|Journal of General Virology current issue|
Development of numerous advanced techniques in recent years have allowed detection of the pathological prion protein (PrPTSE), the unique marker of transmissible spongiform encephalopathies (TSEs, or prion diseases), in the blood of animals and humans; however, an ante mortem screening test that can be used for the routine diagnosis of human prion diseases remains unavailable. A critical, analytical review of all the diagnostic assays developed to date will allow an evaluation of progress in this field and may facilitate the identification of the possible reasons for this delay. Thus, in this review, I provide a detailed overview of the techniques currently available for detecting PrPTSE and other markers of the disease in blood, as well as an analysis of the significance, feasibility, reliability and application spectrum for these methods. I highlight that factors intrinsic and extrinsic to blood may interfere with the detection of PrPTSE/prions, and that this is not yet taken into account in current tests. This may inspire researchers in this field to not only aspire to increase test sensitivity, but also to adopt other strategies in order to identify and overcome the limitations that hamper the development of a successful routine blood test for prion diseases.
Coronaviruses (CoVs) have been studied for over 60 years, but have only recently gained notoriety as deadly human pathogens with the emergence of severe respiratory syndrome CoV and Middle East respiratory syndrome virus. The rapid emergence of these viruses has demonstrated the need for good models to study severe CoV respiratory infection and pathogenesis. There are, currently, different methods and models for the study of CoV disease. The available genetic methods for the study and evaluation of CoV genetics are reviewed here. There are several animal models, both mouse and alternative animals, for the study of severe CoV respiratory disease that have been examined, each with different pros and cons relative to the actual pathogenesis of the disease in humans. A current limitation of these models is that no animal model perfectly recapitulates the disease seen in humans. Through the review and analysis of the available disease models, investigators can employ the most appropriate available model to study various aspects of CoV pathogenesis and evaluate possible antiviral treatments that may potentially be successful in future treatment and prevention of severe CoV respiratory infections.
Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for epidemics of debilitating arthritic disease. The recent outbreak (2004nndash;2014) resulted in an estimated 1.4nndash;6.5 million cases, with imported cases reported in nearly 40 countries. The development of CHIKV-specific diagnostics and research tools is thus highly desirable. Herein we describe the generation and characterization of the first mAbs specific for the capsid protein (CP) of CHIKV. The antibodies recognized isolates representing the major genotypes of CHIKV, as well as several other alphaviruses, and were reactive in a range of assays including ELISA, Western blot, immunofluorescence and immunohistochemistry (IHC). We have also used the anti-CP mAb 5.5G9 in IHC studies to show that capsid antigen is persistently expressed 30 days post-infection in cells with macrophage morphology in a mouse model of chronic CHIKV disease. These antibodies may thus represent useful tools for further research, including investigations into the structure and function of CHIKV CP, and as valuable reagents for CHIKV detection in a range of settings.
Oropouche virus (OROV) is a medically important orthobunyavirus, which causes frequent outbreaks of a febrile illness in the northern parts of Brazil. However, despite being the cause of an estimated half a million human infections since its first isolation in Trinidad in 1955, details of the molecular biology of this tripartite, negative-sense RNA virus remain limited. We have determined the complete nucleotide sequence of the Brazilian prototype strain of OROV, BeAn 19991, and found a number of differences compared with sequences in the database. Most notable were that the S segment contained an additional 204 nt at the 3' end and that there was a critical nucleotide mismatch at position 9 within the base-paired terminal panhandle structure of each genome segment. In addition, we obtained the complete sequence of the Trinidadian prototype strain TRVL-9760 that showed similar characteristics to the BeAn 19991 strain. By using a T7 RNA polymerase-driven minigenome system, we demonstrated that cDNA clones of the BeAn 19991 L and S segments expressed functional proteins, and also that the newly determined terminal untranslated sequences acted as functional promoters in the minigenome assay. By co-transfecting a cDNA to the viral glycoproteins, virus-like particles were generated that packaged a minigenome and were capable of infecting naive cells.
Crimeannndash;Congo hemorrhagic fever virus (CCHFV) is a member of the genus Nairovirus of the family Bunyaviridae, that can cause severe haemorrhagic fever in humans, with mortality rates above 30 %. CCHFV is the most widespread of the tick-borne human viruses and it is endemic in areas of central Asia, the Middle East, Africa and southern Europe. Its viral genome consists of three negative-sense RNA segments. The large segment (L) encodes a viral RNA-dependent RNA polymerase (L protein), the small segment (S) encodes the nucleocapsid protein (N protein) and the medium segment (M) encodes the envelope proteins. The N protein of bunyaviruses binds genomic RNA, forming the viral ribonucleoprotein (RNP) complex. The L protein interacts with these RNP structures, allowing the initiation of viral replication. The N protein also interacts with actin, although the regions and specific residues involved in these interactions have not yet been described. Here, by means of immunoprecipitation and immunofluorescence assays, we identified the regions within the CCHFV N protein implicated in homo-oligomerization and actin binding. We describe the interaction of the N protein with the CCHFV L protein, and identify the N- and C-terminal regions within the L protein that might be necessary for the formation of these Nnndash;L protein complexes. These results may guide the development of potent inhibitors of these complexes that could potentially block CCHFV replication.
Pathogenesis of viral haemorrhagic fevers is associated with alteration of vascular barrier function and haemorrhage. To date, the specific mechanism behind this is unknown. Programmed cell death and regulation of apoptosis in response to viral infection is an important factor for host or virus survival but this has not been well-studied in the case of Crimean-Congo hemorrhagic fever virus (CCHFV). In this study, we demonstrated that CCHFV infection suppresses cleavage of poly(ADP-ribose) polymerase (PARP), triggered by staurosporine early post-infection. We also demonstrated that CCHFV infection suppresses activation of caspase-3 and caspase-9. Most interestingly, we found that CCHFV N can suppress induction of apoptosis by Bax and inhibit the release of cytochrome c from the inner membrane of mitochondria to cytosol. However, CCHFV infection induces activation of Bid late post-infection, suggesting activation of extrinsic apoptotic signalling. Consistently, supernatant from cells stimulated late post-infection was found to induce PARP cleavage, most probably through the TNF-aalpha; death receptor pathway. In summary, we found that CCHFV has strategies to interplay with apoptosis pathways and thereby regulate caspase cascades. We suggest that CCHFV suppresses caspase activation at early stages of the CCHFV replication cycle, which perhaps benefits the establishment of infection. Furthermore, we suggest that the host cellular response at late stages post-infection induces host cellular pro-apoptotic molecules through the death receptor pathway.
Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent throughout the world and has caused major economic losses to the pig industry. Arterivirus non-structural protein 10 (nsp10) is a superfamily 1 helicase participating in multiple processes of virus replication. PRRSV nsp10, however, has not yet been well characterized. In this study, a series of nsp10 mutants were constructed and analysed for functional sites of different enzymic activities. We found that nsp10 could bind both ssDNA and dsDNA, and this binding activity could be inactivated by mutations at Cys25 and His32. These two mutations also abolished unwinding activity without affecting ATPase activity. In addition, substitution of Ala227 by Ser eliminated helicase activity, whilst substitution by Val enhanced unwinding activity. Taken together, our results showed that Cys25 and His32 in PRRSV nsp10 were critical for nucleic acid binding and unwinding, and that Ala227 played an important role in helicase activity.
