes a viral RNA-dependent RNA polymerase (L protein), the small segment (S) encodes the nucleocapsid protein (N) and the medium segment (M), encodes for the envelope proteins. The nucleocapsid protein of bunyaviruses binds genomic RNA forming the viral RiboNucleoProtein (RNP) complex. The L protein interacts with these RNP structures allowing the initiation of viral replication. The nucleocapsid protein also interacts with actin, although the regions and specific residues involved in these interactions have not been yet described. Here, by means of immunoprecipitation and immunofluorescence assays we identify the regions within the CCHFV nucleocapsid protein implicated in homo-oligomerization and actin binding. We describe the interaction of N with the CCHFV L protein, and identify the amino- and carboxy-terminal regions within the L protein that might be necessary for the formation of these N-L protein complexes. These results may guide the development of potent inhibitors of these complexes that could potentially block CCHFV replication.
Development of numerous advanced techniques in recent years have allowed detection of the pathological prion protein (PrPTSE), the unique marker of transmissible spongiform encephalopathies (TSEs, or prion diseases), in the blood of animals and humans; however, an ante-mortem screening test that can be used for the routine diagnosis of human prion diseases remains unavailable. A critical, analytical review of all the diagnostic assays developed to date will allow an evaluation of progress in this field and may facilitate the identification of the possible reason/s for this delay. Thus, in this review, I provide a detailed overview of the techniques currently available for detecting PrPTSE and other markers of the disease in blood, as well as an analysis of the significance, feasibility, reliability, and the application spectrum for these methods. I highlight that factors intrinsic and extrinsic to blood may interfere with the detection of PrPTSE/prions, and that this is not yet taken into account in current tests. This may inspire researchers in this field to not only aspire to increase test sensitivity but also to adopt other strategies in order to identify and overcome the limitations that hamper the development of a successful routine blood test for prion diseases.
In simian virus 40 (SV40) and several other polyomaviruses, the TATA box of the early promoter is embedded in an AT tract that is also an essential part of the replication origin. We generated an "AT trap", an SV40 genome lacking the AT tract and unable to grow in CV-1 monkey cells. Co-transfection of the AT trap with oligonucleotides containing AT tracts of human polyomaviruses, a poly (A:T) tract, or variants of the SV40 wild type sequence all restored infectious virus. In a transfection of the AT trap without suitable oligonucleotide, an AT-rich segment was incorporated, stemming either from bovine (calf serum) or monkey (host cell) DNA. Similarly, when cells were grown with human serum, a human DNA segment was captured by SV40 to substitute for the missing AT stretch. We conclude that the virus is quite opportunistic in accepting heterologous substitutes, and that even low-abundance DNA from serum can be incorporated into the viral genome.
In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2), consistently gained positively charged amino acids in the putative heparan sulfate-binding pocket (VP2 bbeta;E-bbeta;F loop, VP1 C terminus and VP3 bbeta;-B knob), surrounding the five-fold symmetry axis (VP1 bbeta;F-bbeta;G loop) and at other discrete sites on the capsid (VP3 bbeta;G-bbeta;H loop, VP1 C terminus, VP2 bbeta;C strand and VP1 bbeta;G-bbeta;H loop). A lysine insertion in the VP1 bbeta;F-bbeta;G loop of two of the BHK-21 adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line suggesting their possible role in ionic interaction with ubiquitous negatively charged cell surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared to the lower passage viruses with wild-type genotypes. Substitutions fixed in the VP1 bbeta;G-bbeta;H loop (-3, -2 and +2 'RGD' positions) or in the structural element known to be juxtaposed against that loop (VP1 bbeta;B-bbeta;C loop) suggest their possible role in modulating the efficiency and specificity of interaction of 'RGD' motif with aalpha;V-integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production.
Hepatitis E virus (HEV), the causative agent of hepatitis E, is a single-stranded positive-sense RNA virus belonging to the family Hepeviridae. At least four genotypes of the family infect humans: genotypes 1 and 2 are transmitted to humans through contaminated water, while genotypes 3 and 4 are zoonotic and have animal reservoirs. A novel strain of HEV recently identified in rabbits is a distant member of genotype 3, and thus poses a potential risk of zoonotic transmission to humans. The objective of this study was to construct and characterize an infectious cDNA clone of the rabbit HEV. Two full-length cDNA clones of rabbit HEV, pT7g-rabHEV and pT7-rabHEV, were constructed and their infectivity was tested by in vitro transfection of Huh7 human liver cells and by direct intrahepatic inoculation of rabbits with capped RNA transcripts. Results showed that positive signal for rabbit HEV protein was detected by an immunofluorescence assay with a HEV-specific antibody in Huh7 human liver cells transfected with capped RNA transcripts from the two full-length cDNA clones. Rabbits intrahepatically inoculated with capped RNA transcripts from each of the two clones developed active HEV infection as evidenced by seroconversion to anti-HEV antibodies, and detection of rabbit HEV RNA in sera and feces of inoculated animals. The availability of a rabbit HEV infectious cDNA clone now affords us the ability to delineate the mechanism of HEV replication and cross-species infection in a small animal model.
Coronaviruses have been studied for over 60 years, but have only recently gained notoriety as deadly human pathogens with the emergence of severe respiratory syndrome coronavirus and Middle East respiratory syndrome virus. The rapid emergence of these viruses has demonstrated the need for good models to study severe coronavirus respiratory infection and pathogenesis. There are, currently, different methods and models for the study of coronavirus disease. The available genetic methods for the study and evaluation of coronavirus genetics are reviewed here. There are several animal models, both mouse and alternative animals, for the study of severe coronavirus respiratory disease that have been examined, each with different pros and cons relative to the actual pathogenesis of the disease in humans. A current limitation of these models is that no animal model perfectly recapitulates the disease seen in humans. Through the review and analysis of the available disease models investigators can employ the most appropriate available model to study coronavirus various aspects of pathogenesis and evaluate potential antiviral treatments that may potentially be successful in future treatment and prevention of severe coronavirus respiratory infections.