|Journal of General Virology current issue|
Chikungunya virus is a mosquito-borne virus that has been responsible for over 2 million human infections during the past decade. This virus, which previously had a geographical range primarily restricted to sub-Saharan Africa, the Indian subcontinent and South East Asia, has recently moved to subtropical latitudes as well as the western hemisphere. This expansion into novel habitats brings unique risks associated with further spread of the virus and the disease it causes.
In December 1983, a seminal paper appeared on the overexpression of human IFN-bbeta; in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produced massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes were dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years, major improvements were achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells and the scale-up of the cell culture process. To date, thousands of recombinant proteins have been produced in this successful expression system, including several protein-based human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted BEVs are used to deliver and express heterologous genes in mammalian cells, and they may be used for gene therapy and cancer treatment in the future. The purpose of this review is to highlight the thirtieth llsquo;anniversaryrrsquo; of this expression system by summarizing the fundamental research and major technological advances that allowed its development, whilst noting challenges for further improvements.
Bats have been found to harbour a number of new emerging viruses with zoonotic potential, and there has been a great deal of interest in identifying novel bat pathogens to determine the risk to human and animal health. Many groups have identified novel viruses in bats by detection of viral nucleic acid; however, virus isolation is still a challenge, and there are few reports of viral isolates from bats. In recent years, our group has developed optimized procedures for virus isolation from bat urine, including the use of primary bat cells. In previous reports, we have described the isolation of Hendra virus, Menangle virus and Cedar virus in Queensland, Australia. Here, we report the isolation of four additional novel bat paramyxoviruses from urine collected from beneath pteropid bat (flying fox) colonies in Queensland and New South Wales during 2009nndash;2011.
A common critical cellular event that many human enveloped viruses share is the requirement for proteolytic cleavage of the viral glycoprotein by furin in the host secretory pathway. For example, the furin-dependent proteolytic activation of highly pathogenic (HP) influenza A (infA) H5 and H7 haemagglutinin precursor (HA0) subtypes is critical for yielding fusion-competent infectious virions. In this study, we hypothesized that viral hijacking of the furin pathway by HP infA viruses to permit cleavage of HA0 could represent a novel molecular mechanism controlling the dynamic production of fusion-competent infectious virus particles during the viral life cycle. We explored the biological role of a newly identified furin-directed human microRNA, miR-24, in this process as a potential post-transcriptional regulator of the furin-mediated activation of HA0 and production of fusion-competent virions in the host secretory pathway. We report that miR-24 and furin are differentially expressed in human A549 cells infected with HP avian-origin infA H5N1. Using miR-24 mimics, we demonstrated a robust decrease in both furin mRNA levels and intracellular furin activity in A549 cells. Importantly, pretreatment of A549 cells with miR-24 mimicked these results: a robust decrease of H5N1 infectious virions and a complete block of H5N1 virus spread that was not observed in A549 cells infected with low-pathogenicity swine-origin infA H1N1 virus. Our results suggest that viral-specific downregulation of furin-directed microRNAs such as miR-24 during the life cycle of HP infA viruses may represent a novel regulatory mechanism that governs furin-mediated proteolytic activation of HA0 glycoproteins and production of infectious virions.
Newcastle disease virus (NDV) has been developed as a vector for vaccine and gene therapy purposes. However, the optimal insertion site for foreign gene expression remained to be determined. In the present study, we inserted the green fluorescence protein (GFP) gene into five different intergenic regions of the enterotropic NDV VG/GA vaccine strain using reverse genetics technology. The rescued recombinant viruses retained lentogenic pathotype and displayed delayed growth dynamics, particularly when the GFP gene was inserted between the NP and P genes of the virus. The GFP mRNA level was most abundant when the gene was inserted closer to the 3' end and gradually decreased as the gene was inserted closer to the 5' end. Measurement of the GFP fluorescence intensity in recombinant virus-infected cells demonstrated that the non-coding region between the P and M genes is the optimal insertion site for foreign gene expression in the VG/GA vaccine vector.