In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2) consistently gained positively charged amino acids in the putative heparin-sulfate-binding pocket (VP2 bbeta;E-bbeta;F loop, VP1 C-terminus and VP3 bbeta;-B knob) surrounding the fivefold symmetry axis (VP1 bbeta;F-bbeta;G loop) and at other discrete sites on the capsid (VP3 bbeta;G-bbeta;H loop, VP1 C-terminus, VP2 bbeta;C strand and VP1 bbeta;G-bbeta;H loop). A lysine insertion in the VP1 bbeta;F-bbeta;G loop of two of the BHK-21-adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line, suggesting their possible role in ionic interaction with ubiquitous negatively charged cell-surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell-culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared with the lower passage viruses with WT genotypes. Substitutions fixed in the VP1 bbeta;G-bbeta;H loop (nndash;3, nndash;2 and +2 llsquo;RGDrrsquo; positions) or in the structural element known to be juxtaposed against that loop (VP1 bbeta;B-bbeta;C loop) suggest their possible role in modulating the efficiency and specificity of interaction of the llsquo;RGDrrsquo; motif with aalpha;v-integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production.
A salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared with terrestrial alphavirus replicons. In this study, we have compared several modifications to a SAV replicon construct and analysed their influence on foreign gene expression. We found that an insertion of a translational enhancer consisting of the N-terminal 102 nt of the capsid gene, together with a nucleotide sequence encoding the foot-and-mouth disease virus (FMDV) 2A peptide, caused a significant increase in EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 5' end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at the 3' end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production, which can be applied for immunization of salmonid fish in the future.
Pleconaril is a capsid inhibitor used previously to treat enterovirus infections. A pleconaril-resistant echovirus 11 (E11) strain was identified before pleconaril treatment was given in an immunocompromised patient. The patient was also treated with intravenous Ig (IVIg) for a long period but remained unresponsive. The pleconaril-resistant strains could not be neutralized in vitro, confirming IVIg treatment failure. To identify the basis of pleconaril resistance, genetic and structural analyses were conducted. Analysis of a modelled viral capsid indicated conformational changes in the hydrophobic pocket that could prevent pleconaril docking. Substitutions (V117I, V119M and I188L) in the pleconaril-resistant viruses were found in the pocket region of VP1. Modelling suggested that V119M could confer resistance, most probably due to the protruding sulfate side chain of methionine. Although pleconaril resistance induced in vitro in a susceptible E11 clinical isolate was characterized by a different substitution (I183M), resistance was suggested to also result from a similar mechanism, i.e. due to a protruding sulfate side chain of methionine. Our results showed that resistant strains that arise in vivo display different markers from those identified in vitro and suggest that multiple factors may play a role in pleconaril resistance in patient strains. Based on IVIg treatment failure, we predict that one of these factors could be immune related. Thus, both IVIg and capsid inhibitors target the viral capsid and can induce mutations that can be cross-reactive, enabling escape from both IVIg and the drug. This could limit treatment options and should be investigated further.
The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1aalpha; become activated. PERK phosphorylates eIF2aalpha; leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1aalpha; splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2aalpha; and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.
Rotavirus (RV) A is a very common cause of acute diarrhoea in infants and young children worldwide. Most human strains are classified into two major Wa-like and DS-1-like genotype constellations, whilst a minor third strain, AU-1, was described in 1989 among human RV isolates from Japan. AU-1 demonstrates a high degree of homology to a feline RV, FRV-1, which suggests interspecies transmission of feline RV. However, there has been no subsequent report of RVs possessing the AU-1 genotype throughout all 11 genes of the genome. Between March 1997 and December 1999, 157 RV-positive stool samples were collected from Brazilian children, and 16 of the RVs (10.2 %) were P genotype. We analysed eight strains by almost full-genome sequencing. These eight strains were divided into two groups: five AU-1-like and three Wa-like strains. Four of the five AU-1-like strains had the AU-1-like genotype constellation throughout the 11 genes. The remaining AU-1-like strain was considered to be a reassortant strain comprosed of nine, two and one genes from the AU-1-like, Wa-like and G9 strains, respectively. The three Wa-like strains were considered to be reassortants comprising seven to eight genes and three to four genes from Wa-like and non-Wa-like strains, respectively. This report of human G3P RV strains possessing the AU-1 genotype constellation throughout all genes demonstrates the stability and infectivity of the AU-1-like strain with its original genotype over distance and time.
In simian virus 40 (SV40) and several other polyomaviruses, the TATA box of the early promoter is embedded in an AT tract that is also an essential part of the replication origin. We generated an llsquo;AT traprrsquo;, an SV40 genome lacking the AT tract and unable to grow in CV-1 monkey cells. Co-transfection of the AT trap with oligonucleotides containing AT tracts of human polyomaviruses, a poly(A : T) tract or variants of the SV40 WT sequence all restored infectious virus. In a transfection of the AT trap without a suitable oligonucleotide, an AT-rich segment was incorporated, stemming either from bovine (calf serum) or monkey (host cell) DNA. Similarly, when cells were grown with human serum, a human DNA segment was captured by SV40 to substitute for the missing AT stretch. We conclude that the virus is quite opportunistic in accepting heterologous substitutes, and that even low-abundance DNA from serum can be incorporated into the viral genome.
Human papillomaviruses (HPVs) have been detected in urban wastewaters, demonstrating that epitheliotropic viruses can find their way into sewage through the washing of skin and mucous membranes. Papillomavirus shedding through faeces is still an unexplored issue. The objective of the present study was to investigate the presence of HPVs in stool samples. We analysed 103 faecal specimens collected from hospitalized patients with diarrhoea using validated primers able to detect aalpha;, bbeta; and HPVs. PCR products underwent sequencing analysis and sequences were aligned to reference genomes from the Papillomavirus Episteme database. A total of 15 sequences were characterized from the faecal samples. Thirteen samples (12.6 %) were positive for nine genotypes belonging to the aalpha; and bbeta; genera: HPV32 (LR, aalpha;1), HPV39 (HR, aalpha;7), HPV44 (LR, aalpha;10), HPV8 (bbeta;1), HPV9, HPV23, HPV37, HPV38 and HPV120 (bbeta;2). Two putative novel genotypes of the bbeta; genus, species 1 and 2, were also detected. The tissue(s) of origin is unknown, since faeces can collect HPVs originating from or passing through the entire digestive system. To our knowledge, this is the first investigation on the occurrence and diversity of HPVs in faecal samples. Results from this study demonstrate that HPVs can find their way into sewage as a consequence of shedding in the faeces. This highlights the need for further studies aimed at understanding the prevalence of HPV in different water environments and the potential for waterborne transmission.
Hepatitis B virus X protein (HBx) is involved in the development of hepatocellular carcinoma (HCC). The HBx sequence is a preferential site of integration into the human genome, leading to the formation of C-terminal-truncated HBx proteins (Ct-HBx). We previously reported that Ct-HBx proteins were able to potentiate cell transformation in vitro. Our present goal was to compare the ability of Ct-HBx and full-length HBx (FL-HBx) proteins to develop or enhance HCC in transgenic mice. In the absence of treatment, neither Ct-HBx- nor FL-HBx-transgenic mice developed HCC. In young mice treated with diethylnitrosamine (DEN) at 8 months of age, a significantly higher incidence and number of liver lesions were observed in Ct-HBx mice than in FL-HBx and control mice. The earlier development of tumours in Ct-HBx-transgenic mice was associated with increased liver inflammation. At 10 months, macroscopic and microscopic analyses showed that, statistically, FL-HBx mice developed more liver lesions with a larger surface area than control mice. Furthermore, during DEN-induced initiation of HCC, Ct-HBx- and FL-HBx-transgenic mice showed higher expression of IL-6, TNF-aalpha; and IL-1bbeta; transcripts, activation of STAT3, ERK and JNK proteins and an increase in cell apoptosis. In conclusion, in DEN-treated transgenic mice, the expression of Ct-HBx protein causes a more rapid onset of HCC than does FL-HBx protein. HBV genome integration leading to the expression of a truncated form of HBx protein may therefore facilitate HCC development in chronically infected patients.
