|Journal of General Virology current issue|
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 life cycle 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 shall review the recent structural insights nndash; NS5A is predicted to comprise three 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 shall highlight a few pertinent examples from the ever-expanding list of NS5A-binding partners identified over the past decade. Lastly, we shall 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 licensed, or in the late stages of approval for clinical use both in the USA and in the UK/Europe. In combination with other DAAs targeting the viral protease (NS3) and polymerase (NS5B), they are revolutionizing treatment for HCV infection.
The identification of virally encoded microRNAs (miRNAs) has had a major impact on the field of herpes virology. Given their ability to target cellular and viral transcripts, and the lack of immune response to small RNAs, miRNAs represent an ideal mechanism of gene regulation during viral latency and persistence. In this review, we discuss the role of miRNAs in virus latency and persistence, specifically focusing on herpesviruses. We cover the current knowledge on 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 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.
We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here, we showed that the same mutations improved the growth kinetics of a virus containing the A/Vietnam/1203/2004 (H5N1) haemagglutinin and neuraminidase (NA). Total protein yield and NA activity were increased when a chimeric NA was included. These increases indicated that the synergistic effect was due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.
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 revealed that the eight segment-specific NCRs identified could differentially regulate viral RNA synthesis and protein expression at both transcription and translation levels. Interestingly, a highly conserved suboptimal nucleotide at nndash;3 position of the Kozak sequence, which downregulated protein expression at the translation level, was only present in the segment-specific NCR of PB1. By reverse genetics, we demonstrate that recombinant viruses with an optimized Kozak sequence at the nndash;3 position in PB1 resulted in a significant multiple-cycle replication reduction that was independent of PB1-F2 expression. Our detailed dynamic analysis of virus infection revealed that the mutant virus displays slightly altered dynamics from the wild-type virus on both viral RNA synthesis and protein production. Furthermore, we demonstrated that the level of PB1 expression is involved in regulating type I IFN production. Together, these data reveal a novel strategy exploited by influenza A virus to fine-tune virus replication dynamics and host antiviral response through regulating PB1 protein expression.
The human metapneumovirus (HMPV) fusion (F) protein is the most immunodominant protein, yet subunit vaccines containing only this protein do not confer complete protection. The HMPV matrix (M) protein 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 titres, histopathology and cytokine levels were analysed. Mice immunized with F+alum and F+M+alum generated significantly more neutralizing antibodies than mice immunized with F only [titres of 47pplusmn;7 (Pllt;0.01) and 147pplusmn;13 (Pllt;0.001) versus 17pplusmn;2]. Unlike F only [1.6pplusmn;0.5x103 TCID50 (g lung)nndash;1], pulmonary viral titres 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 T-helper Th2/Th1 cytokine ratio. In conclusion, addition of the HMPV-M protein to an F protein-based vaccine modulated both humoral and cellular immune responses to subsequent infection, thereby increasing the protection conferred by the vaccine.
Members of the genus Vesiculovirus, which belongs to the family Rhabdoviridae, can cause great economic loss in fish culture. In the present report, a vesiculovirus [named snakehead fish vesiculovirus (SHVV)] was isolated from diseased hybrid snakehead fish. SHVV shared 94 % nucleotide sequence identity at the genomic level with Siniperca chuatsi rhabdovirus (SCRV), which infects mandarin fish (S. chuatsi). We showed that SHVV was able to replicate and proliferate well in SSN-1 cells, which originate 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 haemorrhage and oedema. Histopathological analysis revealed that extensive inflammation and necrosis were observed in the spleen, kidney, liver, heart and brain of the moribund mandarin fish. These results will shed new light on the epidemic of vesiculovirus infections among fish.
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 humans. However, the parameters that influence hRSV growth and control in this model are still poorly understood. We have documented in the following study 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 IFN-stimulated genes. Animals also developed specific antibodies and were immune to reinfection. Thus, we showed that even in infant macaques, intranasal hRSV infection induced both local and systemic immune responses to efficiently control the virus.
Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally transmitted by mosquitoes. During infection, JEV generally enters host cells via receptor-mediated clathrin-dependent endocytosis that requires the 70 kDa heat-shock protein (Hsp70). Heat-shock cognate protein 70 (Hsc70) is one member of the Hsp70 family and is constitutively expressed; thus, it may be expressed under physiological conditions. In C6/36 cells, Hsc70 is upregulated 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 the 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 these, isoform D 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.
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 titres 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 primenndash;boost immunization of AAD 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 the induction of 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 and as a vaccine platform for the development of DNA-based recombinant YFV vaccines.
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 faecal 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.
Infection with hepatitis C virus (HCV) is characterized by systemic oxidative stress that is caused by either viral core protein or chronic inflammation. It is well recognized that reactive oxygen species (ROS) such as H2O2 can induce apoptotic cell death and can therefore function as anti-tumorigenic species. However, the detailed mechanisms by which ROS induce apoptotic cell death and HCV copes with the oxidative conditions are largely unknown. In the present study, we found that H2O2 induced apoptotic cell death in p53-positive human hepatocytes, but not in p53-negative human hepatocytes. For this effect, H2O2 upregulated 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 p53-dependent apoptotic pathways. Interestingly, HCV core repressed p14 expression via promoter hypermethylation to abolish the potential of H2O2 to activate the p14nndash;MDM2nndash;p53 pathway. As a consequence, HCV core-expressing cells could overcome p53-mediated apoptosis provoked by H2O2. Taken together, HCV core could contribute to hepatocellular carcinoma formation by removing deleterious roles of ROS inducing cell death.
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 FCS. A previously unknown species, BoPyV2, occupies the same clade as human Merkel cell polyomavirus and raccoon polyomavirus, 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.
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 herpes simplex virus (HSV) as a helper virus for human AAV5 infections, underlining the in vivo relevance of the AAVnndash;herpesvirus relationship. In this study we analysed, for the first time, 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), helicasenndash;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 co-localized with ICP8 in nuclear replication compartments, and HSV alkaline exonuclease (UL12) enhanced AAV5 replication, similarly 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.
Little is known about viruses associated with Antarctic animals, although they are probably widespread. We recovered a novel polyomavirus from Adeeacute;lie penguin (Pygoscelis adeliae) faecal matter sampled in a subcolony at Cape Royds, Ross Island, Antarctica. The 4988 nt Adeeacute;lie penguin polyomavirus (AdPyV) has a typical polyomavirus genome organization with three ORFs that encoded capsid proteins on the one strand and two non-structural protein-coding ORFs on the complementary strand. The genome of AdPyV shared ~60 % pairwise identity with all avipolyomaviruses. Maximum-likelihood phylogenetic analysis of the large T-antigen (T-Ag) amino acid sequences showed that the T-Ag of AdPyV clustered with those of avipolyomaviruses, sharing between 48 and 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.
During productive infection with Epsteinnndash;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.
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 ASF virus (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 haemadsorption inhibition (HAI) serotype-specific. A better understanding of ASFV HAI serological groups and their diversity in nature, as well as improved methods to serotype ASFV isolates, is needed. Here, we demonstrated that the genetic locus encoding ASFV CD2v and C-type lectin proteins mediates HAI serological specificity and that CD2v/C-type lectin genotyping provides a simple method to group ASFVs by serotype, thus facilitating study of ASFV strain diversity in nature, and providing information necessary for eventual vaccine design, development and efficacious use.
IFN-induced restriction factors can significantly affect the replicative capacity of retroviruses in mammals. TRIM5aalpha; (tripartite motif protein 5, isoform aalpha;) 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 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 human immunodeficiency virus type 1 (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 showed that efficient restriction of HIV-1 by omkTRIMCyp was 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 small interfering RNA-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 had a significant effect on the restriction of HIV-1 in an owl monkey cell line. These results suggested the existence of cell-specific functional interactions between MTs/dynein and TRIMCyp.
APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3) proteins are cellular DNA deaminases that restrict a broad spectrum of lentiviruses. This process is counteracted by Vif (viral infectivity factor) of lentiviruses, which binds APOBEC3s and promotes their degradation. CBF-bbeta; (core binding factor subunit 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 determined the sequence of feline CBFB and performed phylogenetic analyses. These analyses revealed that mammalian CBFB is under purifying selection. Moreover, we demonstrated that CBF-bbeta; is dispensable for feline immunodeficiency virus Vif-mediated degradation of APOBEC3s of its host. These findings suggested 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.
Analysing the evolution of feline immunodeficiency virus (FIV) at the intra-host level is important in order to address whether the diversity and composition of viral quasispecies affect 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 a rate of 1.16x10nndash;3 substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3nndash;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 V3nndash;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 maximum 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 with human immunodeficiency virus might explain why many naturally infected cats do not progress rapidly to AIDS.
A granulovirus (GV) producing occlusion bodies (OBs) with an unusual appearance was isolated from Adoxophyes spp. larvae in the 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 this new isolate 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 Adoxophyes honmai. However, AdorGV-M retained more infectivity towards larvae after irradiation with UV light than did 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 AdorGV isolates and other Japanese AdorGV isolates revealed differences that may account for the unusual OB morphology of AdorGV-M.
Over recent decades, metagenomic studies have expanded the number of newly described, often unclassified, viruses within the family Circoviridae. Using broad-spectrum circovirus and cyclovirus PCRs, we characterized a novel circo-like virus in Aedes vexans mosquitoes from Germany whose main putative ORFs shared very low amino acid identity with those of previously characterized circoviruses and cycloviruses. Phylogenetic and genetic distance analysis revealed that this new virus species defined, together with previously described mosquito- and bat faeces-derived circo-like viruses, a different genus, tentatively called Krikovirus, within the family Circoviridae. We further demonstrated that viruses of the putative genus Krikovirus all shared a genomic organization that was unique among the family 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 family Circoviridae.
As a member of the newly established Betaflexiviridae family, grapevine rupestris stem pitting-associated virus (GRSPaV) has an RNA genome containing five 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 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 towards 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 acids in the methyl-transferase domain responsible for the formation of these punctate structures. The 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 is likely through an amphipathic aalpha; 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.
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 pathogennndash;insect specificity are poorly understood. Rice dwarf virus (RDV), a plant reovirus, is transmitted mainly by the leafhopper species Nephotettix cincticeps but is transmitted ineffectively by the leafhopper Recilia dorsalis. Here, we demonstrated that virus-containing tubules composed of viral non-structural 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 is 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 pathogennndash;vector specificity.
|Journal of General Virology Publish Ahead of Print|
The capsid genes from fourteen pigeon circovirus (PiCV) strainssequences, collected from around Taiwan between 2009 and 2010, were sequenced and compared with 14 PiCV capsid sequences from Genbank. Based on pairwise comparison, PiCV strains from Taiwan shared 73.9% - 100% nucleotide identity and 72% -- 100% amino acid identity with those of the 14 reported PiCV sequencesstrains. Phylogenic analyses revealed that Taiwanese PiCV isolates can be grouped into 2 clades - clade 1 comprising Belgium, Australia, USA, Italy, and China isolates and clade 2 showed close relation to Germany and France isolates. Recurrent positive selection was detected at clade 1 PiCV lineages, which may contribute to the diversification of predominant PiCV sequencesstrains in Taiwan. Further observations suggest that synonymous codon usage variations between PiCV clade 1 and clade 2 may reflect the adaptive divergence on translation efficiency of capsid genes in infectious hosts. Variation in selective pressures acting on the evolutionary divergence and codon usage bias of both clades explains the regional coexistence of virus strainsequences congeners prevented from competitive exclusion within an island such as Taiwan. Our genotyping results also provide insight into the etiological agents of PiCV outbreak in Taiwan and we present a comparative analysis of the central coding region of PiCV genome. From the sequence comparison results of 28 PiCVs which differs in regard to the geographical origin and columbid species, we identified conserved regions within the Cap capsid gene that are likely to be suitable for primer selection and vaccine development.
The human papillomavirus (HPV) E2 protein is a multifunctional protein essential for the control of virus gene expression, genome replication and persistence. E2 is expressed throughout the differentiation-dependent virus life cycle and is functionally regulated by association with multiple viral and cellular proteins. Here, we show for the first time that HPV16 E2 directly associates with the major capsid protein L1, independently of other viral or cellular proteins. We have mapped the L1 binding region within E2 and show that the aalpha;-2 helices within the E2 DNA binding domain mediate L1 interaction. Using cell-based assays, we show that co-expression of L1 and E2 results in enhanced transcription and virus origin-dependent DNA replication. Upon co-expression in keratinocytes, L1 reduces nucleolar association of E2 protein and when co-expressed with E1 and E2, L1 is partially recruited to viral replication factories. Furthermore, co-distribution of E2 and L1 was detected in the nuclei of upper suprabasal cells in stratified epithelia of HPV16 genome-containing primary human keratinocytes. Taken together, our findings suggest that the interaction between E2 and L1 is important for the regulation of E2 function during the late events of the HPV life cycle.
Japanese encephalitis (JE) remains the leading cause of viral encephalitis in children in Asia and is expanding its geographic range to larger areas in Asia and Australasia. Five genotypes of Japanese encephalitis virus (JEV) co-circulate in its geographically affected areas. Especially, the emergence of genotype I (GI) JEV has displaced genotype III (GIII) as dominant circulating strains in many Asian regions. However, all approved vaccine products are derived from GIII strains. In the present study, bioinformatic analysis revealed GI and GIII JEV strains shared two distinct amino acid residues within envelope (E) protein (E222 and E327). By using reverse genetic approaches, A222S and S327T mutations were demonstrated to decrease the live-attenuated vaccine (LAV) SA14-14-2 induced neutralizing antibody in human, without altering viral replication. Then, A222S or S327T mutations were rationally engineered into the infectious clone of SA14-14-2, respectively, and the resulting mutant strains remained the same genetic stability and attenuation characteristics as their parent strain. More importantly, immunization of mice with LAV-A222S or LAV-S327T elicited increased neutralizing antibodies against GI strains. Together, these results demonstrated that E222 and E327 are potential genotype-related neutralization determinants, and are critical in determining the protective efficacy of live JE vaccine SA14-14-2 against circulating GI strains. Our findings will aid in the rational design of next generation of live-attenuated JE vaccine capable of providing broad protection against all JEV strains belonging to different genotypes.
Glycoprotein gH is essential for herpesvirus-induced membrane fusion during entry and cell-to-cell spread. Structural analyses of gH homologues revealed a conserved syntaxin-like bundle motif composed of three -helices. Previous studies showed that targeted disruption of any of these helices strongly impaired maturation, cell surface expression, and fusion activity of pseudorabies virus gH, as well as formation and spread of infectious virus. After passaging of one corresponding mutant (pPrV-gH-V275P) these replication defects were widely corrected by an adjacent, spontaneous amino acid substitution (V271A). Although the doubly mutated gH was still non-functional in fusion assays, its targeted reinsertion into the cloned virus genome (pPrV-gH-V275P-V271A) led to 200-fold increased plaque sizes, and 10000-fold higher virus titers, compared to pPrV-gH-V275P. Thus, our results demonstrate that structural requirements for gH function in in vitro assays and virus replication are different, and that minor amounts of mature gH in virions are sufficient for productive replication.
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.
The hepatitis B virus (HBV) remains to be a global health threat since the chronic HBV infection may lead to liver cirrhosis or cancer. Current antiviral therapies with nucleoside analogues can inhibit the replication of HBV, but do not disrupt the already existing HBV cccDNA. The newly developed clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated 9 (Cas9) system is a powerful tool to target cellular genome DNA for gene editing. In order to investigate the possibility of using CRISPR/Cas9 system to disrupt the HBV DNA templates, we designed eight gRNAs that target the conserved regions of different HBV genotypes, which could significantly inhibit HBV replication both in vitro and in vivo. Moreover, the HBV-specific gRNA/Cas9 could inhibit the replication of HBV of different genotypes in cells and the viral DNA is significantly reduced by single gRNA/Cas9 and cleared by combination of different gRNAs/Cas9.
Low pathogenic avian influenza A viruses have a natural host reservoir in wild waterbirds and the potential to spread to other host species. Here we investigate the evolutionary, spatial and temporal dynamics of avian influenza A viruses in Eurasian wild birds. We use whole genome sequences collected as part of an intensive long-term Eurasian wild bird surveillance study and combine this genetic data with temporal and spatial information to explore the virus evolutionary dynamics. Frequent reassortment and co-circulating lineages were observed for all eight genomic RNA segments over time. There was no apparent species-specific effect on the diversity of the avian influenza A viruses. There was a spatial and temporal relationship between the Eurasian sequences and significant viral migration of avian influenza A viruses from West towards Central Asia. The observed viral migration patterns differed between segments. Further we discuss the challenges faced when analysing these surveillance and sequence data and the caveats to be borne in mind when drawing conclusions from the apparent results of such analyses.
Bats harbour a diverse array of viruses, including significant human pathogens. The extensive metagenomic study of material from bats, in particular guano, has revealed a large number of novel or divergent viral taxa that were previously unknown. New Zealand has only two extant indigenous terrestrial mammals which are both bats, Mystacina tuberculata (lesser short-tailed bat) and Chalinobus tuberculatus (long-tailed bat). Until the human introduction of exotic mammals, these species had been isolated from all other terrestrial mammals for over one million years (potentially over 16 million years for M. tuberculata). Four bat guano samples were collected from M. tuberculata roosts on the isolated offshore island of Whenua hou (Codfish Island). Metagenomic analysis reveals that this species still hosts a plethora of divergent viruses. While the majority of viruses detected were likely to be of dietary origin, some putative vertebrate virus sequences were identified. Papillomavirus, polyomavirus, calicivirus and hepevirus were found in the metagenomic data and then subsequently confirmed using independent PCR assays and sequencing. The new hepevirus and calicivirus sequences may represent new genera within these viral families. Our findings may provide an insight into the origins of viral families given their detection in an isolated host species.
Epstein-Barr virus (EBV) expresses two immediate-early proteins, Rta and Zta, which are key transcription factors that can form a complex with MCAF1 at Zta-responsive elements (ZREs) to synergistically activate several viral lytic genes. Our previous research indicated that RanBPM interacts with Rta and enhances Rta sumoylation. Here we show that RanBPM binds to Zta in vitro and in vivo, and acts as an intermediary protein in Rta-Zta complex formation. The Rta-RanBPM-Zta complex was observed to bind with ZREs in the transcriptional activation of key viral genes, such as BHLF1 and BHRF1, while the introduction of RanBPM short hairpin RNA (shRNA) subsequently reduced the synergistic activity of Zta and Rta. RanBPM was found to enhance Zta-dependent transcriptional activity via the inhibition of Zta sumoylation. Interestingly, Z-K12R, a SUMOylation-defective mutant of Zta, demonstrated transcriptional activation capabilities that were stronger than Zta and apparently unaffected by RanBPM modulation. Finally, RanBPM silencing inhibited the expression of lytic proteins. Taken together, these results shed light on the mechanisms by which RanBPM regulates Zta-mediated transcriptional activation, and point to an important role for RanBPM in EBV lytic progression.
Influenza B viruses fall in two antigenically distinct lineages (B/Victoria/2/1987 and B/Yamagata/16/1988 lineage) that co-circulate with influenza A viruses of the H3N2 and H1N1 subtype during seasonal epidemics. Infections with influenza B viruses contribute considerably to morbidity and mortality in the human population. Influenza B virus neutralizing antibodies, elicited by natural infections or vaccination, poorly cross-react with viruses of the opposing influenza B lineage. Therefore, there is an increased interest in identifying other correlates of protection which could aid the development of broadly-protective vaccines. BLAST analysis revealed high sequence identity of all viral proteins. With two online epitope prediction algorithms, putative conserved epitopes relevant for study subjects used in the present study, were predicted. The cross-reactivity of influenza B virus-specific polyclonal CD8+ T lymphocyte populations, obtained from HLA-typed healthy study subjects, with intra-lineage drift variants and viruses of the opposing lineage was determined by assessing their in vitro interferon gamma (IFN-) response and lytic activity. Here, we show for the first time, that CD8+ T lymphocytes directed to viruses of the B/Victoria lineage cross-react with viruses of the B/Yamagata lineage and vice versa.
Viroid systemic spread involves cell-to-cell movement from initially infected cells via plasmodesmata, long-distant movement within the phloem, and again cell-to-cell movement to invade distal tissues including the mesophyll. Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid (CDVd), Citrus bark cracking viroid (CBCVd) and Citrus viroid V (CVd-V) remained phloem-restricted when singly infecting C. karna, C. aurantium and Poncirus trifoliate, but not Etrog citron, where they were additionally detected in mesophyll protoplasts. However, when CEVd-infected C. karna was side-grafted with Etrog citron mmdash;with the resulting plants being composed of a C. karna stock and an Etrog citron branch- the viroid was detected in mesophyll protoplasts of the former, thus indicating that the ability of Etrog citron to support viroid invasion of non-vascular tissues was transferred to the stock. Further results suggest that a translocatable factor from Etrog citron mediates this viroid trafficking.
Hepatitis C virus (HCV) is classified into 7 genotypes based on genetic diversity and most genotypes have been found in Africa. Infections with HCV genotype 2 (HCV2) are most prevalent in West Africa, and it was suggested that HCV2 originated in West Africa. To better understand the evolutionary epidemiology of HCV2 in Africa, we examined new NS5b sequences of HCV2 strains obtained from Coocirc;te d'Ivoire, Ghana and Nigeria sequenced in this laboratory with those available from West, North and Central Africa. Bayesian phylogeographic analysis using a discrete trait model showed that Ghana is the most likely geographic region for origin of HCV2. Spread of HCV2 from Ghana does not appear to be through diffusion to adjacent countries along the coast. Rather, it was transmitted from Ghana to many distant countries in Africa, suggesting that certain routes of geographic dissemination were historically more efficient than mere proximity and that the HCV2 epidemic history in West Africa is extremely complex.
The placenta of domestic sheep plays a key role in horizontal transmission of classical scrapie. Domestic goats are frequently raised with sheep and are susceptible to classical scrapie, yet potential routes of transmission from goats to sheep are not fully defined. Sparse accumulation of disease-associated prion protein in cotyledons casts doubt about the role of the goat's placenta. Thus, relevant to mixed-herd management and scrapie-eradication efforts worldwide, we determined if the goat's placenta contains prions orally infectious to goat kids and lambs. A pooled cotyledon homogenate, prepared from the shed placenta of a goat with naturally acquired classical scrapie disease, was used to orally inoculate scrapie-naiiuml;ve prion genotype-matched goat kids and scrapie-susceptible lambs raised separately in a scrapie-free environment. Transmission was detected in all four goats and in two of four sheep which importantly identifies the goat's placenta as a risk for horizontal transmission to sheep and other goats.
The conserved alphaherpesviral serine/threonine kinase US3 causes dramatic changes in the actin cytoskeleton, consisting of actin stress fiber breakdown and protrusion formation, associated with increased viral spread. In this report, we show that US3 expression leads to RhoA phosphorylation at serine 188 (S188), one of the hallmarks of suppressed RhoA signaling, and that expression of a non-phosphorylatable RhoA variant interferes with the ability of US3 to induce actin rearrangements. Furthermore, inhibition of cellular protein kinase A (PKA) abrogates the ability of US3 to induce S188 RhoA phosphorylation, pointing to a role for PKA in US3-induced RhoA phosphorylation. Hence, the US3 kinase leads to PKA-dependent S188 RhoA phosphorylation, which contributes to US3-mediated actin rearrangements. Our data suggest that US3 efficiently usurps the antagonistic RhoA and Cdc42/Rac1/PAK signaling branches to rearrange the actin cytoskeleton.
Hepatitis C virus (HCV) is an RNA virus which exists as swarms of closely related viruses known as quasispecies (QS). A number of studies have demonstrated associations between QS hypervariable region 1 (HVR1) characteristics (diversity and complexity) and treatment success. We investigated HCV QS change in chronic infection over intervals of 2-4 weeks in 23 chronically infected individuals to describe the natural history of virus evolution and establish whether HCV QS characteristics could be used to individualise treatment regimens at a molecular level.
HVR1 QS diversity, complexity, and divergence continue to change in an unpredictable fashion in chronic infection even where there is little phylogenetic change which is likely to preclude the use of these features in treatment individualisation. Our phylogenetic analysis identified no change in the HVR1 QS in 12 subjects, minor change in 4 subjects and we describe a time order phylogeny for the first time over a period as short as 16 weeks in 7 subjects. We identify the existence of multiple subpopulation infections and illustrate how subpopulations are sequentially replaced in a number of subjects We illustrate marked variation in the nucleotide substitution per codon position between patients with sequence change and those without change in the phylogenetic tree. Analysis of codon specific selection pressures identifies a number of codons under purifying selection suggesting that these code for structurally conserved amino acids. We also identify sections of the HVR1 under positive selection with marked sequence heterogeneity suggesting that these may be potential epitope binding sites.
The PA-X protein, arising from ribosomal frame-shift during PA translation, was recently discovered in influenza A virus. The C-terminal domain "X" of PA-X proteins in influenza A viruses can be classified as full length (61 amino acids) or truncated (41 amino acids). In the main, avian influenza viruses express full length PA-X proteins, while 2009 pandemic H1N1 (pH1N1) influenza viruses harbor truncated PA proteins. The truncated form lacks amino acids 232-252 of the full length PA-X protein. The significance of PA-X length in virus function remains unclear. To address this issue, we constructed a set of contemporary influenza viruses (pH1N1, avian H5N1 and H9N2) with full and truncated PA-X by reverse genetics to compare their replication and host pathogenicity. All full length PA-X viruses in human A549 cells conferred 10-100 fold increase in viral replication and 5-8% increase in apoptosis relative to corresponding truncated PA-X viruses. Full length PA-X viruses were more virulent and caused more severe inflammatory responses in mice. Furthermore, amino acids 233-252 at the C-terminus of PA-X strongly suppressed co-transfected gene expression by around 50%, suggesting that these terminal 20 amino acids could play a role in enhancing viral replication and contribute to virulence.