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epsteinnndash;Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.
Marekrrsquo;s disease virus (MDV) is an important oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts. The Meq-clustered miRNAs encoded by MDV have been suggested to play potentially critical roles in the induction of lymphomas. Using the technique of bacterial artificial chromosome mutagenesis, we have presently constructed a series of specific miRNA-deleted mutants and demonstrate that these miRNAs are not essential for replication of MDV and have no effects on the early cytolytic or latent phases of the developing disease. However, compared to the parental GX0101, mortality of birds infected with the mutants GXmiR-M2, GXmiR-M3, GXmiR-M5, GXmiR-M9 and GXmiR-M12 was reduced from 100 % to 18 %, 30 %, 48 %, 24 % and 14 %, coupled with gross tumour incidence reduction from 28 % to 8 %, 4 %, 12 %, 8 % and 0 %, respectively. Our data confirm that except for mdv1-miR-M4, the other Meq-clustered miRNAs also play critical roles in MDV oncogenesis. Further work will be needed to elucidate the miRNA-mediated regulatory mechanisms that trigger the development of MD lymphomas.
Mouse mammary tumour virus (MMTV) is a member of the genus Betaretrovirus, infects rodent cells and uses mouse tranferrin receptor 1 for cell entry. Several MMTV strains have been shown to productively infect, in addition to murine cells, various heterologous cell lines including those of human origin, albeit less efficiently than murine cells. Here, we analysed whether MMTV from C3H mice [MMTV(C3H)], reported previously to be incapable of infecting human cells, could productively infect human cells. Using a recently described high-titre MMTV-based vector carrying MMTV(C3H) envelope protein (Env), we successfully transduced cells of human origin. Furthermore, WT MMTV(C3H) was able to infect human cells, albeit less efficiently than mouse cells. The established infection was, however, sufficient to enable virus spread to every cell in culture. The infectivity of WT MMTV(C3H) and MMTV-based vectors carrying MMTV(C3H)Env was blocked by heat inactivation, an inhibitor of reverse transcription (3'-azido-3'-deoxythymidine) and pre-incubation with neutralizing anti-MMTV antibodies that did not neutralize vectors pseudotyped with amphotropic murine leukemia virus Env, providing evidence for an authentic, receptor-mediated and reverse transcriptase-dependent infection process. Persistently infected human Hs578T cells produced infectious virions capable of infecting naiiuml;ve human breast cells in culture, the infectivity of which could also be blocked by neutralizing anti-MMTV antibodies, demonstrating that virus particles released by the persistently infected Hs578T cells were related antigenically to the virus produced from murine cells. Taken together, our results show that MMTV(C3H), like MMTV(GR) and MMTV(RIII), is able not only to infect but also to replicate in cultured human breast cells.
Chronic hepatitis B (CHB) is treated with nucleos(t)ide analogues (NAs). The reverse transcriptase (RT) region in the hepatitis B virus (HBV) genome mutates to resist NA treatment, yet the RT mutations have not been well characterized. Furthermore, the HBV genotype might influence RT sequence evolution, NA resistance (NAr) mutation patterns and drug resistance development. We examined 42 NAr mutation sites in 169 untreated and 131 NA-treated CHB patient samples. Patients were identified with HBV-B and HBV-C genotype infections, with a higher prevalence and mutation frequency of HBV-C than HBV-B. Seventeen reported NAr mutation sites and 13 novel mutations were detected. NAr-related mutation prevalence was significantly higher in NA-treated versus untreated patients. Primary antiviral-resistant mutants only existed in NA-treated patients. Sequencing data revealed seven HBV-C-specific mutations and three HBV-B-specific mutations. In conclusion, NA treatment and HBV genotype might constitute the selection basis and promote NA-resistant HBV strain evolution under antiviral therapy.
Neutralizing antibodies (NAbs) are believed to comprise an essential component of the protective immune response induced by vaccines against feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) infections. However, relatively little is known about the role of NAbs in controlling FIV infection and subsequent disease progression. Here, we present studies where we examined the neutralization of HIV-luciferase pseudotypes bearing homologous and heterologous FIV envelope proteins (n = 278) by sequential plasma samples collected at 6 month intervals from naturally infected cats (n = 38) over a period of 18 months. We evaluated the breadth of the NAb response against non-recombinant homologous and heterologous clade A and clade B viral variants, as well as recombinants, and assessed the results, testing for evidence of an association between the potency of the NAb response and the duration of infection, CD4+ T lymphocyte numbers, health status and survival times of the infected cats. Neutralization profiles varied significantly between FIV-infected cats and strong autologous neutralization, assessed using luciferase-based in vitro assays, did not correlate with the clinical outcome. No association was observed between strong NAb responses and either improved health status or increased survival time of infected animals, implying that other protective mechanisms were likely to be involved. Similarly, no correlation was observed between the development of autologous NAbs and the duration of infection. Furthermore, cross-neutralizing antibodies were evident in only a small proportion (13 %) of cats.
T-lymphotropic feline leukemia virus (FeLV-T), a highly pathogenic variant of FeLV, induces severe immunosuppression in cats. FeLV-T is fusion defective because in its PHQ motif, a gammaretroviral consensus motif in the N terminus of an envelope protein, histidine is replaced with aspartate. Infection by FeLV-T requires FeLIX, a truncated envelope protein encoded by an endogenous FeLV, for transactivation of infectivity and Pit1 for binding FeLIX. Although Pit1 is present in most tissues in cats, the expression of FeLIX is limited to certain cells in lymphoid organs. Therefore, the host cell range of FeLV-T was thought to be restricted to cells expressing FeLIX. However, because FeLIX is a soluble factor and is expressed constitutively in lymphoid organs, we presumed it to be present in blood and evaluated its activities in sera of various mammalian species using a pseudotype assay. We demonstrated that cat serum has FeLIX activity at a functional level, suggesting that FeLIX is present in the blood and that FeLV-T may be able to infect cells expressing Pit1 regardless of the expression of FeLIX in vivo. In addition, FeLIX activities in sera were detected only in domestic cats and not in other feline species tested. To our knowledge, this is the first report to prove that a large amount of truncated envelope protein of endogenous retrovirus is circulating in the blood to facilitate the infection of a pathogenic exogenous retrovirus.
Baculoviruses are arthropod-specific pathogens, and find extensive applications in pest control strategies and recombinant protein expression. Spodoptera litura nucleopolyhedrovirus (SpltNPV) infects the tropical armyworm Spodoptera litura, which is an important polyphagous crop pest widely distributed in regions of Asia and Oceania. Using next-generation sequencing, we report stage-specific profiling of SpltNPV-encoded microRNAs (miRNAs) at different time intervals post-infection (p.i.) of Sf21 cells. Sequence length distribution analysis of the small RNA libraries revealed a significant increase in 20 nt reads and a reduction of other size fractions during late phases of infection. In silico miRNA prediction tools identified 48 novel SpltNPV-encoded miRNAs, of which 10 were validated experimentally in Sf21 cells using Northern blot analysis and TaqMan quantitative real-time (qRT)-PCR. The viral miRNAs were also found to be expressed in fat-body and mid-gut tissues of infected fifth-instar S. litura larva. qRT-PCR analysis confirmed that expression of most viral miRNAs was triggered 12 h p.i. and continued thereafter. Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway annotation of computationally predicted targets of the reported miRNAs suggested a major impact of these miRNAs on cell signalling, protein translation and metabolic processes.