Genetic instability is intimately associated with tumor development. In particular, liver cancers associated with hepatitis B virus (HBV) exhibit high genetic instability; however, our understanding of the underlying molecular mechanisms remains limited. In this study, we found that -H2AX, a marker of DNA double-strand breaks (DSBs), and the levels of phospho-Chk2 (p-Chk2, the activated form) were significantly elevated in HBV-associated hepatocellular carcinomas and neighboring regenerating nodules. Likewise, introduction of the pHBV or pMyc-HBx genes into cells induced accumulation of -H2AX foci and increased the p-Chk2 level. In these cells, inhibitory phosphorylation of Cdc25C phosphatase (Ser216) and CDK1 (Tyr15) was elevated; consequently, cell-cycle progression was delayed at G2/M phase, suggesting that activation of the ATM-Chk2 pathway by HBx induces cell-cycle delay. Accordingly, inhibition of ataxia telangiectasia mutated (ATM) by caffeine or siRNA abolished the increase in the p-Chk2 level and restored the delayed CDK1 kinase activity in ChangX cells. We also found that cytoplasmic HBx, but not nuclear HBx, induced ROS production and led to the accumulation of -H2AX foci and the p-Chk2 level. Together, these data indicate that HBx-induced ROS accumulation induces DNA damage that activates the ATM-Chk2 pathway. Our findings provide insight into the mechanisms of HBV pathogenesis.
Oncogenic human papillomaviruses (HPVs) attach predominantly to extracellular matrix (ECM) components during infection of cultured keratinocytes and in the rodent vaginal challenge model in vivo. However, the mechanism of virion transfer from the ECM to receptors that mediate entry into host cells has not been determined. In this work we strove to assess the role of heparan sulfate (HS) chains in HPV16 binding to the ECM and determine how HPV16 release from the ECM is regulated. We also assessed the extent to which capsids released from the ECM are infectious. We show that a large fraction of HPV16 particles binds to the ECM via HS chains, and that syndecan-1 (snd-1) molecules present in the ECM are involved in virus binding. Inhibiting the normal processing of snd-1 and HS molecules via matrix metalloproteinases and heparanase dramatically reduces virus release from the ECM, cellular uptake, and infection. Conversely, exogenous heparinase activates each of these processes. We confirm that HPV16 released from the ECM is infectious in keratinocytes. Use of a specific inhibitor shows furin is not involved in HPV16 release from ECM attachment factors and corroborates other studies showing only the intracellular activity of furin is responsible for modulating HPV infectivity. These data suggest that our recently proposed model, describing the action of HS proteoglycan processing enzymes in releasing HPV16 from the cell surface in complex with the attachment factor snd-1, is also relevant to the release of HPV16 particles from the ECM to promote efficient infection of keratinocytes.
HLA-B27 allele is over-represented among HIV-1 infected long term non-progressors (LNTPs). In these patients, strong CTL responses targeting HLA-B27 restricted viral epitopes have been associated with long term asymptomatic survival. Indeed loss of control of viremia in HLA-B27 patients has been associated with CTL escape at position 264 in the immunodominant KK10 epitope. This CTL escape mutation in the viral Gag protein has been associated with severe viral attenuation and may require the presence of compensatory mutations before emerging.
We here studied sequence evolution within HLA-B27-restricted CTL epitopes in the viral Gag protein during the course of infection of seven HLA-B27+ patients. Longitudinal gag sequences obtained at different time points around AIDS diagnosis were obtained and analysed for the presence of mutations in epitopes restricted by HLA-B27, and for potential compensatory mutations. Sequence variations were observed in the HLA-B27-restricted CTL epitopes IK9, DR11 and the immunodominant KK10 epitope. However, the presence of sequence variations in the HLA-B27-restricted CTL epitopes could not be associated with an increase in viremia in the majority of the patients studied. Furthermore, we observed low genetic diversity in the gag region of the viral variants throughout the course of infection, which is indicative of low viral replication and corresponds to the low viral load observed in the HLA-B27+ patients. These data indicate that control of viral replication can be maintained in HLA-B27+ patients despite the emergence of viral mutations in HLA-B27-restricted epitopes.
To date, proteomic studies have been performed on occlusion-derived virus (ODV) from five members of the Baculoviridae genus Alphabaculovirus but only a single member of Betabaculovirus (Pieris rapae granulovirus, PrGV). In this study, liquid chromatography-tandem mass spectrometry was used to analyze the ODV proteins of Clostera anachoreta granulovirus (ClanGV), another Betabaculovirus member. The results indicated that 73 proteins, including the products of 27 baculovirus core genes, are present in ClanGV-ODV. This is the largest number of ODV proteins identified in baculoviruses to date. To our knowledge, twenty-four of these proteins were newly identified as ODV associated proteins. Twelve of the proteins are shared by all seven of the other baculoviruses that have been analyzed by proteomic techniques, including P49, PIF-2, ODV-EC43, P74, P6.9, P33, VP39, ODV-EC27, VP91, GP41, VLF-1 and VP1054. ClanGV shared between 20 and 36 ODV proteins with each of the other six baculoviruses that have been analyzed by proteomics. Ten proteins were identified only as the ODV components of ClanGV and PrGV: Clan22, Clan27, Clan69, Clan83, Clan84, Clan90, Clan116, Clan94, FGF-3 and ME53, the first 7 of which were encoded by betabaculovirus-specific genes. These findings may provide novel insights into baculovirus structure as well as reveal similarities and differences between members of Betabaculovirus and Alphabaculovirus.
Lymphocyte proliferation, mobility and longevity makes them prime targets for viral infection. Myeloid cells that process and present environmental antigens to lymphocytes are consequently an important line of defence. Subcapsular sinus macrophages (SSM) filter the afferent lymph and communicate with B cells. How they interact with B cell-tropic viruses is unknown. We analyzed their encounter with Murid Herpesvirus-4 (MuHV-4), an experimentally accessible gamma-herpesvirus related to the Kaposi's Sarcoma-associated Herpesvirus. MuHV-4 disseminates via lymph nodes, and intranasally or subcutaneously inoculated virions readily infected SSM. However this infection was poorly productive. SSM depletion with clodronate-loaded liposomes or with diphtheria toxin in CD169-diphtheria toxin receptor transgenic mice increased B cell infection and hastened viral spread to the spleen. Dendritic cells provided the main route to B cells, and SSM slowed host colonization apparently by absorbing virions non-productively from the afferent lymph.
Hepatitis C virus (HCV) establishes a persistent infection that in many cases leads to cirrhosis and hepatocellular carcinoma (HCC). The non-structural 5A protein (NS5A) has been implicated in this process as it contains a C-terminal polyproline motif (termed P2) that binds to Src homology 3 (SH3) domains to regulate cellular signalling and trafficking pathways. We have previously shown that NS5A impairs epidermal growth factor receptor (EGFR) endocytosis, thereby inhibiting EGF-stimulated EGFR degradation by a mechanism that remained unclear. As EGFR has been implicated in HCV cell entry, and trafficking of the receptor involves several SH3-domain containing proteins, we investigated in more detail the mechanisms by which NS5A perturbs EGFR trafficking.
We demonstrate that the P2 motif is required for the NS5A-mediated disruption to EGFR trafficking. We further demonstrate that the P2 motif is required for an interaction between NS5A and CMS, a homologue of CIN85 that has previously been implicated in EGFR endocytosis. We provide evidence that CMS is involved in the NS5A-mediated perturbation of EGFR trafficking. We also show that NS5A effects a loss of EGFR ubiquitination in a P2-motif dependent fashion. These data provide clues to the mechanism by which NS5A regulates the trafficking of a key cellular receptor, and demonstrate for the first time the ability of NS5A to regulate host cell ubiquitination pathways.
In the present study, we developed a novel approach for foreign gene expression by Newcastle disease virus (NDV) from a second open reading frame (ORF) through an internal ribosomal entry site (IRES). Six NDV LaSota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein (RFP) gene behind the NP, P, M, F, HN, or L gene ORF were generated using reverse genetics technology. The insertion of the 2nd ORF slightly attenuated the virus pathogenicity, but did not affect virus growth ability. Quantitative measurements of the RFP expression in virus-infected DF-1 cells revealed that the abundance of viral mRNAs and red fluorescence intensity were positively correlated with the gene order of NDV, 3'NP-P-M-F-HN-L, proving the sequential transcription mechanism for NDV. The results suggested that the level of foreign gene expression could be regulated by selecting the 2nd ORF insertion site to maximize the efficacy of vaccine and gene therapy.
Chikungunya virus (CHIKV) is a rapidly spreading, enveloped alphavirus causing fever, rash and debilitating polyarthritis. No specific treatment or vaccines are available to treat or prevent infection. For the rational design of vaccines and antiviral drugs, it is imperative to understand the molecular mechanisms involved in CHIKV infection. A critical step in the life cycle of CHIKV is fusion of the viral membrane with a host cell membrane. Here, we elucidate this process using ensemble-averaging liposome-virus fusion studies, in which the fusion behavior of a large virus population is measured, and a newly developed microscopy-based single-particle assay, in which the fusion kinetics of an individual particle can be visualized. The combination of these approaches allowed us to obtain detailed insight in the kinetics, lipid dependency, and pH dependency of hemifusion. We found that CHIKV fusion is strictly dependent on low pH, with a threshold of pH 6.2 and optimal fusion efficiency below pH 5.6. At this pH, CHIKV fuses rapidly with target membranes, with typically half of the fusion occurring within less than two seconds after acidification. Cholesterol and sphingomyelin in the target membrane were found to strongly enhance the fusion process. By analysing our single-particle data using kinetic models, we were able to derive that the number of rate-limiting steps occurring before hemifusion equals about three. To explain these data, we propose a mechanistic model in which multiple E1 fusion trimers are involved in initiating the fusion process.
Hepatitis C virus nonstructural protein 5A (HCV NS5A) is essential for viral replication, however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3 untranslated region (UTR), where NS5A binds to the polyU/UC sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing renilla luciferase gene flanked by the HCV UTRs. We found that NS5A down-regulates viral RNA translation in a dose-dependent manner. This down-regulation requires both the 5' and 3 UTRs of HCV because substitution of either sequence with the 5' and 3 UTRs of the enterovirus 71 or a cap structure at the 5' end abrogates the effects of NS5A on translation. Translation of the HCV genomic RNA is also down-regulated by NS5A. The inhibition of HCV translation by NS5A requires the polyU/UC sequence in the 3 UTR since NS5A does not affect translation when it is deleted. In addition, we showed that, while the amphipathic aalpha;-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently also dependent on the presence of the polyU/UC sequence in the 3 UTR. These results suggest that NS5A down-regulates HCV RNA translation through a mechanism involving the polyU/UC sequence in the 3'UTR.
CCR5 serves as an essential coreceptor for HIV-1 entry and individuals with a CCR532 variant appears to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, that functions as a naturally-existed adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4+ T cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNA (sgRNA) targeting the open reading frame of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analyzed, no significant off-target effects were detected at the 15 top-scored potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4+ T lymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4+ T cells utilizing adenovirus-delivered CRISPR/Cas9.
After a primary lytic infection at the epithelia, herpes simplex virus type 1 (HSV-1) enters the innervating sensory neurons and translocates to the nucleus, where it establishes a quiescent latent infection. Periodically, the virus can reactivate and the progeny viruses spread back to the epithelium. In this article we introduce an embryonic mouse dorsal root ganglion (DRG) culture system, which can be used to study the mechanisms that control the establishment, maintenance and reactivation from latency. Use of acyclovir is not necessary in our model. We have examined different phases of HSV-1 lifecycle in DRG neurons and showed that wild-type HSV-1 can establish both lytic and latent form of infection in the cells. After reactivating stimulus, the wild-type viruses show all markers of true reactivation. In addition, we show that deletion of the 134.5 gene renders the virus incapable of reactivation, even though the virus clearly is able to replicate and persist in a quiescent form in the DRG neurons.
Infectious myonecrosis virus (IMNV) causes significant economic losses in farmed shrimp, where associated mortality in ponds can reach 70%. To explore host-pathogen interactions, a next generation sequencing approach (NGS) using lymphoid organ (LO) tissue from IMNV-infected Litopenaeus vannamei shrimp was conducted. Preliminary sequence assembly of just the virus showed that there were at least an additional 639 base pairs (bp) at the 5' terminus and 23 nt at 3' terminus as compared to the original description of the IMNV genome (7561 nt). Northern blot and RT-PCR analysis confirmed the presence of novel sequence at both ends of the genome. Using 5' RACE, 4 additional nucleotides were discovered; 3' RACE confirmed the presence of 22, rather than 23 bp of sequence. Based on these data, the IMNV genome is 8226 bp in length. Double-stranded RNA was used to trigger RNA interference (RNAi) and suppress expression of the newly revealed genome sections at the 5' end of the IMNV genome in IMNV-infected L. vannamei. An RNAi trigger targeting a 376 bp length of the 5'UTR did not improve survival of infected shrimp. In contrast, an RNAi trigger that targets a 381 bp sequence in ORF1 improved survival to 82.2% as compared to 2.2% survival in positive control animals. These studies revealed the importance of the new genome sections to produce high titer infection and associated disease and mortality, in infected shrimp.
Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid-binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. In this report we show that BRO-B and BRO-E associate with cellular TIA-1 homolog (BmTRN-1), a translational regulator, and other cellular translation-related proteins in silkworm cells during viral infection. We created BM-N cells that express BRO-B/E to study molecular interactions between BmTRN-1 and BRO-B/E and how they influence protein synthesis. Fluorescent microscopy revealed that BmTRN-1 is localized in cytoplasmic foci during BmNPV infection. Immunofluorescence studies confirmed that BmTRN-1 and BRO-B/E are colocalized in the amorphous conspicuous cytoplasmic foci. Reporter gene studies revealed that co-expression of BRO-B and BRO-E synergistically led to a significant decrease in protein synthesis from a designed transcript carrying the 5UTR of a cellular mRNA with no significant change of the transcript abundance. Additionally, RNAi-mediated knockdown of BmTRN-1 resulted in a marked inhibition of the ability of BRO-B/E to regulate the transcript. These results suggest that the association of BmTRN-1 with BRO-B/E is responsible for the inhibitory regulation of certain mRNAs at the post-transcriptional level, and add an additional mechanism for how baculovirus control protein synthesis during infection.
Vaccination with live attenuated SIV in non-human primate species provides a means of characterising the protective processes of retroviral superinfection and may lead to novel advances of HIV/AIDS vaccine design. The minimally attenuated SIVmacC8 vaccine has been demonstrated to elicit early potent protection against pathogenic re-challenge with genetically diverse viral isolates in cynomolgus macaques (Macaca fascicularis). In this study, we have characterised further the biological breadth of this vaccine protection by assessing the ability of both the nef-disrupted SIVmacC8 and its nef-intact counterpart SIVmacJ5 viruses to prevent superinfection with the macrophage/neurotropic SIVmac239/17E-Fr (SIVmac17E-Fr) isolate. Inoculation with either SIVmacC8 or SIVmacJ5 and subsequent detailed characterisation of the viral replication kinetics revealed a wide range of virus-host outcomes. Both nef-disrupted and nef-intact immunising viruses were able to prevent establishment of SIVmac17E-Fr in peripheral blood and secondary lymphoid tissues. Differences in virus kinetics, indicative of an active process, identified uncontrolled replication in one macaque which although able to prevent SIVmac17E-Fr superinfection led to extensive neuropathological complications. The ability to prevent a biologically heterologous, CD4-independent/CCR5+ viral isolate and the macrophage-tropic SIVmac316 strain from establishing infection support the hypothesis that direct target cell blocking is unlikely to be a central feature of live lentivirus vaccination. These data provide further evidence to demonstrate that inoculation of a live retroviral vaccine can deliver broad spectrum protection, against both macrophage-tropic as well as lymphocytotropic viruses. These data add to our knowledge of live attenuated SIV vaccines but further highlight potential safety concerns of vaccinating with a live retrovirus.
Hepatitis B virus (HBV) infection is associated with a wide spectrum of clinical manifestations including cirrhosis and hepatocellular carcinoma (HCC). Endoplasmic reticulum (ER) stress and subsequent oxidative stress have been implicated in liver carcinogenesis and disease progression with chronic inflammation. In our previous study, several mutations in the preC/C region of HBV genotype C were identified from 70 Korean chronic patients, and the mutations were associated with HCC and/or HBeAg serostatus. Here, we found that the naturally occurring mutations P5T/H/L of the HBV core region (HBcAg) induced endoplasmic reticulum (ER) stress. The upregulation of ER stress resulted in higher reactive oxygen species production, intracellular calcium concentration, inflammatory cytokines, as well as surface antigen production and apoptosis of cells. This study suggests that these mutations may contribute to the progression of liver disease in chronic patients.
Porcine epidemic diarrhea virus (PEDV) is a coronavirus that induces persistent diarrhea in swine, resulting in severe economic losses in swine-producing countries. Insights into the interplay between PEDV infection and innate immune system are necessary for understanding the associated mechanism of pathogenesis. The transcription factor nuclear factor kappa B (NF-B) plays an important role in regulating host immune responses. Here, we elucidated for the first time the potential mechanism of PEDV-mediated NF-B activation in porcine small intestinal epithelial cells (IECs). During PEDV infection, NF-B p65 was found to translocate from the cytoplasm to the nucleus, and PEDV-dependent NF-B activity was associated with viral dose and active replication. Using small interfering RNAs to screen different mRNA components of the Toll-like receptor (TLR) or RIG-I-like receptor signaling pathways, we demonstrated that TLR2, TLR3, and TLR9 contribute to NF-B activation in response to PEDV infection, but not RIG-I. By screening PEDV structural proteins for their abilities to induce NF-B activities, we found that PEDV nucleocapsid protein (N) could activate NF-B and that the central region of N was essential for NF-B activation. Furthermore, TLR2 was involved in PEDV N-induced NF-B activation in IECs. Collectively, these findings provide new avenues of investigation into the molecular mechanisms of NF-B activation induced by PEDV infection.
The viral protein Npro is unique to the pestiviruses within the Flaviviridae family. After autocatalytic cleavage from the nascent polyprotein, Npro suppresses type I interferon (IFN-aalpha;/bbeta;) induction by mediating proteasomal degradation of interferon regulatory factor 3 (IRF-3). Previous studies found that the Npro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of Npro coordinating zinc by means of the amino acid residues C112, C134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-aalpha;/bbeta; induction despite the presence of an intact TRASH domain. By systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of Npro of these four isolates were related to the lack of IRF-3 degrading activity. Restoring a histidine at position 40 or both, a proline at position 17 and a lysine at position 61 based on the sequence of a functional Npro contributed to higher stability of the reconstructed Npro compared with the Npro from the Thai isolate. This leaded to enhanced interaction of Npro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of Npro are involved in the stability of Npro, in interaction of Npro with IRF-3 and subsequent degradation of IRF-3, leading to down-regulation of IFN-aalpha;/bbeta; production.
The actin rearrangement inducing factor-1 gene (arif-1) is a baculoviral early gene conserved in most alphabaculoviruses. Previous studies reported that Autographa californica nucleopolyhedrovirus (NPV) ARIF-1 protein induces filamentous actin concentration on the plasma membrane during the early stage of infection in Trichoplusia ni TN-368 cells, but its role in larval infection remains unknown. In this study, we performed behavioral screening using Bombyx mori larvae infected with B. mori NPV (BmNPV) mutants and found that larvae infected with arif-1-mutated BmNPVs did not show locomotor hyperactivity that was normally observed in BmNPV-infected larvae. arif-1-deficient BmNPVs also showed reduced pathogenicity and total viral propagation in B. mori larvae, whereas viral propagation of arif-1-deficient viruses was comparable to that of control viruses in B. mori cultured cells. An arif-1-defective BmNPV expressing green fluorescent protein (gfp) was used to monitor the progression of infection in B. mori larvae. GFP expression and quantitative reverse transcription-polymerase chain reaction analyses revealed that the infection by the arif-1-disrupted virus was significantly delayed in trachea, fat body, suboesophageal ganglion, and brain. These results indicate that BmNPV ARIF-1 enhances systemic infection in B. mori larvae.
The recent emergence of Middle East Respiratory Syndrome Coronavirus from the Middle East and the discovery of the virus from dromedary camels have boosted interest in the search of novel viruses in dromedaries. While picornaviruses are known to infect various animals, their existence in dromedaries was unknown. We describe the discovery of a novel dromedary camel enterovirus (DcEV) from dromedaries in Dubai. Among 215 dromedaries, DcEV was detected in fecal samples of four dromedaries [one (0.5%) adult dromedary and three (25%) dromedary calves] by RT-PCR. Analysis of two DcEV genomes showed that DcEV was clustered with other species of Enterovirus and were most closely related and possessed highest amino acid identities to Enterovirus E and Enterovirus F found in cattle. The G+C contents of DcEV were 45%, which differ from those of Enterovirus E and Enterovirus F (49-50%) by 4-5%. Similar to other members of Enterovirus, the 5'-UTR of DcEV possessed a putative type I internal ribosome entry site/segment. The low Ka/Ks ratios of various coding regions supported that dromedaries were the natural reservoir that DcEV was stably evolving in. These results suggest that DcEV is a novel species of the genus Enterovirus in Picornaviridae. Western blot analysis using recombinant DcEV VP1 polypeptide showed a high seroprevalence of 52% among serum samples from 172 dromedaries for IgG, concurring with its much higher infection rates in dromedary calves than adults. Further studies are important to understand the pathogenicity, epidemiology and genetic evolution of DcEV in this unique group of animals.
Classical swine fever (CSF) is one of the most important swine diseases worldwide and has tremendous socioeconomic impact. In this study, we focused on the signaling pathways of Toll-like receptors (TLRs) because of their roles in the detection and response to viral infections. To this end, two CSFV strains, namely the highly virulent CSFV Shimen strain and the avirulent C strain (a vaccine strain), were employed, and the expression of 19 immune effector genes were analyzed by real-time PCR, western blot analyses, enzyme-linked immunosorbent assay (ELISA) and flow cytometry analyses. In vitro experiments were conducted with porcine monocyte-derived macrophages (pMDMs). The results showed that the mRNA and protein levels of TLR2, TLR4 and TLR7 were upregulated in response to CSFV infection, but TLR3 remained unchanged and was downregulated after infection with the C strain and the Shimen virus, respectively. Furthermore, TLR3-mediated innate immune responses were inhibited in Shimen-strain-infected pMDMs by stimulation with poly (I:C). Accordingly, comprehensive analyses were performed to detect TLR-dependent cytokine responses and the activation of TLR signaling elements. CSFV infection induced MAPKs activation but did not elicit NF-B activation, thereby affecting the production of pro-inflammatory cytokines. The Shimen strain infection resulted in a significant activation of IRF7 and suppression of IRF3. These data provide clues for understanding the effect of CSFV infection on the TLR-mediated innate immune response and associated pathological changes.
In most forms of prion diseases blood is infectious, but the detection by immunochemistry techniques of the only available marker of infection (the misfolded prion protein, PrPTSE) in blood remains elusive. We developed a novel method for the detection of PrPTSE in blood of prion-infected rodents based on the finding that PrPTSE is associated with plasma exosomes. However, further purification of exosome on sucrose gradient was necessary for removing plasma immunoglobulins, which interfere with PrPTSE masking its detection by immunochemistry. Finally, we report that about 20% of plasma infectivity is associated with exosomes.
Herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) are human neurotropic alphaherpesviruses that cause lifelong infections in ganglia. Following primary infection and establishment of latency, HSV-1 reactivation typically results in herpes labialis (cold sores), but can occur frequently elsewhere on the body at the site of primary infection (e.g. whitlow), particularly at the genitals. Rarely, HSV-1 reactivation can cause encephalitis; however, 1/3 of the cases of HSV-1 encephalitis are associated with HSV-1 primary infection (Whitley aamp; Gnann, 2002). Primary VZV infection causes varicella (chickenpox) following which latent virus may reactivate decades later to produce herpes zoster (shingles), as well as an increasingly recognized number of subacute, acute and chronic neurological conditions. Following primary infection, both viruses establish a latent infection in neuronal cells in human peripheral ganglia. However, the detailed mechanisms of viral latency and reactivation have yet to be unraveled. In both cases latent viral DNA exists in an "end-less" state where the ends of the virus genome are joined to form structures consistent with unit length episomes and concatemers, from which viral gene transcription is restricted. In latently infected ganglia, the most abundantly detected HSV-1 RNAs are the spliced products originating from the primary Latency Associated Transcript (LAT). This primary LAT is an 8.3 kb unstable transcript from which two stable (1.5 and 2.0 kb) introns are spliced. Transcripts mapping to 12 VZV genes have been detected in human ganglia removed at autopsy; however,
Puumala virus (PUUV, carried by Myodes glareolus) co-circulates with Seewis virus (SWSV, carried by Sorex araneus) in Finland. While PUUV causes annually 1000-3000 nephropathia epidemica (NE) cases, the pathogenicity of SWSV to man is unknown. To study the prevalence of SWSV antibodies in hantavirus fever-like patients sera, we used recombinant SWSV nucleocapsid (N) protein as the antigen in enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and immunoblotting (IB). While characterizing the recombinant SWSV N protein, we observed that a polyclonal rabbit antiserum against PUUV N cross-reacts with SWSV N, and vice versa. We initially screened 486 (450 PUUV-seronegative and 36 PUUV-seropositive) samples sent to Helsinki University Hospital Laboratory for PUUV serodiagnosis during 2002 and 2007 in SWSV N IgG ELISA. In total 4.2% (19/450) of PUUV-seronegative were reactive in SWSV N IgG ELISA, and none of the tested samples (43 PUUV-seronegative, weakly reactive in SWSV IgG ELISA, and 15 random) were reactive in SWSV N IgM ELISA. None of the IgG reactions could be confirmed by IFA or IB. Furthermore, among 36 PUUV-seropositive samples, three were reactive in SWSV N IgG and ten in SWSV N IgM ELISA. One PUUV-seropositive sample reacted with SWSV N protein in IFA and four in IB. Finally, we applied competitive ELISA to confirm that the observed reactivity is actually cross-reaction rather than a true SWSV response. In conclusion, no evidence of SWSV infection was found among the 486 samples studied; however, we could demonstrate PUUV antiserum to cross-react with shrew-borne hantavirus N protein.
A novel double-stranded RNA virus was identified from the arboreal ant Camponotus yamaokai. The complete nucleotide sequence analysis of the virus revealed that the virus consists of 5,704 bp with two open reading frames (ORFs). ORF1 (3,084 nt) encodes a putative capsid protein. ORF2 (1,977 nt) encodes a viral RNA-dependent RNA polymerase (RdRp). ORF2 could be translated as a fusion with the ORF1 product by a -1 frameshift in the overlapping ORF1. Phylogenetic analyses based on the RdRp revealed that the virus from C. yamaokai is most likely a novel totivirus, but it is not closely related to the previously known totiviruses in arthropods. Transmission electron microscopy revealed isometric virus particles of approximately 30 nm in diameter in the cytoplasm, which is consistent with the characteristics of the family Totiviridae. The virus was detected by RT-PCR in all caste members and developmental stages of ants, including eggs, larvae, pupae, adult workers, alates (male and female) and queens. This study is the first report of a totivirus in a hymenopteran, and the virus was designated as Camponotus yamaokai virus (CYV).