Turnip mosaic virus (TuMV) is a potyvirus that is transmitted by aphids and infects a wide range of plant species. We investigated the evolution of this pathogen by collecting 32 isolates of TuMV, mostly from Brassicaceae plants, in Australia and New Zealand. We performed a variety of sequence-based phylogenetic and population genetic analyses of the complete genomic sequences and of three non-recombinogenic regions of those sequences. The substitution rates, divergence times and phylogeographical patterns of the virus populations were estimated. Six inter- and seven intralineage recombination-type patterns were found in the genomes of the Australian and New Zealand isolates, and all were novel. Only one recombination-type pattern has been found in both countries. The Australian and New Zealand populations were genetically different, and were different from the European and Asian populations. Our Bayesian coalescent analyses, based on a combination of novel and published sequence data from three non-recombinogenic protein-encoding regions, showed that TuMV probably started to migrate from Europe to Australia and New Zealand more than 80 years ago, and that distinct populations arose as a result of evolutionary drivers such as recombination. The basal-B2 subpopulation in Australia and New Zealand seems to be older than those of the world-B2 and -B3 populations. To our knowledge, our study presents the first population genetic analysis of TuMV in Australia and New Zealand. We have shown that the time of migration of TuMV correlates well with the establishment of agriculture and migration of Europeans to these countries.
Analysis of virus-derived small RNAs with high-throughput sequencing has been successful for detecting novel viruses in plants and invertebrates. However, the applicability of this method has not been demonstrated in fungi, although fungi were among the first organisms reported to utilize RNA silencing. Here, we used virus-infected isolates of the fungal species complex Heterobasidion annosum sensu lato as a model system to test whether mycovirus genome segments can be detected with small RNA deep sequencing. Species of the genus Heterobasidion are some of the most devastating forest pathogens in boreal forests. These fungi cause wood decay and are commonly infected with species of the family Partitiviridae and the unassigned virus species Heterobasidion RNA virus 6. Small RNA deep sequencing allowed the simultaneous detection of all eight double-stranded RNA virus strains known to be present in the tested samples and one putative mitovirus species (family Narnaviridae) with a single-stranded RNA genome, designated here as Heterobasidion mitovirus 1. Prior to this study, no members of the family Narnaviridae had been described as infecting species of Heterobasidion. Quantification of viral double- and single-stranded RNA with quantitative PCR indicated that co-infecting viral species and viruses with segmented genomes can be detected with small RNA deep sequencing despite vast differences in the amount of RNA. This is the first study demonstrating the usefulness of this method for detecting fungal viruses. Moreover, the results suggest that viral genomes are processed into small RNAs by different species of Heterobasidion.
|Journal of General Virology Publish Ahead of Print|
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.
The serpin family of serine proteinase inhibitors plays key roles in a variety of biochemical pathways. In insects, one of the important functions carried out by serpins is regulation of the phenoloxidase cascade, a pathway that produces melanin and other compounds that are important in insect humoral immunity. Recent sequencing of the baculovirus Hemileuca sp. nucleopolyhedrovirus (HespNPV) genome revealed the presence of a gene, hesp018, with homology to insect serpins. To our knowledge hesp018 is the first viral serpin homolog to be characterized outside of the chordopoxviruses. The Hesp018 protein was found to be a functional serpin with inhibitory activity against a subset of serine proteinases. Hesp018 also inhibited phenoloxidase activation when mixed with lepidopteran hemolymph. The Hesp018 protein was secreted when expressed in lepidopteran cells, and a baculovirus expressing Hesp018 exhibited accelerated production of viral progeny during in vitro infection. Expression of Hesp018 also reduced caspase activity induced by baculovirus infection, but caused increased cathepsin activity. In infected insect larvae, expression of Hesp018 resulted in faster larval melanization, consistent with increased activity of viral cathepsin. Finally, expression of Hesp018 increased the virulence of a prototype baculovirus by 4-fold in orally-infected neonate Trichoplusia ni larvae. Based on our observations, we hypothesize that the hesp018 may have been retained in HespNPV due to its ability to inhibit the activity of select host proteinases, possibly including proteinases involved in the phenoloxidase response, during infection of host insects.
Three (MoCAV/F2, MoCAV/F8, MoCAV/F11) of 4 mouse monoclonal antibodies (mAbs) established against the A2/76 strain of chicken anemia virus (CAV) showed neutralization activity. Immunoprecipitation showed a band at approximately 50 kDa in A2/76-infected cell lysates by neutralizing mAbs, corresponding to the 50-kDa capsid protein (VP1) of CAV, and the mAbs reacted with recombinant VP1 proteins expressed in Cos7 cells. MoCAV/F2 and MoCAV/F8 neutralized the 14 CAV strains tested, whereas MoCAV/F11 did not neutralize 5 of the strains, indicating distinct antigenic variation among the strains. In blocking immunofluorescence tests with the A2/76-infected cells, binding of MoCAV/F11 was not inhibited by the other mAbs. MoCAV/F2 inhibited the binding of MoCAV/F8 to the antigens and vice versa, suggesting that the 2 mAbs recognized the same epitope. However, mutations were found in different parts of VP1 of the escape mutants of each mAb: EsCAV/F2 (deletion of T89+A90), EsCAV/F8 (I261T), and EsCAV/F11 (E144G). Thus, the epitopes recognized by MoCAV/F2 and MoCAV/F8 seemed to be topographically close in the VP1 structure, suggesting that VP1 has at least 2 different neutralizing epitopes. However, MoCAV/F8 did not react to EsCAV/F2 or to EsCAV/F8, suggesting that binding of MoCAV/F8 to the epitope requires coexistence of the epitope recognized by MoCAV/F2. In addition, MoCAV/F2, with a titer of 1:12,800 to the parent strain, neutralized EsCAV/F2 and EsCAV/F8 with low titers of 32 and 152, respectively. The similarity of the reactivity of MoCAV/F2 and MoCAV/F8 to VP1 may also suggest the existence of a single epitope recognized by these mAbs.
Epidemiological studies have suggested that consumption of beef may correlate with an increased risk of colorectal cancer. One hypothesis to explain this proposed link might be the presence of a carcinogenic infectious agent capable of withstanding cooking. Polyomaviruses are a ubiquitous family of thermostable non-enveloped DNA viruses that are known to be carcinogenic. Using virion enrichment, rolling circle amplification (RCA), and next-generation sequencing, we searched for polyomaviruses in meat samples purchased from several supermarkets. Ground beef samples were found to contain three polyomavirus species. One species, bovine polyomavirus 1 (BoPyV1), was originally discovered as a contaminant in laboratory fetal calf serum. A previously unknown species, BoPyV2, occupies the same clade as human Merkel cell polyomavirus and raccoon polyomavirus 1, both of which are carcinogenic in their native hosts. A third species, BoPyV3, is related to human polyomaviruses 6 and 7. Examples of additional DNA virus families, including herpesviruses, adenoviruses, circoviruses, and gyroviruses were also detected either in ground beef samples or in comparison samples of ground pork and ground chicken. The results suggest that the virion enrichment/RCA approach is suitable for random detection of essentially any DNA virus with a detergent-stable capsid. It will be important for future studies to address the possibility that animal viruses commonly found in food might be associated with disease.