Infection of cultured mammalian cells with African horse sickness virus (AHSV) is known to induce cell death. To date the trigger(s) of this response, the apoptotic pathways activated during AHSV infection and the functional consequences of apoptosis on the virus replication cycle have yet to be characterized. This study demonstrates that extracellular treatment of BHK-21 cells with both of the AHSV4 outer capsid proteins, VP2 and VP5, is sufficient to trigger apoptosis. Whether steps in AHSV4 replication subsequent to viral attachment are required for AHSV4-induced apoptosis was also investigated. Apoptosis was induced in BHK-21 cells infected with UV-inactivated AHSV4 and in ribavirin-treated cells infected with AHSV4. However, both AHSV4- and VP2-VP5-stimulated apoptotic responses were inhibited in the presence of the endosomal acidification inhibitors ammonium chloride and chloroquine. These results indicate that uncoating of AHSV4 virions, but not viral transcription or subsequent steps in viral replication, is required for AHSV4 to induce apoptosis in BHK-21 cells. Furthermore, this study shows that both the extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic pathways are induced following AHSV4 infection. The inhibition of caspase activity in AHSV4-infected cells does not diminish AHSV4 replication, but reduces the release and dissemination of progeny viral particles. Taken together, the data indicate that uncoating of AHSV virions is required for apoptosis induction and that apoptosis enhances virus spread and release.
The open reading frame 45 (ORF45) of the Kaposi's sarcoma-associated herpesvirus is an immediate-early phosphorylated tegument protein critical for viral escape from host immune surveillance. Its expression is up regulated by the viral replication and transcription activator (RTA), a key protein that controls the switch from latency to lytic replication. We report here that ORF45 expression was not only up regulated by RTA but ORF45 can also be degraded by RTA in a proteasome-dependent manner. The ORF45 was activated by RTA via activation of the ORF45 promoter, and the promoter region from nt 69271 to nt 69026 was involved. In chronic KSHV infected TRE-BCBL-1 RTA cells, the endogenous ORF45 protein increased dramatically after the induction of RTA expression, but then decreased rapidly after 8 h post induction. Our study suggests that RTA might control the kinetics of viral replication through fine-tuning of the level of ORF45 and other viral/host proteins.
Rotavirus strains with the uncommon genotype G10 have been detected sporadically in cases of acute gastroenteritis in humans and are thought to be transmitted zoonotically.
During 2009, ten G10P rotavirus strains were detected in the stools of children hospitalized with acute diarrhea in several pediatric hospitals in Italy. The phylogenetic analysis of the VP7 gene of the Italian G10P strains analyzed revealed nucleotide identities ranging from 94% to 99%. Molecular characterization of the 11 genomic segments was performed for one of the G10 strains, which displayed a complete genomic constellation 1 for the non-G genes. The analysis of the deduced amino acid sequences of the G10 VP7 epitopes revealed low amino acid identity with common human strains of different G genotype and with the VP7 proteins included in both anti-rotavirus commercial vaccines (Rotarix and RotaTeq). Among the common G genotypes, the VP7 amino acid sequence of the G10 strains showed a high similarity with sequences from G9 strains.
A hydrophobic cluster analysis (HCA) of the VP7 protein including amino acids from 20 to 298 was performed for the G10 Italian sequences in comparison with the major human RVA G genotypes. The HCA analysis confirmed the findings obtained previously by amino acid analysis of the VP7 epitopes, detecting a genotype-specific pattern of hydrophobicity in the hypervariable regions of the major outer capsid protein.
The 5' leader region of the HIV-1 RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA hairpin influences splicing efficiency. In addition, splicing may be modulated by binding of splicing-regulatory proteins, in particular SR proteins SF2/ASF (SRSF1), SC35 (SRSF2), SRp40 (SRSF5) and SRp55 (SRSF6), to sequence elements in the SD region. The role of RNA structure and SR protein binding in splicing control was previously studied by functional analysis of mutant SD sequences. The interpretation of these studies was complicated by the fact that most mutations simultaneously affect both structure and sequence elements. We therefore tried to disentangle the contribution of these two variables by designing more precise SD region mutants with a single effect on either the sequence or the structure. The current analysis indicates that HIV-1 splicing at the major 5'ss is modulated by both the stability of the local RNA structure and the binding of splicing regulatory proteins.
Persistent infection with oncogenic Human Papillomavirus (HPV) is a prerequisite for cervical disease development yet data regarding the host immune response to infection at the genotype level are quite limited. We created pseudoviruses bearing the major (L1) and minor (L2) capsid proteins and L1 virus-like particles (VLP) representing the reference sequence and a consensus of thirty-four European sequences of HPV51. Despite the formation of similarly-sized particles, motifs in the reference L1 and L2 genes had a profound impact on the immunogenicity, antigenicity and infectivity of these antigens. The antibody status of women exhibiting low-grade disease was similar between HPV16 and consensus HPV51 but both demonstrated discrepancies between binding and neutralizing antibody responses. These data support the use of pseudoviruses as the preferred target antigen in studies of natural HPV infection and the need to consider variation in both the L1 and L2 proteins for the appropriate presentation of antibody epitopes.
All human-immunodeficiency-virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently the viral envelope CD4-binding-site (CD4bs) is relatively conserved, making it a logical neutralizing antibody target. It is important to understand how CD4-binding site variation allows for escape from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites, whereas escape mutant selectionI dentifies viable mutants. We selected HIV-1 to escape CD4bs neutralizing MAbs b12, A12 and HJ16. Viruses that escape from A12- and b12- remained susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12-resistant virus proved that HIV-1 can escape multiple BNMAbs, but still retain sensitivity to VRC01 and the llama derived J3 nanobody. This antigenic variability could reflect that occurring in circulating viruses so studies like this could predict immunologically relevant antigenic forms of the CD4bs for inclusion in HIV-1 vaccines.
Rift Valley fever virus (RVFV) is the most pathogenic member of the genus phlebovirus within the family Bunyaviridae, and can cause severe disease in humans and livestock. Until recently, limited information has been published on the cellular host response elicited by RVFV, particularly in macrophage and dendritic cells, which play critical roles in stimulating adaptive and innate immune responses to viral infection. In an effort to define the initial response of host immunomodulatory cells to infection, primary mouse bone marrow derived macrophages (BMDM) were infected with the pathogenic RVFV strain ZH501, or attenuated strains MP-12 or MP-12 based Clone13 type (rMP12-C13 type), and cytokine secretion profiles examined. The secretion of Th1-associated antiviral cytokines, chemokines and various interleukins increased rapidly after infection with the attenuated rMP12-C13 type RVFV, which lacks a functional NSs virulence gene. In comparison, infection with live-attenuated MP-12 encoding a functional NSs gene appeared to cause a delayed immune response, while pathogenic ZH501 ablates the immune response almost entirely. These data demonstrate that NSs can inhibit components of the BMDM antiviral response and supports previous work indicating that NSs can specifically regulate type I interferon response in macrophages. Furthermore, our data demonstrate that genetic differences between ZH501 and MP-12 reduce the ability of MP-12 to inhibit antiviral signaling and subsequently reduce virulence in BMDM demonstrating that viral components other than NSs play a critical role in regulating the host response to RVFV infection.
The immunological effect of porcine reproductive and respiratory syndrome disease virus (PRRSV) vaccines is thought to be influenced by a variety of host factors, in which ADE (antibody-dependent enhancement) of infection is one crucial factor. Here we assessed the mechanism of ADE of PRRSV infection. First we found that subneutralizing serum can induce ADE of PRRSV infection in porcine alveolar macrophages (PAMs). Quantitative PCR, Western-blot, and flow cytometry revealed that CD16 is the most abundant Fc receptor (FcR) expressed on the surface of PAMs; thus, the role of CD16 in ADE of PRRSV infection was examined in PAMs. By using functional blocking antibodies, we demonstrated that CD16 is involved in the enhanced virus production in PRRSV-antibody immune complex-infected PAMs. Because PAMs co-express different FcR isoforms, we evaluated the effects of CD16 in FcR-nonbearing cells by transfection. Using these engineered cells, we found that CD16 can specifically bind to the PRRSV-antibody immune complex, subsequently mediate the internalization of the virus and result in the generation of progeny virus. We also showed that the efficient expression of CD16 requires the association of the FcR -chain. Altogether, our findings provide significant new insights into PRRSV infection that can be enhanced by CD16-mediated PRRSV-antibody immune complex. This CD16-mediated ADE may induce a shift in PRRSV tropism towards CD16-expressing cells, distributing virus to more organs during virus infection.
In recent years bats have been identified as a natural reservoir for a diverse range of viruses. Nelson Bay orthoreovirus (NBV) was first isolated from the heart blood of a fruit bat (Pteropus poliocephalus) in 1968. While the pathogenesis of NBV remains unknown, other related members of this group have caused acute respiratory disease in humans. Thus the potential for NBV to impact human health appears plausible. Here, to increase our knowledge of NBV we examined the replication and infectivity of NBV using different mammalian cell lines derived from bat, human, mouse and monkey. All cell lines supported the replication of NBV, however L929 cells showed a greater than 2-log reduction in virus titre compared to the other cell lines. Furthermore, NBV did not induce major cytopathic effects in the L929 cells, as was observed in other cell lines. Interestingly, the related Pteropine orthoreovirus, Pulau virus (PulV) and Melaka virus (MelV) in L929 were able to replicate to high titres in L929 cells but infection resulted in reduced cytopathic effect. Our study demonstrates a unique virus-host interaction between NBV and L929 cells, where cells effectively control viral infection/replication and limit the formation of syncytia. By elucidating the molecular mechanisms that control this unique relationship, important insights will be made into the biology of this fusogenic virus.
Grass carp reovirus (GCRV) is a member of the Aquareovirus genus in the family Reoviridae, and contains five core proteins (VP1-VP4, VP6) and two outer capsid proteins (VP5 and VP7) in its particle. Previous studies have revealed that the outer capsid proteins of reovirus are responsible for initiating infection, but the mechanism is poorly understood. Using baculovirus-expressed VP5 and VP7 to recoat purified cores, in vitro assembly of GCRV was achieved in this study. Recoated GCRV (R-GCRV) closely resembled native GCRV (N-GCRV) in particle morphology, protein composition and infectivity. Similar to N-GCRV, the infectivity of R-GCRV could be inhibited by treating cells with the weak base NH4Cl. In addition, recoated particles (VP5N42A/VP7 R-GCRV) carrying an Asn to Ala substitution at residue 42 of VP5 were no longer infectious. These results provide strong evidence that autocleavage of VP5 is critical for aquareovirus to initiate efficient infection.
Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus prM and E genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examine the physicochemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37˚C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate (BMP) and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on the timescale of 7-20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process.
Hepatitis C virus (HCV) represents a significant global health burden. Viral replication is thought to occur in close association with remodelled host cell membranes with non-structural protein 4B (NS4B) being a key player in this process. NS4B is a poorly characterized integral membrane protein, which has been reported to be palmitoylated at its carboxy-terminal end. In order to extend this observation and to establish a functional role for NS4B palmitolylation, we sought to determine the status of this post-translational modification when the protein was expressed as part of a functional viral replicase. We performed direct metabolic labelling and polyethylene glycol-maleimide palmitoylation reporter assays on NS4B expressed in cells containing subgenomic replicons and infectious viral RNA. In a vaccinia virus-based expression system NS4B palmitoylation was detected in a genotype-dependent manner. However, in spite of the high sensitivity of the methods used, no NS4B palmitoylation was found in physiologically more relevant systems. Thus, NS4B palmitoylation is most likely dispensable for HCV RNA replication.
In the genus Orthoreovirus, five species, Mammalian orthoreovirus, Avian orthoreovirus (ARV), Nelson Bay orthoreovirus (NBV), Baboon orthoreovirus and Reptilian orthoreovirus, have been identified. Their genomes each consist of ten double-stranded RNA segments. A novel orthoreovirus was isolated from the hemorrhagic intestine of a dead brown-eared bulbul in Japan. The virus formed syncytia in Caco-2 and Vero cells. Electron microscopy revealed non-enveloped capsids of approximately 70 nm in diameter, which are characteristic of reoviruses. Complete genomic sequences were determined. The S1 segment was tricistronic and encoded three proteins, p10, p17 and C, as in the two species ARV and NBV. Sequence and phylogenetic analyses showed that the virus was similar to ARV and NBV but was located on a phylogenetic branch different from that of the two species. The virus had the closest phylogenetic relationship to two reovirus strains, SSRV from a Steller sea lion in Canada and PsRV Ge01 from a psittaciform bird in Europe. The ten RNA segments had a 3' pentanucleotide sequence (UCAUC-3') conserved among all members of the genus Orthoreovirus and a unique 5' terminal heptasequence (5'-GCUUUUC) that was the same as those of SSRV and PsRV Ge01. These results suggest that the novel virus might form a new species with the two strains in the genus Orthoreovirus.