The human cytomegalovirus (HCMV) UL112-113 gene is implicated in lytic viral replication. The UL112-113 proteins p34, p43, p50, and p84 are expressed via alternative splicing. However, the mechanism for the generation of three additional virus-associated proteins (p20, p26, and p28), which share the UL112 reading frame, remains unknown. Bioinformatic analyses indicated that p34, p43, p50, and p84 contain potential PEST-like degradation motifs. In this study, inhibitors of calpains, lysosomes, and proteasomes reduce p20, p26, and p28 levels in virus-infected cells, suggesting the involvement of proteolytic modification. Moreover, maitotoxin, which increases intracellular calcium levels and activates calpain activity, induces the intracellular proteolysis of p34 into p20, p26, and p28 and the cleavage of p43, p50, and p84 into p38 and a novel protein, p34c. Proteolytic assays further indicated that p34, p43, p50, and p84 were substrates of calpain-1 and calpain-2 and that they generated proteolytic products that corresponded to those detected during the HCMV infectious period. Furthermore, substitution mutations in the putative calpain cleavage sites of p34 reduced accumulation of proteolytic products. The knockdown of endogenous calpain-1 and calpain-2 by RNA interference reduced accumulation of p20, p26, and p28 and concurrently increased levels of nascent p43, p50, and p84 during the infectious cycle. Intriguingly, the calpain depletion enhanced viral genome synthesis. Moreover, HCMV-permissive cells that stably expressed p20, p26 or p28 exhibited reduced viral genome synthesis and mature virus production. Our findings suggest that cognate UL112-113 proteins derived from calpain-catalysed proteolysis are involved in the HCMV replication process.
The members of Vesiculovirus which belongs to the family of Rhabdoviridae can cause great economic loss in fish culture. In the present report, a vesiculovirus (named as SHVV) was isolated from diseased hybrid snakehead fish. The SHVV shared 94% nucleotide sequence identity at the genomic level with Siniperca chuatsi rhabdovirus (SCRV) which infected Mandarin fish (Siniperca chuatsi). We showed that SHVV was able to replicate and proliferate very well in SSN-1 cells which were original from striped snakehead fish (Channa striatus). Furthermore, Mandarin fish was susceptible to SHVV by bath exposure as well as by intraperitoneal injection. The infected fish showed typical clinical signs of rhabdovirus infection, including hemorrhage and edema. Histopathological analysis revealed that extensive inflammation and necrosis were observed in the spleen, kidney, liver, heart and brain of the moribund Mandarin fish. The present results will shed new lights on the epidemic of vesiculoviruses among fish.
Little is known about viruses associated with Antarctic animals, though they are likely widespread. We recovered a novel polyomavirus from Adeeacute;lie penguin (Pygoscelis adeliae) faecal matter sampled in a sub-colony at Cape Royds, Ross Island, Antarctica. The 4988 nt Adeeacute;lie penguin polyomavirus (AdPyV) has a typical polyomavirus genome organisation with three open reading frames (ORFs) that encode capsid proteins on the one strand and two non-structural protein coding ORFs on the complementary strand. The genome of AdPyV shares ~60% pairwise identity with all avipolyomaviruses. Maximum likelihood phylogenetic analysis of the large T-antigen (T-Ag) amino acid sequences shows that the T-Ag of AdPyV clusters with those of avipolyomaviruses sharing between 48-52% identities. Only three viruses associated with Adeeacute;lie penguins have been identified at a genomic level, Avian influenza virus subtype H11N2 from the Antarctic Peninsula and, respectively, Pygoscelis adeliae papillomavirus and AdPyV from capes Crozier and Royds on Ross Island.
There is no large-scale therapy available against Human Respiratory Syncytial Virus (hRSV), a major pathogen responsible for acute respiratory diseases. Macaques represent an interesting animal model to evaluate potential treatments because of their genetic, anatomical and immunological proximity with human. However, the parameters that influence hRSV growth and control in this model are still poorly understood. We have documented in the following the influence of age as well as repeated infections on the virological, clinical and immunological parameters of this animal model. Following intranasal inoculation, hRSV replicated in the upper respiratory tract for less than 15 days with no clinical signs regardless of age. Interestingly, we observed the induction of a local immune response at the nasal mucosa as assessed by expression profiles of inflammatory and interferon-stimulated genes. Animals also developed specific antibodies, and were immune to re-infection. Thus, we showed that even in infant macaques, intranasal hRSV infection induced both local and systemic immune responses to efficiently control the virus.
Infection with hepatitis C virus (HCV) is characterized by systemic oxidative stress that is caused by either viral Core or chronic inflammation. It is well recognized that reactive oxygen species (ROS) like H2O2 can induce apoptotic cell death and can therefore function as anti-tumorigenic species. However, the detailed mechanisms by which ROS induces apoptotic cell death and HCV copes with the oxidative condition are largely unknown. In the present study, we found that H2O2 induced apoptotic cell death in the p53-positive human hepatocytes but not in the p53-negative ones. For this effect, H2O2 up-regulated levels of p14, increased ubiquitin-dependent degradation of mouse double minute 2 (MDM2), and reduced the interaction between MDM2 and p53 to prevent p53 degradation, resulting in accumulation of p53 and subsequent activation of the p53-dependent apoptotic pathways. Interestingly, HCV Core repressed p14 expression via promoter hypermethylation to abolish the potential of H2O2 to activate the p14-MDM2-p53 pathway. As a consequence, the HCV Core-expressing cells could overcome the p53-mediated apoptosis provoked by H2O2. Taken together, HCV Core can contribute to hepatocellular carcinoma formation by removing deleterious roles of ROS inducing cell death.
During the last decades, metagenomic studies expanded the numbers of newly described, often unclassified, viruses within the Circoviridae family. Using broad-spectrum circo-/cyclovirus PCRs, we characterized a novel circo-like virus in Aedes vexans mosquitoes from Germany whose main putative open reading frames (ORFs) shared very low amino acid identity with those of previously characterized circo-/cycloviruses. Phylogenetic and genetic distance analysis revealed that this new virus species defines, with previously described mosquito- and bat feces-derived circo-like viruses, a different genus, tentatively called "krikovirus", within Circoviridae. We further demonstrated that viruses of the putative krikovirus genus all share a genomic organization which is unique among Circoviridae. Further investigations are needed to determine the host range, tissue tropism and transmission route(s). This report increases the current knowledge of the genetic diversity and evolution of the members of the Circoviridae family.
Analysing the evolution of FIV on the intra-host level is important, in order to address whether the diversity and composition of viral quasispecies affects disease progression.
We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at the rate of 1.16 x 10-3 substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3-V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3-V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation.
Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximal evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared to HIV might explain why many naturally infected cats do not progress to AIDS rapidly.