The genome of the Rat Cytomegalovirus (RCMV) English isolate (MuHV-8) differs significantly from the RCMV Maastricht isolate (MuHV-2) and other Cytomegaloviruses in its size as well as base composition and genomic content. The analysis of a newly isolated MuHV-8, the RCMV-Berlin isolate, revealed that the two MuHV-8 isolates are highly similar in size and gene content, indicating that the smaller genome size (202,946 bp) compared to other known CMVs is not the result of an accidental deletion during passage in tissue culture. Surprisingly, MuHV-8 proteins show overall greater similarity to their mouse CMV (MuHV-1) than to their rat CMV (MuHV-2) orthologues. Phylogenetic analyses of conserved viral genes showed that the two MuHV-8 isolates are from the same species and represent a unique clade that is distinct from other rodent CMVs.
Oropouche virus (OROV) is a public health threat in South America, and in particular Northern Brazil, causing frequent outbreaks of febrile illness. Using a combination of deep sequencing and Sanger sequencing approaches we have determined complete genome sequences of eight clinical isolates that were obtained from patient sera during an Oropouche fever outbreak in Amapa state, northern Brazil in 2009. We also report complete genome sequences of two OROV reassortants isolated from two marmosets in Minas Gerais state, southeast Brazil in 2012 that contain a novel M genome segment. Interestingly, all ten isolates posses a 947 nucleotide long S segment that lacks 11 residues in the S segment 3' UTR compared to the recently redetermined Brazilian prototype OROV strain BeAn19991. OROV maybe circulating more widely in Brazil and in the non-human primate population than previously appreciated and the identification of yet another reassortant highlights the importance of bunyavirus surveillance in South America.
HIV prevalence has decreased in Uganda since the 1990s, but remains substantial within high-risk groups. Here, we reconstruct the history and spread of HIV subtypes A1 and D in Uganda and explore the transmission dynamics in high-risk populations. We analysed HIV pol sequences from female sex workers in Kampala (n=42), Lake Victoria fisher-folk (n=46), and a rural clinical cohort (n=74), together with publicly available sequences from adjacent regions in Uganda (n=412) and newly generated sequences from samples taken in Kampala in 1986 (n=12). Of the sequences from the 3 Ugandan populations, 60 (37.1%) were classified as subtype D, 54 (33.3%) as subtype A1, 31 (19.1%) as A1/D recombinants, 6 (3.7%) as subtype C, 1 (0.6%) subtype G and 10 (6.2%) other recombinants. Among the A1/D recombinants we identified a new candidate circulating recombinant form. Phylodynamic and phylogeographic analyses using BEAST indicated that the Ugandan epidemics originated in 1960 (1950-1968) for subtype A1 and 1973 (1970-1977) for D, in rural southwestern Uganda with subsequent spread to Kampala. They also showed extensive interconnection with adjacent countries. The sequence analysis shows both epidemics grew exponentially during the 1970-1980s and decreased from 1992; which agrees with HIV prevalence reports in Uganda. Inclusion of sequences from the 1980s indicated the origin of both epidemics was more recent than expected and substantially narrowed the confidence intervals in comparison to previous estimates. We identified 3 transmission clusters and 10 pairs, none of them including patients from different populations, suggesting active transmission within a structured transmission network.
Lymphoma is the most common haematopoietic malignancy in dogs but little is known about the aetiology of this heterogeneous group of cancers. In humans, the Epstein-Barr virus (EBV) is associated with several lymphoma subtypes. Recently, it has been suggested that EBV or an EBV-like virus is circulating in dogs. We therefore investigated whether EBV, or a novel herpesvirus, is associated with canine lymphoma using both serological and molecular techniques. In an assay designed to detect antibodies to EBV viral capsid antigens, 41% of dogs were positive. Dogs with cancers, including lymphoma, were more frequently positive than controls, but no particular association with B-cell lymphoma was noted. EBV-specific RNA and DNA sequences were not detected in lymphoma tissue by in situ hybridisation or PCR and herpesvirus genomes were not detected using multiple degenerate PCR assays with the ability to detect novel herpesviruses. We therefore found no evidence that herpesviruses are directly involved in common types of canine lymphoma although cannot exclude the presence of an EBV-like virus in the canine population.
Lassa virus (LASV) small zinc-finger protein (Z), which contains two L-domain motifs, plays a central role in virus budding. Here, we report that coexpression of glycoprotein (GPC) alters the requirements of cholesterol, but not the L-domains and host factor, Tsg101, for Z-induced virus-like particle (VLP) production. Especially, the cholesterol requirement for VLP production was cell type-dependent. In addition, GPC was important for colocalization of Z with CD63, a late endosome marker. We also found that the N-terminal region (amino acids 3 - 10) of Z was critical for its myristoylation and VLP production. These findings will contribute to our understanding of LASV assembly and budding.
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the leading swine pathogens causing tremendous economic loss to the global swine industry due to its virulence, pathogenesis, infectivity and transmissibility. Although formally recognized only two and half decades ago, molecular dating estimation indicates a more ancient evolutionary history which involved divergence into two genotypes (type 1 and type 2) prior to the "initial" outbreaks of the late 1980s. Type 2 PRRSV primarily circulates in North America and Asia. The relatively greater availability of sequence data from this genotype from widespread geographic territories has enabled a better understanding of the evolving genotype. However, there are a number challenges in terms of the vastness of data available and what it indicates in the context of viral diversity. Accordingly, we revisit the mechanisms by which PRRSV generates variability, describe a means of organizing type 2 diversity captured in voluminous ORF5 sequences in a phylogenetic framework and provide a holistic view of known global type 2 diversity in the same setting. The consequences of the expanding diversity for control measures like vaccination are discussed as well as the contribution of modified live vaccines to the circulation of field isolates. We end by highlighting some limitations of current molecular epidemiology studies in relation to inferring PRRSV diversity and what steps can be taken to overcome these and additionally enable PRRSV sequence data to be informative of viral phenotypic traits like virulence.
The viral tegument is a layer of proteins between the herpesvirus's capsid and its outer envelope. According to phylogenetic studies, only a third of these proteins are conserved among the three herpesvirus subfamilies. Although some of these tegument proteins have been studied in more detail, the structure and function for the majority of them are still poorly characterized.
VP22 from herpes simplex virus 1 (HSV-1), an alpha herpesvirus, is a highly interacting tegument protein that has been associated with tegument assembly. We have determined the crystal structure of the conserved core domain of VP22, which reveals an elongated dimer with several potential protein-protein interaction regions and a peptide-binding site. The structure provides us with the structural basics to understand the numerous functional mutagenesis studies of VP22 found in the literature. It also establishes an unexpected structural homology to the tegument protein ORF52 from the murine herpesvirus 68 (MHV-68), a gamma herpesvirus. Homologs for both VP22 and ORF52 have been identified in their respective subfamilies. Although there is no obvious sequence overlap in the two subfamilies, this structural conservation provides compelling structural evidence for shared ancestry and functional conservation.
In Escherichia coli, the major poly(A) polymerase (PAP I), is encoded by the pcnB gene. In this report, a significant impairment of lysogenization by Shiga toxin-converting (Stx) bacteriophages (Phi24B, 933W, P22, P27, and P32) is demonstrated in host cells with a mutant pcnB gene. Moreover, lytic development of these phages after both, infection and prophage induction, was significantly less efficient in the pcnB mutant than in the wild-type host. The increase in DNA accumulation of the Stx phages was lower under conditions of defective RNA polyadenylation. Although shortly after prophage induction the levels of mRNAs of most phage-borne early genes were higher in the pcnB mutant, at subsequent phases of the lytic development a drastically decreased abundance of certain mRNAs, including those derived from the N, O and Q genes, was observed in PAP I-deficient cells. All of these effects observed in the pcnB cells were significantly more strongly pronounced in the Stx phages than in bacteriophage lambda. Abundance of mRNA derived from the pcnB gene was drastically increased shortly (20 min) after prophage induction by mitomycin C, and decreased back after next 20 min, while no such changes were observed in non-lysogenic cells treated with this antibiotic. This prophage induction-dependent transient increase in pcnB transcript abundance may explain the polyadenylation-caused coordinated regulation of expression of phage genes.
Human cytomegalovirus (CMV) is the most common infectious cause of congenital birth defects in developed countries. Studies of infected amniotic fluid and placentae show CMV infection leads to a pro-inflammatory shift in cytokine profiles with implications for pathogenesis of fetal disease. Enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and real time PCR assays were used to investigate CCL2 (Monocyte chemotactic protein-1 or MCP-1) and tumour necrosis factor alpha (TNF-aalpha;) changes following CMV infection of human fibroblasts, as well as following transient expression of CMV gene products in HeLa cells. Infection of human fibroblasts with CMV AD169 resulted in increased cytoplasmic and extracellular expression of CCL2 during early stages of infection, followed by marked downregulation of the chemokine at late times. Induction of CCL2 was not observed with CMV clinical strain Merlin, consistent with postulated immune evasion potential of this genetically intact wild type strain. Comparison between live and UV-irradiated virus infections showed that changes in CCL2 levels were a direct response to active CMV replication. There were no significant changes in TNF-aalpha; expression during a parallel time course of CMV infection. In transient transfection assays, overexpression of CMV tegument protein pp71 resulted in intracellular and extracellular upregulation of CCL2 protein. Messenger RNA analysis showed pp71-induced elevation in CCL2 was mediated through transcriptional upregulation. These data show CMV-induced upregulation of CCL2 during early stages of infection is mediated, at least in part, by stimulation of viral pp71, which may contribute to viral pathogenesis through enhanced virus dissemination.
Human cerebral microvascular endothelial cells (hCMEC/D3 cell line) form a steady polarized barrier when cultured in vitro on a permeable membrane. Their susceptibility to enterovirus (EV) strains was analysed to investigate how these viruses may cross the blood-brain barrier. A sample of 88 virus strains was selected on phylogenetic features among 44 epidemiologically relevant types of the four EV species A-D. The EV-A71 genome was replicated at substantial rates while the infectious virus was released at extremely low but sustained rates at both barrier sides for at least 4 days. EV-A71 antigens were detected in a limited number of cells. The properties of the endothelial barrier (structure and permeability) remained intact throughout infection. The chronic EV-A71 infection was in sharp contrast with the productive infection of cytolytic EVs (e.g. echoviruses 6 and 30). The hCMEC/D3 barriers infected with the latter EVs exhibited elevated proportions of apoptotic and necrotic cells, which resulted in major injuries to the endothelial barriers with dramatic increase of paracellular permeability and virus crossing to the abluminal side. The following intracellular rearrangements were also seen: early destruction of the actin cytoskeleton, remodelling of intracellular membranes, and reorganization of the mitochondrion network in a small cluster near the perinuclear space.
The oldest porcine circovirus type 2 (PCV2) sequence dates back to 1962 and is among several hundreds of publically available PCV2 sequences. Despite this resource, limited studies have investigated the global genetic diversity of PCV2. To evaluate the phylogenetic relationship of PCV2 strains, 1680 PCV2 open reading frame 2 (ORF2) sequences were compared and analyzed by methods of Neighbor-Joining, Maximum Likelihood, Bayesian inference and network analysis. Four distinct clades were consistently identified and included PCV2a, PCV2b, PCV2c and PCV2d; the p-distance between PCV2d and PCV2b was 0.055pplusmn;0.008, larger than the PCV2 genotype-definition cut-off of 0.035, supporting PCV2d as an independent genotype. Among the 1680 sequences, 278-285 (16.5-17%) were classified as PCV2a, 1007-1058 (59.9-63%) as PCV2b, three (0.2%) as PCV2c, and 322-323 (19.2%) as PCV2d with the remaining 12-78 sequences (0.7-4.6%) classified as intermediate clades or strains by the various methods. Classification of strains to genotypes differed based on the number of sequences used for the analysis indicating that sample size is important when determining classification and assessing PCV2 trends and shifts. PCV2d was initially identified in 1999 samples in Switzerland, now appears to be widespread in China and has been present in North America since 2012. During 2012-2013, 37% of all investigated PCV2 sequences from U.S. pigs were classified as PCV2d and overall data analysis suggests an ongoing genotype shift from PCV2b towards PCV2d. The present analyses further indicate that PCV2d has emerged approximately 20 years ago.
Hepatocellular carcinoma (HCC) carries a dismal prognosis, with advanced disease being resistant to both radiotherapy and conventional cytotoxic drugs, whilst anti-angiogenic drugs are marginally efficacious. Oncolytic viruses (OV) offer the promise of selective cancer therapy through direct and immune-mediated mechanisms. The premise of OV lies in their preferential genomic replication, protein expression and productive infection of malignant cells. Numerous oncolytic viruses are being tested in pre-clinical models of HCC, with good evidence of direct and immune-mediated anti-tumour efficacy. Efforts to enhance the performance of these agents have concentrated on engineering OV cellular specificity, immune evasion, enhancing anti-tumour potency and improving delivery. The lead agent in HCC clinical trials, JX-594, a recombinant Wyeth strain Vaccinia virus has demonstrated evidence for significant benefit and earned orphan drug status. Thus, JX-594 appears to be transcending the barrier between novel laboratory science and credible clinical therapy. Otherwise, relatively few other OV have entered clinical testing, a hurdle that must be overcome if significant progress is to be made in this field.
This review summarises the pre-clinical and clinical experience of OV therapy in the difficult-to-treat area of HCC.
Flaviviruses are a group of single-stranded positive sense RNA viruses that generally circulate between arthropod vectors and susceptible vertebrate hosts, producing significant human and veterinary disease burdens. Intensive research efforts have broadened scientific understanding of the replication cycles of these viruses and have revealed several elegant and tightly coordinated post-translational modifications that regulate the activity of viral proteins. The three structural proteins in particular - capsid (C), pre-membrane (prM), and envelope (E) - are subjected to strict regulatory modifications as they progress from translation through virus particle assembly and egress. The timing of proteolytic cleavage events at the C-prM junction directly influences the degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E proteins are also important for particle assembly as prM acts as a chaperone facilitating correct conformational folding of E. It is only once prM/E heterodimers form that these proteins may be efficiently secreted. The addition of branched glycans to the prM and E proteins during virion transit also plays a key role in modulating the rate of secretion, pH sensitivity, and infectivity of flavivirus particles. The insights gained from research into post-translational regulation of structural proteins are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make attractive targets for the development of novel therapeutics.
Non-structural protein 1 (NS1) binds siRNAs and suppresses RNA silencing in plants, but the underlying mechanism of this suppression is not well understood. Therefore, here we characterized NS1 encoded by the avian influenza virus H9N2. The NS1 protein was able to suppress RNA silencing induced by either sense RNA or double-stranded RNA (dsRNA). Using deletion and point mutants, we discovered that the first 70 residues of NS1 can suppress RNA silencing triggered by sense transgene, but this sequence is not sufficient to block dsRNA-induced silencing. Any mutations of two arginine residues (35R and 46R) of NS1, which contribute to its homodimeric structure, cause the loss of its silencing suppression activity. These results indicate that the region after residue 70 of NS1 is essential for the repression activity on dsRNA-induced RNA silencing, and that the dimeric structure of NS1 plays a critical role in its RNA silencing suppression function.
Understanding the diversity of henipaviruses and related viruses is important in determining the viral ecology within flying-fox populations and assessing the potential threat posed by these agents. This study sought to identify the abundance and diversity of previously unknown paramyxoviruses in Australian flying-fox species (Pteropus alecto, P. scapulatus, P. poliocephalus and P. conspicillatus) and in the Christmas Island species P. melanotus natalis. Using a degenerative RT-PCR specific for the L gene of known species of Henipavirus and two closely related paramyxovirus genera Respirovirus and Morbillivirus, we identified an abundance and diversity of previously unknown paramyxoviruses (UPV), with a representative 31 UPVs clustering in eight distinct groups (100 UPV/495 samples). No new henipaviruses were identified. The findings are consistent with a hypothesis of co-evolution of paramyxoviruses and their flying-fox hosts. Quantification of the degree of co-speciation between host and virus (beyond the scope of this study) would strengthen this hypothesis.
In 2011, neurological disease was reported in a herd of goats (Capra hircus) in Asturias, Spain. Initial sequencing identified the causative agent as louping ill virus (LIV). Subsequently, with the application of whole genome sequencing and phylogenic analysis, empirical data demonstrates that the LIV-like virus detected is significantly divergent from LIV and Spanish sheep encephalitis virus (SSEV). This virus encoded an amino acid sequence motif at the site of a previously identified marker for differentiating tick-borne flaviviruses, that was shared with a virus previously isolated in Ireland in 1968. The significance of these observations reflects the diversity of tick-borne flaviviruses in Europe. These data also contribute to our knowledge of the evolution of tick-borne flaviviruses and could reflect the movement of viruses throughout Europe. Based on these observations, the proposed name for this virus is Spanish goat encephalitis virus (SGEV), to distinguish it from SSEV.
Central to the development of new treatments for human immunodeficiency virus 1 (HIV-1) is a more thorough understanding of the viral lifecycle and the cellular co-factors upon which this depends. Targeting cellular proteins and their interaction with HIV-1 has the potential to reduce the problem of emerging viral resistance to drugs since mutational escape is more difficult. We performed a short interfering RNA library screen targeting 59 cellular RNA helicases, assessing the effect on both viral capsid protein production and infectious virion formation. Five RNA helicases were identified which, when knocked down, reproducibly decreased infectious particle production - DDX5, DDX10, DDX17, DDX28 and DDX52. Two of these proteins (DDX5 and DDX17) have known roles in HIV-1 replication. A further helicase (DDX10) was a positive hit from a previous genome-wide siRNA screen, however DDX28 and DDX52 have not previously been implicated as essential cofactors for HIV-1.
Hepatitis C virus (HCV) entry is a sequential and multi-step process that includes receptor interactions followed by pH-dependent membrane fusion. Specific and conserved histidine residues on the viral envelope proteins are involved in most pH-induced virus entries. In the case of HCV, some conserved histidines on the E1 and E2 proteins have been investigated in HCV pseudotype particle (HCVpp) systems. However, the roles of these histidines in cell-culture derived HCV particle (HCVcc) systems remain unclear due to the different aspects of the viral life cycle emphasized by the two systems. In this study, the role of two conserved histidines (His490 and His621, located in domain II and domain III of E2, respectively) in HCV infection was evaluated in the context of JFH-1-based HCVcc using alanine substitutions. The infectivity of the H490A mutant decreased in spite of comparable initial RNA replication, protein expression, and assembly efficiency as wild-type virus. The H621A mutant did not affect viral proteins expression, but exhibited no obvious infectivity; there were fewer core proteins in the culture supernatant compared with wild-type virus, indicating the partially deficient virus assembly. The HCV receptor CD81-binding ability of the two mutant E2s was assessed further using enzyme immunoassays. The CD81-binding activity of H490A-E2 was reduced, and H621A-E2 was unable to bind to CD81. These data reveal the crucial role played by His490 and His621 in HCV infection, particularly during CD81 binding in cell entry. These results also contribute to the mechanical identification of the histidines involved in pH-dependent HCV entry.
Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV replication and contribute to the hepatic reservoir. We screened cholangiocytes along with a panel of cholangiocarcinoma-derived cell lines for their ability to support HCV entry and replication. While primary cholangiocytes were refractory to infection and lacked expression of several entry factors, two cholangiocarcinoma lines, CC-LP-1 and Sk-ChA-1, supported efficient HCV entry; furthermore, Sk-ChA-1 cells supported full virus replication. In vivo cholangiocarcinomas expressed all of the essential HCV entry factors; however, cholangiocytes adjacent to the tumor and in normal tissue showed a similar pattern of receptor expression to ex vivo isolated cholangiocytes, lacking SR-BI expression, explaining their inability to support infection. This study provides the first report that HCV can infect cholangiocarcinoma cells and suggests that these heterogeneous tumors may provide a reservoir for HCV replication in vivo.
Rotavirus is a leading cause of severe gastroenteritis in infants worldwide. Rotavirus nonstructural protein 1 (NSP1) is a virulence factor that inhibits innate host immune responses. NSP1 from some rotaviruses targets host interferon response factors (IRFs), leading to inhibition of type I interferon expression. A few rotaviruses encode an NSP1 that inhibits the NF-B pathway by targeting bbeta;-TrCP, a protein required for IB degradation and NF-B activation. Available evidence suggests that these NSP1 properties involve proteosomal degradation of target proteins. We show here that NSP1 from several human rotaviruses and porcine rotavirus CRW-8 inhibits the NF-B pathway, but cannot degrade IRF3. Furthermore, bbeta;-TrCP levels were much reduced in cells infected with these rotaviruses. This provides strong evidence that bbeta;-TrCP degradation is required for NF-B pathway inhibition by NSP1 and demonstrates the relevance of bbeta;-TrCP degradation to rotavirus infection. C-terminal regions of NSP1, including a serine-containing motif resembling the bbeta;-TrCP recognition motif of IB, were required for NF-B inhibition. CRW-8 infection of HT-29 intestinal epithelial cells induced significant levels of IFN-bbeta; and CCL5 but not IL-8. This contrasts with monkey rotavirus SA11-4F, whose NSP1 inhibits IRF3 but not NF-B. Substantial amounts of IL-8 but not IFN-bbeta; or CCL5 were secreted from HT-29 cells infected with SA11-4F. Our results show that human rotaviruses commonly inhibit the NF-B pathway by degrading bbeta;-TrCP and thus stabilising IB. They suggest that NSP1 plays an important role during human rotavirus infection by inhibiting the expression of NF-B-dependent cytokines, such as IL-8.
The reverse zoonotic events that introduced the 2009 pandemic influenza virus into pigs have drastically increased the diversity of swine influenza viruses in Europe. The pandemic potential of these novel reassortments is still unclear, necessitating enhanced surveillance of European pigs with additional focus on risk assessment of these new viruses. In this study, four European swine influenza viruses were assessed for their zoonotic potential. Two of the four viruses were enzootic viruses of subtype H1N2 (with avian-like H1) and H3N2 and two were new reassortants, one with avian-like H1 and human-like N2 and one with 2009 pandemic H1 and swine-like N2. All viruses replicated to high titers in nasal wash- and nasal turbinate samples from inoculated ferrets and transmitted efficiently by direct contact. Only the H3N2 virus transmitted to naiiuml;ve ferrets via the airborne route. Growth kinetics using a differentiated human bronchial epithelial cell line showed that all four viruses were able to replicate to high titers. Further, the viruses revealed preferential binding to the aalpha;2,6-silalylated glycans and investigation of the antiviral susceptibility of the viruses revealed that all were sensitive to neuraminidase inhibitors. These findings suggest that these viruses have the potential to infect humans and further underline the need for continued surveillance as well as biological characterization of new influenza A viruses.
Parapoxvirus (PPV) infections are of worldwide importance particularly in sheep and goat herds. Due to the zoonotic potential of all PPV species they are a permanent threat to human health as well. The virus is also known to affect wildlife as reported for pinnipeds, red deer and several other wild ruminants. PPVs found in red deer have been claimed as a unique species according to certain genomic features. So far affection of wildlife has been recognized because of clinical manifestation such as inflammation, stomatitis or typical pox-like lesions in the skin or mucous membranes. Here we report about targeted molecular diagnostics for the presence of PPV genomes in tonsil swabs of apparently healthy red deer in the Bavarian Alps. Out of 1764 swabs, 0.79% were tested positive for PPV genome presence. From one sample PPV was successfully isolated in cell culture. This virus became subject of complete genome characterization using next generation sequencing (NGS) and various subsidiary PCR protocols. Strikingly, about a quarter of all ORFs were found to be larger than the corresponding ORFs in the reference PPV genome sequences used for comparison. To our knowledge this is the first genome-wide analysis that confirms red deer PPV as a unique species within the PPV genus in Europe. Persistence of PPV in Alpine red deer indicates a source for virus transmission to susceptible livestock and hunters. The findings provide a further example for wildlife animals playing an important role as an inconspicuous reservoir of zoonotic diseases.
Epizootic hemorrhagic disease virus (EHDV), an Orbivirus not previously reported in Israel, was isolated from Israeli cattle during a "bluetongue like" disease outbreak in 2006. To ascertain the origin of this new virus, three isolates from the outbreak were fully sequenced and compared with available sequences. While the L2 gene segment clustered with the Australian EHDV serotype 7 (EHDV-7) reference strain, most of the other segments were clustered with EHDV isolates of African/Middle East origin specifically, Bahrain, Nigeria and South Africa. The M6 gene had genetic relatedness to the Australian/Asian strains but with the limited data available the significance of this relationship is unclear. Only one EHDV-7 L2 sequence is available and since this gene encodes the serotype specific epitope, the relationship of these EHDV-7 L2 genes to an Australian EHDV-7 reflects the serotype association, not necessarily the origin. The genetic data indicates that the strains affecting Israel in 2006 may have been related to similar outbreaks that occurred in North Africa that same year. This finding also supports the hypothesis that EHDV entered Israel during 2006 and was not present there before this outbreak.
The knowledge of viral shedding patterns and viremia in the reservoir host species is a key factor in assessing the human risk of zoonotic viruses. The shedding of hantaviruses (family Bunyaviridae) by their host rodents has widely been studied experimentally, but rarely in natural settings. Here we present the dynamics of Puumala (PUUV) hantavirus shedding and viremia in naturally infected, wild bank voles (Myodes glareolus). In a monthly capture-mark-recapture study, we analyzed 18 bank voles for the presence and relative quantity of PUUV RNA in the excreta and blood from 2 months before up to 8 months after seroconversion.
The proportion of animals shedding PUUV RNA in saliva, urine, and feces peaked during the first month after seroconversion, but continued throughout the study period with only a slight decline. The quantity of shed PUUV in RT-PCR positive excreta was constant over time. In blood, PUUV RNA was present for up to 7 months but both the probability of viremia and the virus load declined by time.
Our findings contradict the current view of a decline in virus shedding after the acute phase and a short viremic period in hantavirus infection - an assumption widely adopted into current epidemiological models. We suggest the life-long shedding as a means of hantaviruses to survive over host population bottlenecks, and to disperse in fragmented habitats where local host and/or virus populations face temporary extinctions. Our results indicate that the kinetics of pathogens in wild hosts may considerably differ from those observed in laboratory settings.
Hantaviruses are emerging zoonotic pathogens that can cause severe disease in humans. Clinical observations suggest that human immune components contribute to hantavirus-induced pathology. To address this issue we generated mice with a humanized immune system. Hantavirus infection of these animals resulted in systemic infection associated with weight loss, decreased activity, ruffled fur and inflammatory infiltrates of lung tissue. Intriguingly, after infection humanized mice harboring Human Leukocyte Antigen (HLA) class I-restricted human CD8+ T cells started to lose weight earlier (day 10) than HLA class I-negative humanized mice (day 15). Moreover, in these mice the number of human platelets dropped by 77% whereas the number of murine platelets did not change, illustrating how differences between rodent and human hemato-lymphoid systems may contribute to disease development. To our knowledge this is the first description of a humanized mouse model of hantavirus infection, and our results indicate a role for human immune cells in hantaviral pathogenesis.