Adeno-associated virus (AAV) type 5 represents the genetically most distant AAV serotype and the only one isolated directly from human tissue. Seroepidemiological evidence suggests HSV as helper virus for human AAV5 infections underlining the in vivo relevance of the AAV-herpesvirus relationship. In this study we analyzed for the first time herpes simplex virus (HSV) helper functions for productive AAV5 replication and compared these to AAV2. Using a combination of HSV strains and plasmids for individual genes, the previously defined HSV helper functions for AAV2 replication were shown to induce AAV5 gene expression, DNA replication and production of infectious progeny. The helper functions comprise the replication genes for ICP8 (UL29), helicase-primase (UL5/8/52), and DNA polymerase (UL30/42). HSV immediate-early genes for ICP0 and ICP4 further enhanced AAV5 replication, mainly by induction of rep gene expression. In the presence of HSV helper functions AAV5 Rep colocalized with ICP8 in nuclear replication compartments and HSV alkaline exonuclease (UL12) enhanced AAV5 replication, similar to AAV2. UL12 in combination with ICP8 was shown to induce DNA strand exchange on partially double-stranded templates to resolve and repair concatemeric HSV replication intermediates. Similarly, concatemeric AAV replication intermediates appeared to be processed to yield AAV unit-length molecules, ready for AAV packaging. Taken together, our findings show that productive AAV5 replication is promoted by the same combination of HSV helper functions as AAV2.
Various herpesviruses have been discovered in marine mammals, associated with a wide spectrum of disease. In the present study we describe the detection and phylogenetic analysis of a novel gammaherpesvirus, tentatively called phocine herpesvirus 7 (PhHV-7), that was detected in samples collected during an outbreak of ulcerative gingivitis and glossitis from juvenile harbor seals (Phoca vitulina) at the Seal Rehabilitation and Research Centre, the Netherlands. The presence of this novel gammaherpesvirus was confirmed by viral metagenomics, while no other viruses except four novel anelloviruses were detected. However, PhHV-7 DNA was also detected in harbor and grey seals (Halichoerus grypus) without gingivitis or glossitis. Genetic analysis of the partial polymerase gene of PhHV-7 detected in both species revealed limited sequence variation. Additional studies are needed to elucidate whether the discovered viruses played a role in the observed disease.
A granulovirus (GV) producing occlusion bodies (OBs) with an unusual appearance was isolated from Adoxophyes spp. larvae in a field. Ultrastructural observations revealed that its OBs were significantly larger and cuboidal in shape, rather than the standard ovo-cylindrical shape typical of GVs. N-terminal amino acid sequence analysis of the OB matrix protein from this virus suggested that it was a variant of Adoxophyes orana granulovirus (AdorGV). Bioassays of this GV (termed AdorGV-M) and an English isolate of AdorGV (termed AdorGV-E) indicated that the two isolates were equally pathogenic against larvae of A. honmai. However, AdorGV-M retained more infectivity towards larvae after irradiation with UV light than AdorGV-E. Sequencing and analysis of the AdorGV-M genome revealed little sequence divergence between this isolate and AdorGV-E. Comparison of selected genes among the two AdorGVs isolates and other Japanese AdorGV isolates revealed differences that may account for the unusual OB morphology of the AdorGV-M.
African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens responsible for protection in the pig. Available data from vaccination/challenge experiments in pigs indicate ASF protective immunity is hemadsorption inhibition (HAI) serotype-specific. A better understanding of ASFV HAI serologic groups and their diversity in nature, as well as improved methods to serotype ASFV isolates, is needed. Here, we demonstrate that the genetic locus encoding ASFV CD2v and C-type lectin proteins mediates HAI serologic specificity and that CD2v/C-type lectin genotyping provides a simple method to group ASF viruses by serotype, thus facilitating study of ASFV strain diversity in nature and providing information necessary for eventual vaccine design, development and efficacious use.
The HMPV fusion protein (F) is the most immunodominant protein, yet subunit vaccines containing only this protein do not confer complete protection. The HMPV matrix protein (M) induces the maturation of antigen presenting cells in vitro. The inclusion of the M protein into an F protein subunit vaccine might therefore provide an adjuvant effect. We administered the F protein twice intramuscularly, adjuvanted with alum, the M protein or both, to BALB/c mice at 3-week intervals. Three weeks after the boost, mice were infected with HMPV and monitored for 14 days. At day 5 post challenge, pulmonary viral titers, histopathology and cytokine levels were analyzed. Mice immunized with F+alum and F+M+alum generated significantly more neutralizing antibodies than mice immunized with F only (titers of 47pplusmn;7 (pllt;0.01) and 147pplusmn;13 (pllt;0.001) vs 17pplusmn;2). Unlike F only (1.6pplusmn;0.5x103 TCID50/g lung), pulmonary viral titers in mice immunized with F+M and F+M+alum were undetectable. Mice immunized with F+M presented the most important reduction in pulmonary inflammation and the lowest Th2/Th1 cytokine ratio. In conclusion, addition of the HMPV M protein to an F protein-based vaccine modulates both humoral and cellular immune responses to subsequent infection, thereby increasing the protection conferred by the vaccine.
The segment-specific non-coding regions (NCRs) of influenza A virus RNA genome play important roles in controlling viral RNA transcription, replication and genome packaging. In this report, we present, for the first time to our knowledge, a full view of the segment-specific (NCRs) of all influenza A viruses by bioinformatics analysis. Our systematic functional analysis reveal that the eight segment-specific NCRs could differently regulate viral RNA synthesis and protein expression at both transcription and translation levels. Interestingly, a highly conserved suboptimal nucleotide at -3 position of Kozak sequence, that could down-regulate protein expression at translation level, is only present in the segment-specific NCR of PB1. By reverse genetics, we demonstrate that recombinant viruses with an optimized Kozak sequence at -3 position in PB1 results in a significant multiple-cycle replication reduction that is independent of PB1-F2 expression. Our detailed dynamic analysis of the virus infection reveals that, the mutant virus displays slightly altered dynamics from the wild type virus on both viral RNA synthesis and protein production. Furthermore, we demonstrate that the level of PB1 expression is involved in regulating type I interferon production. Together, these data reveal a novel strategy exploited by influenza A virus to fine-tune virus replication dynamics and host anti-viral response through regulating PB1 protein expression.
Bats are important reservoirs of many viruses with zoonotic potential worldwide, including Europe. Among bat viruses, members of the Picornaviridae family remain a neglected group. We performed viral metagenomic analyses on Miniopterus schreibersii bat fecal samples, collected in Hungary in 2013. In the present study we report the first molecular data and genomic characterization of a novel picornavirus from the bat species M. schreibersii in Europe. Based on phylogenetic analyses, the novel bat picornaviruses unambiguously belong to the Mischivirus genus and were highly divergent from other bat-derived picornaviruses of the Sapelovirus genus. Although the Hungarian viruses were most closely related to Mischivirus A, they formed a separate monophyletic branch within the genus.
Yellow fever virus (YFV)-17D is an empirically developed, highly effective live-attenuated vaccine that has been administered to human beings for almost a century. YFV-17D has stood as a paradigm for a successful viral vaccine, and has been exploited as a potential virus vector for the development of recombinant vaccines against other diseases. In this study, a DNA-launched YFV-17D construct (pBeloBAC-FLYF) was explored as a new modality to the standard vaccine to combine the commendable features of both DNA vaccine and live attenuated viral vaccine. The DNA-launched YFV-17D construct was characterized extensively both in cell culture and in mice. High titers of YFV-17D were generated upon transfection of the DNA into cells, whereas a mutant with deletion in the capsid coding region (pBeloBAC-YF/C) was restricted to a single round of infection, with no release of progeny virus. Homologous prime-boost immunization of mice with both pBeloBAC-FLYF and pBeloBAC-YF/C elicited specific dose-dependent cellular immune response against YFV-17D. Vaccination of A129 mice with pBeloBAC-FLYF resulted in induction YFV-specific neutralizing antibodies in all vaccinated subjects. These promising results underlined the potential of the DNA-launched YFV both as an alternative to standard YFV-17D vaccination but also as a vaccine platform for the development of DNA-based recombinant YFV vaccines.
Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cellular DNA deaminases that restrict a broad spectrum of lentiviruses. This process is counteracted by the viral infectivity factor (Vif) of lentiviruses, which binds APOBEC3s and promotes their degradation. Core binding factor subunit bbeta; (CBF-bbeta;) is an essential co-factor for the function of human immunodeficiency virus type 1 Vif to degrade human APOBEC3s. However, the requirement for CBF-bbeta; in Vif-mediated degradation of other mammalian APOBEC3 proteins is less clear. Here we determine the sequence of feline CBFB and perform phylogenetic analyses. These analyses reveal that mammalian CBFB is under purifying selection. Moreover, we demonstrate that CBF-bbeta; is dispensable for feline immunodeficiency virus Vif-mediated degradation of APOBEC3s of its host. These findings suggest that primate lentiviruses have adapted to use CBF-bbeta;, an evolutionary stable protein, to counteract APOBEC3 proteins of their hosts after diverging from other lentiviruses.
Many insect-transmissible pathogens are transmitted by specific insect species and not by others, even if the insect species are closely related. The molecular mechanisms underlying such strict pathogen-insect specificity are poorly understood. Rice dwarf virus (RDV), a plant reovirus, is mainly transmitted by the leafhopper species Nephotettix cincticeps, but is ineffectively transmitted by the leafhopper Recilia dorsalis. Here, we demonstrated that virus-containing tubules composed of viral nonstructural protein Pns10 of RDV, associated with the intestinal microvilli of N. cincticeps, but not with those of R. dorsalis. Furthermore, Pns10 of RDV specifically interacted with cytoplasmic actin, the main component of microvilli of N. cincticeps, but not with that of R. dorsalis, suggesting that the interaction of Pns10 with insect cytoplasmic actin was consistent with the transmissibility of RDV by leafhoppers. All these results suggested that the interaction of Pns10 of RDV with insect cytoplasmic actin may determine pathogen-vector specificity.
We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here we show that the same mutations improve the growth kinetics of a virus containing the A/VietNam/1203/2004 (H5N1) HA and NA. Total protein yield and neuraminidase activity were increased when a chimeric NA was included. These increases indicate that the synergistic effect is due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.
During productive infection with Epstein-Barr virus (EBV), a dramatic suppression of cellular protein expression is caused by the viral alkaline exonuclease BGLF5. Among the proteins downregulated by BGLF5 are multiple immune components. Here, we show that shutoff reduces expression of the innate EBV-sensing Toll-like receptor-2 and the lipid antigen-presenting CD1d molecule, thereby identifying these proteins as novel targets of BGLF5.
To silence BGLF5 expression in B cells undergoing productive EBV infection, we employed an shRNA approach. Viral replication still occurred in these cells, albeit with reduced late gene expression. Surface levels of a group of proteins, including immunologically relevant molecules such as CD1d and HLA class I and class II, were only partly rescued by depletion of BGLF5, suggesting that additional viral gene products interfere with their expression. Our combined approach thus provides a means to unmask novel EBV (innate) immune evasion strategies that may operate in productively infected B cells.
Human noroviruses are one of the major causes of acute gastroenteritis worldwide. Due to the lack of an efficient human norovirus cell culture system coupled with an animal model, human norovirus research mainly relies on human volunteer studies and surrogate models. Current models either utilize human norovirus infected animals including the gnotobiotic pig or calf and the chimpanzee models, or employ other members of the Caliciviridae family including cell culture propagable surrogate caliciviruses such as the feline calicivirus, murine norovirus and most recently the Tulane virus. One of the major features of human noroviruses is their extreme biological diversity, including genetic, antigenic and histo-blood group antigen binding diversity, and possible differences of virulence and environmental stability. This extreme biological diversity and its effect on intervention/prevention strategies cannot be modeled by uniform groups of surrogates much less by single isolates. Tulane virus, the prototype recovirus strain, was discovered in 2008. Since then, several other novel recoviruses have been described and cell culture adapted. Recent studies indicate that the epidemiology, the biological features and diversity of recoviruses and the course of infection and clinical disease in recovirus infected macaques more closely reflect those properties of human noroviruses than any of the current surrogates. This review aims to summarize what is currently known about recoviruses, highlight their biological similarities to human noroviruses and discuss applications of the model in addressing questions relevant for human norovirus research.
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.
As a member of the newly established Betaflexiviridae family, Grapevine rupestris stem pitting-associated virus (GRSPaV) has a RNA genome containing five open reading frames (ORFs). ORF1 encodes a putative replicase polyprotein typical of the Alphavirus superfamily of positive-strand, ssRNA viruses. Several viruses of this superfamily have been demonstrated to replicate in structures designated as viral replication complexes associated with intracellular membranes. However, structure and cellular localization of the replicase complex have not been studied for members of Betaflexiviridae, a family of mostly woody plant viruses. As a first step toward the elucidation of the replication complex of GRSPaV, we investigated the subcellular localization of full-length and truncated versions of its replicase polyprotein via fluorescent tagging, followed by fluorescence microscopy. We found that the replicase polyprotein formed distinctive punctate bodies in both Nicotiana benthamiana leaf cells and tobacco protoplasts. We further mapped a region of 76 amino acid in the methyl-transferase domain responsible for the formation of these punctate structures. These punctate structures are distributed in close proximity to the endoplasmic reticulum network. Membrane flotation and biochemical analyses demonstrate that the N-terminal region responsible for punctate structure formation associated with cellular membrane likely through an amphipathic a helix serving as an in-plane anchor. The identity of this membrane is yet to be determined. This is, to our knowledge, the first report on the localization and membrane association of the replicase proteins of a member of the family Betaflexiviridae.
Interferon-induced restriction factors can significantly affect the replicative capacity of retroviruses in mammals. Tripartite motif protein 5, isoform aalpha; (TRIM5aalpha;) is a restriction factor that acts at early stages of the virus life cycle by intercepting and destabilizing incoming retroviral cores. Sensitivity to TRIM5aalpha; maps to the N-terminal domain of the retroviral capsid (CA) proteins. In several New World and Old World monkey species, independent events of retrotransposon-mediated insertion of the cyclophilin A (CypA) coding sequence in the trim5 gene have given rise to TRIMCyp (also called TRIM5-CypA), a hybrid protein that is active against some lentiviruses in a species-specific fashion. In particular, TRIMCyp from the owl monkey (omkTRIMCyp) very efficiently inhibits HIV-1. Previously, we showed that disrupting the integrity of microtubules (MTs) and of cytoplasmic dynein complexes partially rescued replication of retroviruses, including HIV-1, from restriction mediated by TRIM5aalpha;. Here we show that efficient restriction of HIV-1 by omkTRIMCyp is similarly dependent on the MT network and on dynein complexes, but in a context-dependent fashion. When omkTRIMCyp was expressed in human HeLa cells, restriction was partially counteracted by pharmacological agents targeting MTs or by siRNA-mediated inhibition of dynein. The same drugs (nocodazole and paclitaxel) also rescued HIV-1 from restriction in cat CRFK cells, although to a lesser extent. Strikingly, neither nocodazole, paclitaxel nor depletion of the dynein heavy chain (DHC) had a significant effect on the restriction of HIV-1 in an owl monkey cell line. These results suggest the existence of cell-specific functional interactions between MTs/dynein and TRIMCyp.