Feline coronavirus (FCoV) infections are endemic amongst cats worldwide. The majority of infections are asymptomatic, or result only in mild enteric disease. However, approximately 5% of cases develop feline infectious peritonitis (FIP), a systemic disease that is a frequent cause of death in young cats. In this study, we report the complete coding genome sequences of six FCoVs; three from fecal samples from healthy cats and three from tissue lesion samples from cats with confirmed FIP. The six samples were obtained over a period of eight weeks at a single-site cat rescue and rehoming center in the UK. We found amino acid differences are located at 44 positions across an alignment of the six virus translatomes and, at 21 of these positions, the differences fully or partially discriminate between the genomes derived from the fecal samples and the genomes derived from tissue lesion samples. In this study, two amino acid differences fully discriminate the two classes of genomes; these are both located in the S2 domain of the virus surface glycoprotein gene. We also identified deletions in the 3c protein ORF of genomes from two of the FIP samples. Our results support previous studies that implicate S protein mutations in the pathogenesis of FIP.
The coding sequences of five human enterovirus (HEV)-C genotype 105 strains recovered in Italy, Romania and Burundi from patients with upper and lower respiratory tract infections were analyzed and phylogenetically compared with other circulating HEV-C strains. The EV-C105 was closely related with EV-C109 and EV-C118 strains. The European strains were similar to other circulating EV-C105 strains, while the two African EV-C105 clustered in a separate bootstrap-supported (ggt;0.90) branch of P2 and P3 region trees. Minor inconsistencies in the clustering pattern of EV-C105 in the capsid region (P1) and non-capsid region (P3) suggest that recombination may have occurred in EV-C105 group B viruses. In conclusion, phylogenetic analysis revealed the circulation of two distinct EV-C105 lineages in Europe and Africa. A different pattern of evolution could be hypothesized for the two EV-C105 lineages.
Human Pegivirus (HPgV; originally called GB virus C/hepatitis G virus) is an RNA virus within the Pegivirus genus of the Flaviviridae that commonly causes persistent infection. Worldwide, approximately 750 million people are actively infected (viremic) and an estimated 0.75 to 1.5 billion people have evidence of prior HPgV infection. No causal association between HPgV and disease has been identified; however, several studies describe a beneficial relationship between persistent HPgV infection and survival in HIV-infected individuals. The beneficial effect appears to be related to a reduction in host immune activation. HPgV replicates well in vivo (ggt; 1x107 genome copies detected per ml plasma); however, the virus grows poorly in vitro and systems to study this virus are limited. Consequently, mechanisms of viral persistence and host immune modulation remain poorly characterized, and the primary permissive cell type(s) has not yet been identified. HPgV RNA is found in liver, spleen, bone marrow, and peripheral blood mononuclear cells (PBMCs), including T and B lymphocytes, NK cells, and monocytes, although the mechanism of cell-to-cell transmission is unclear. HPgV RNA is also present in serum microvesicles with properties of exosomes. These microvesicles are able to transmit viral RNA to PBMCs in vitro, resulting in productive infection. This review summarizes existing data on HPgV cellular tropism, the effect of HPgV on immune activation in various PBMCs and discusses how this may influence viral persistence. We conclude that an increased understanding of HPgV replication and immune modulation may provide insights into persistent RNA viral infection of humans.
The major cell type supporting hepatitis C virus (HCV) infection is the hepatocyte, however, most reports studying viral entry and replication utilize transformed hepatoma cell lines. We demonstrate that HCV pseudoparticles (HCVpp) infect primary hepatocytes with comparable rates to hepatoma cells, demonstrating the limited variability in donor hepatocytes to support HCV receptor-glycoprotein dependent entry. In contrast, we observed a 2-log range in viral replication between the same donor hepatocytes. We noted that cell proliferation augments pseudoparticle reporter activity and arresting hepatoma cells yields comparable levels of infection to hepatocytes. This study demonstrates comparable rates of HCVpp entry into primary hepatocytes and hepatoma cells, validating the use of transformed cells as a model system to study HCV entry.
There are three conserved N-linked glycosites, namely, Asn10, Asn23, and Asn286, at the stem region of hemagglutinin (HA) in H5N1 avian influenza viruses (AIVs). To understand the effect of glycosylation in the stem domain of HA on the biological characteristics of H5N1 AIVs, we used site-directed mutagenesis to generate different patterns of stem glycans on the HA of A/Mallard/Huadong/S/2005. The results indicated that these three N-glycans were dispensable for the generation of replication-competent influenza viruses. However, when N-glycans at Asn10 plus either Asn23 or Asn268 were removed, the cleavability of HA was almost completely blocked, leading to a significant decrease of the growth rates of the mutant viruses in MDCK and CEF in comparison with that of the wild-type (WT) virus. Moreover, the mutant viruses lacking these oligosaccharides, particularly the N-glycan at Asn10, revealed a significant decrease in thermostability and pH stability compared with the WT virus. Interestingly, the mutant viruses induced a lower level of neutralizing antibodies against the WT virus, and most of the mutant viruses were more sensitive to neutralizing antibodies than the WT virus. Taken together, these data strongly suggest that the HA stem glycans play a critical role on HA cleavage, replication, thermostability, pH stability, and antigenicity of H5N1 AIVs.
Human metapneumovirus (hMPV) and its close family member respiratory syncytial virus (RSV) are two major causes of lower respiratory tract infection in the pediatrics population. hMPV is also a common cause of morbidity and mortality worldwide in the immunocompromised patients and older adults. Repeated infections occur often demonstrating a heavy medical burden. However, there is currently no hMPV-specific prevention treatment. This review focuses on the current literatures on hMPV vaccine development. We believe that a better understanding of the role(s) of viral proteins in host responses might lead to efficient prophylactic vaccine development.
Aleutian mink disease virus (AMDV) can cause severe immune complex-mediated disease in American mink. AMDV has also been detected in several other mustelid species with potential negative impact on their health and population. A molecular and cross-sectional epidemiologic study was conducted to gain data on the prevalence, distribution, transmission, and diversity of AMDV strains in Finnish free-ranging mustelids and risk factors associated with infection. The presence of anti-AMDV antibodies and/or AMDV DNA was tested from 308 samples representing eight mustelid species and 17 administrative regions. Positive samples were detected across the country and in 54% (31/57) of feral American mink, 27% (7/26) of European badgers, and 7% (1/14) of European polecats. Samples from Eurasian otters, European pine martens, least weasels, stoat, and wolverine were negative. Major risk factors for infection were the species American mink with 335 and badger with 74 times higher odds than other species and the years 2006-2009 with 5 times higher odds than years 2010-2014. No clustering according to species, geographical origin, or year was evident in phylogeny, except for four divergent sequences from Estonian badgers that formed a separate phylogroup distinct from other AMDV strains. This study showed that AMDV is prevalent in certain species of Finnish free-ranging mustelids and widely distributed across the country. Furthermore, the free-ranging mustelids carry both strains similar to those found in farmed mink, but also distinct strains that may represent novel amdoparvoviruses.
Infectious bronchitis virus (IBV) is a coronavirus of chickens that causes great economic losses to the global poultry industry. The present study focuses on South American IBVs and their genetic relationships with global strains. We obtained full-length sequences of the S1 coding region and N gene of IBV field isolates from Uruguay and Argentina, and performed phylodynamics analysis to characterize the strains and estimate the time of the most recent common ancestor. We identified two major South American genotypes that were here denoted South America I (SAI) and Asia/South America II (A/SAII). The SAI genotype is an exclusive South American lineage that emerged in the 1960s. The A/SAII genotype may have emerged in Asia in approximately 1995 before being introduced in South America. Both SAI and A/SAII genotype strains clearly differ from the Massachusetts strains that are included in the vaccine formulations being used in most South American countries.
West Nile Virus (WNV), an important global human pathogen, targets neurons to cause lethal encephalitis (WNE) primarily in elderly and immunocompromised patients. Currently there are no approved therapeutic agents or vaccines to treat WNV encephalitis (WNE). Recent studies have suggested that inflammation is a major contributor to WNE morbidity. In this study we evaluated the use of intravenous immunoglobulins (IVIG), a clinical product comprised of pooled human IgG, as an anti-inflammatory treatment in a model of lethal WNV infection. We report here that IVIG and pooled human WNV convalescent sera (WNV-IVIG) inhibited development of lethal WNE by suppressing CNS infiltration by CD45high leukocytes. Pathogenic Ly6Chigh CD11b+ monocytes were the major infiltrating subset in the CNS of infected control mice, whereas IVIG profoundly reduced infiltration of these pathogenic Ly6Chigh monocytes into the CNS of infected mice. Interestingly, WNV-IVIG was more efficacious than IVIG in controlling CNS inflammation when mice were challenged with a high inoculum (105 compared to 104 PFU) of WNV. Importantly, adsorption of WNV E-glycoprotein neutralizing antibodies did not abrogate IVIG protection, consistent with virus neutralization not being essential for IVIG protection. These findings confirm potent immunomodulatory activity of generic IVIG and emphasize its potential as an effective immunotherapeutic drug for encephalitis and other virus induced inflammatory diseases.
Hendra virus (HeV) is lethal to humans and horses and little is known about its epidemiology. Biosecurity restrictions impede advances, particularly on understanding pathways of transmission. Quantifying HeV's environmental survival can be used for making decisions and to infer transmission pathways. We estimated HeV survival with a Weibull distribution and calculated parameters from data generated in laboratory experiments. HeV survival rates based on air temperatures 24 h after excretion ranged from 2 to 10% in summer and from 12 to 33% in winter. Simulated survival across the distribution of Pteropus alecto, a key reservoir host, does not predict spillover events. Based on our analyses we conclude that the most likely pathways of transmission do not require long periods of virus survival and are likely to involve relatively direct contact with flying fox excreta shortly after excretion.
The Thai trial (RV144) indicates that a prime/boost vaccine combination that induces both T-cell and antibody responses may be desirable for an effective HIV vaccine. We have previously shown that immunisation with synthetic long peptides (SLP), covering the conserved parts of SIV, induced strong CD4 T-cell and antibody responses, but only modest CD8 T-cell responses. To generate a more balanced CD4/CD8 T-cell and antibody response, this study evaluated a pox-vector prime/SLP boost strategy in rhesus macaques. Priming with a replication competent NYVAC, encoding HIV-1 clade C gag, pol, nef, induced modest IFN T-cell immune responses, predominantly directed against HIV-1 gag. Booster immunization with SLP, covering the conserved parts of HIV-1 gag, pol, env, resulted in a more than 10 fold increase in IFN ELISpot responses in 4 of 6 animals, which were predominantly HIV-1 Pol-specific. The animals showed a balanced polyfunctional CD4 and CD8 T-cell response and high Ab titers.
The host and viral factors that influence disease outcome during flavivirus infections are not fully understood. Using the live attenuated yellow fever virus (YFV) vaccine strain 17D as a model system we evaluated how viral dose, inoculation route, and immunopathogenesis contribute to disease outcome in mice deficient in the type I IFN response. We found that YFV-17D infection of interferon aalpha;/bbeta; receptor knockout mice resulted in three distinct disease outcomes: no clinical signs of disease, fatal viscerotropic disease or fatal neurotropic disease. Interestingly, viral load at disease onset did not correlate with disease outcome. However, we found increased immune infiltrates in the brain tissues of mice that developed neurotropic disease. Additionally, mice that developed viscerotropic disease, as characterized by liver and spleen pathology and/or intestinal hemorrhage, had significantly elevated levels of alanine aminotransferase, monocyte chemotactic protein, and interferon-inducible protein (IP)-10 as compared to mice with no clinical signs of disease or neurotropic disease. Furthermore, mice treated with recombinant IP-10 throughout YFV-17D infection had increased mortality and an increased percentage of mice with viscerotropic disease. Our results demonstrate that viral load does not correlate with pathogenesis and the host immune response plays a pivotal role in disease outcome and contributes to YFV-17D pathogenesis in mice.
Rat hepatitis E virus (ratHEV) is related to human HEV and has been detected in wild rats worldwide. Here, the complete genome of ratHEV strain R63/DEU/2009 was cloned downstream a T7 RNA polymerase promotor and capped genomic RNA generated by in vitro transcription was injected into nude rats. RNA of ratHEV could be subsequently detected in serum and faeces of rats injected intrahepatically, but not in those injected intravenously. The ratHEV RNA-positive faecal suspension was intravenously inoculated into nude rats and Wistar rats leading to ratHEV RNA detection in serum and faeces of nude rats and to seroconversion in Wistar rats. In addition, ratHEV was isolated in PLC/PRF/5 cells from the ratHEV RNA-positive faecal suspension of nude rats, and passaged subsequently. The cell culture supernatant was infectious for nude rats. Genome analysis identified 9 point mutations of the cell culture-passaged virus in comparison to the originally cloned ratHEV genome. The results indicate that infectious ratHEV could be generated from the cDNA clone. Since rats are widely used and well characterized laboratory animals, studies on genetically engineered ratHEV may give novel insights into organ tropism, replication and excretion kinetics as well as immunological changes induced by hepeviruses.
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.
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.
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.
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.
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.
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.
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.
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.