The division of viruses into orders, families, genera and species provides a classification framework that seeks to organise and make sense of the diversity of viruses infecting animals, plants and bacteria. Classifications are based on similarities in genome structure and organisation, the presence of homologous genes and sequence motifs, and, at lower levels such as species, host range, nucleotide and antigenic relatedness and epidemiology. Classification below the level of family must also be consistent with phylogeny and virus evolutionary histories. Recently developed methods such as PASC, DEMaRC and NVR offer alternative strategies for genus and species assignments that are based purely on degrees of divergence between genome sequences. They offer the possibility of automating classification of the vast number of novel virus sequences being generated by next generation metagenomic sequencing. However, distance-based methods struggle to deal with the complex evolutionary history of virus genomes which are shuffled by recombination and re-assortment and where taxonomic lineages evolve at different rates. In biological terms, classifications based on sequences distances alone are also arbitrary whereas the current system of virus taxonomy is of utility precisely because it is primarily based upon phenotypic characteristics. However, a separate system is clearly needed by which virus variants that lack biological information might be incorporated in to the ICTV classification even if based solely on sequence relationships to existing taxa. For these, simplified taxonomic proposals and naming conventions represent a practical way to expand the existing virus classification and catalogue our rapidly increasing knowledge of virus diversity.
The human genome is comprised of 8% endogenous retroviruses (ERVs), the majority of which are defective due to deleterious mutations. Nonetheless, transcripts of ERVs are found in most tissues and these transcripts could either be reverse transcribed to generate single-stranded DNA or expressed to generate proteins. Thus, the expression of ERVs could produce nucleic acids or proteins with viral signatures, much like the pathogen-associated molecular patterns (PAMPs) of exogenous viruses, which would enable them to be detected by the innate immune system. The activation of some pattern recognition receptors (PRRs) in response to ERVs has been described in mice and in the context of human autoimmune diseases. Here, we review the evidence for detection of ERVs by PRRs and the resultant activation of innate immune signalling. This is an emerging area of research within the field of innate antiviral immunity, showing how ERVs could possibly initiate immune signalling pathways and might have implications for numerous inflammatory diseases.
Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally transmitted by mosquitoes. During the infection, JEV generally enters host cells via receptor-mediated clathrin-dependent endocytosis that requires the involvement of the 70 kDa heat shock protein (Hsp70). Heat shock cognate protein 70 (Hsc70) is one member of the Hsp70 family and is mainly constitutive; thus, it may be expressed at physiological condition. In C6/36 cells, Hsc70 is up-regulated in response to JEV infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface, it probably does not serve as the receptor according to our results in the present study. In contrast, Hsc70 is evidently associated with virus penetration into the cell and resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly involved in clathrin-mediated endocytosis, particularly at a late stage of viral entry into host cells. Furthermore, we found that Hsc70 is composed of at least three isoforms, including B, C, and D. Of them, the isoform D is the one that helps JEV to penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant evidence that sheds light on the regulatory mechanisms of JEV infection in host cells, especially on the process of clathrin-mediated endocytosis.
Since one of us co-authored a review on NS5A a decade ago the hepatitis C virus (HCV) field has changed dramatically, primarily due to the advent of the JFH-1 cell culture infectious clone which allowed the study of all aspects of the virus lifecycle from entry to exit. This review will describe advances in our understanding of NS5A biology over the past decade, highlighting how the JFH-1 system has allowed us to determine that NS5A is essential not only in genome replication but also in the assembly of infectious virions. We will review the recent structural insights - NS5A is predicted to comprise 3 domains, X-ray crystallography has revealed the structure of domain I but there is a lack of detailed structural information about the other two domains which are predicted to be largely unstructured. Recent insights into the phosphorylation of NS5A will be discussed, and we will highlight a few pertinent examples from the ever expanding list of NS5A binding partners identified over the past decade. Lastly, we will review the literature showing that NS5A is a potential target for a new class of highly potent small molecules that function to inhibit virus replication. These direct acting antivirals (DAAs) are now either licenced, or in the late stages of approval, for clinical use both in the USA and UK/Europe. In combination with other DAAs targeting the viral protease (NS3) and polymerase (NS5B), they are revolutionising treatment for HCV infection.
A fundamental aspect of herpesviruses is their ability to establish a life long latent infection within the host. Current research would suggest that small regulatory RNAs called microRNAs play a central role in all three aspects of virus latency and persistence. In this review we will discuss the role of miRNAs in virus persistence and latency, specifically focusing on herpesviruses. We will cover the current knowledge on the role of miRNAs in establishing and maintaining virus latency and promoting survival of infected cells through targeting of both viral and cellular transcripts, highlighting key publications in the field. We will also discuss potential areas of future research and how novel technologies may aid in determining how miRNAs shape virus latency in the context of herpesvirus infections.
The stability and conservation of the sequences of RNA viruses in the field and the high error rates measured in vitro are paradoxical. The field stability indicates that there are very strong selective constraints on sequence diversity. The nature of these constraints is discussed. Apart from constraints on variation in cis-acting RNA and the amino acid sequences of viral proteins, there are other ones relating to the presence of specific dinucleotides such CpG and UpA as well as the importance of RNA secondary structures and RNA degradation rates. Recent other constraints identified in other RNA viruses such as effects of secondary RNA structure on protein folding or modification of cellular tRNA complements are also discussed. Using the family Paramyxoviridae I show that the codon usage pattern (CUP) is (i) specific for each virus species and (ii) that it is markedly different from the host - it does not vary even in vaccine viruses that have been derived by passage in a number of inappropriate host cells. The CUP might thus be an additional constraint on variation and I propose the concept of codon constellation to indicate the informational content of the sequences of RNA molecules relating not only to stability and structure but also to the efficiency of translation of a viral mRNA resulting from the CUP and the numbers and position of rare codons.
Hepatitis E virus (HEV), the causative agent of hepatitis E, is a single-stranded positive-sense RNA virus belonging to the family Hepeviridae. At least four genotypes of the family infect humans: genotypes 1 and 2 are transmitted to humans through contaminated water, while genotypes 3 and 4 are zoonotic and have animal reservoirs. A novel strain of HEV recently identified in rabbits is a distant member of genotype 3, and thus poses a potential risk of zoonotic transmission to humans. The objective of this study was to construct and characterize an infectious cDNA clone of the rabbit HEV. Two full-length cDNA clones of rabbit HEV, pT7g-rabHEV and pT7-rabHEV, were constructed and their infectivity was tested by in vitro transfection of Huh7 human liver cells and by direct intrahepatic inoculation of rabbits with capped RNA transcripts. Results showed that positive signal for rabbit HEV protein was detected by an immunofluorescence assay with a HEV-specific antibody in Huh7 human liver cells transfected with capped RNA transcripts from the two full-length cDNA clones. Rabbits intrahepatically inoculated with capped RNA transcripts from each of the two clones developed active HEV infection as evidenced by seroconversion to anti-HEV antibodies, and detection of rabbit HEV RNA in sera and feces of inoculated animals. The availability of a rabbit HEV infectious cDNA clone now affords us the ability to delineate the mechanism of HEV replication and cross-species infection in a small animal model.