The outbreak of human infections caused by the novel avian-origin H7N9 influenza viruses in China since March 2013 underscores the urgent need to find an effective treatment strategy against H7N9 infection in humans. In this study, we assessed the effectiveness of combinations of oseltamivir and two immunomodulators (simvastatin and fenofibrate) against H7N9 infection in a mouse model. Mice treated with oseltamivir plus fenofibrate exhibited the longest mean survival time, the largest reduction of viral titre in lung tissue, the highest levels of CD4+ and CD8+ T-lymphocytes, and the greatest decrease in pulmonary inflammation. Thus, the combination of oseltamivir plus fenofibrate improved the outcomes of mice infected with H7N9 virus by simultaneously reducing viral replication and normalizing the aberrant immune response. This drug combination should be considered in randomized controlled trials of treatments for H7N9 patients.
The antibody response plays a crucial role against hepatitis C virus (HCV) infection, and our understanding of this intricate progress in vivo is far from complete. We previously reported a novel and robust technique based on a large combinatorial viral antigen library displayed on the surface of the yeast Saccharomyces cerevisiae, allowing comprehensive profiling of polyclonal antibody responses in vivo in both qualitative and quantitative terms. Here, we report the generation and application of a combinatorial library of HCV strain JFH1 envelope glycoprotein to profile the antibody response in four HCV chronically infected individuals. By systematic analysis of the location and frequency of antigenic fragments along the JFH1 envelope glycoprotein, we showed that the major binding antibody response was targeted to E2 (80.9nndash;99.8 %), whilst that against E1 was relatively small (0.3nndash;19.0 %). A total of five major antigenic domains (D1nndash;D5) were identified: one was within E1 and an additional four within E2, despite substantial variability among the different individuals. However, serum absorption with the yeast clones containing the antigenic domain D1 resulted in more reduction in neutralizing antibody activity against pseudotyped HCV than those in E2, suggesting that E1 contains additional neutralizing epitopes. Our results have provided additional insights into the HCV-specific antibody response in humans and should assist in a better understanding of protective antibody immunity and in guiding the development of effective vaccines and therapeutics against HCV infection.
In contrast to the current wealth of structural information concerning dicistrovirus particle structure, very little is known about their morphogenetic pathways. Here, we describe the expression of the two ORFs encoded by the Triatoma virus (TrV) genome. TrV, a member of the Cripavirus genus of the Dicistroviridae family, infects blood-sucking insects belonging to the Triatominae subfamily that act as vectors for the transmission of Trypanosoma cruzi, the aetiological agent of the Chagas disease. We have established a baculovirus-based model for the expression of the NS (non-structural) and P1 (structural) polyproteins. A preliminary characterization of the proteolytic processing of both polyprotein precursors has been performed using this system. We show that the proteolytic processing of the P1 polyprotein is strictly dependent upon the coexpression of the NS polyprotein, and that NS/P1 coexpression leads to the assembly of virus-like particles (VLPs) exhibiting a morphology and a protein composition akin to natural TrV empty capsids. Remarkably, the unprocessed P1 polypeptide assembles into quasi-spherical structures conspicuously larger than VLPs produced in NS/P1-coexpressing cells, likely representing a previously undescribed morphogenetic intermediate. This intermediate has not been found in members of the related Picornaviridae family currently used as a model for dicistrovirus studies, thus suggesting the existence of major differences in the assembly pathways of these two virus groups.
Porcine sapovirus (PSaV) of the family Caliciviridae, is the only member of the genus Sapovirus with cell culture and reverse genetics systems. When combined with the piglet model, these approaches provide a system to understand the molecular basis of sapovirus pathogenesis. The replication of PSaV in cell culture is, however, restricted, displaying an absolute requirement for bile acids and producing lower levels of infectious virus than other caliciviruses. The effect of bile acids has previously been linked to a reduction in the signal transducer and activator of transcription (STAT1)-mediated signalling pathway. In the current study, we observed that even in the presence of bile acids, PSaV replication in cell culture was restricted by soluble factors produced from infected cells. This effect was at least partially due to secreted IFN because treatment of cells with recombinant porcine IFN-bbeta; resulted in significantly reduced viral replication. Moreover, IFN-mediated signalling pathways (IFN, STAT1 and the 2',5'-oligoadenylate synthetase) were activated during PSaV infection. Characterization of PSaV growth in cell lines deficient in their ability to induce or respond to IFN showed a 100nndash;150-fold increase in infectious virus production, indicating that the primary role of bile acids was not the inactivation of the innate immune response. Furthermore, the use of IFN-deficient cell lines enabled more efficient recovery of PSaV from cDNA constructs. Overall, the highly efficient cell culture and reverse genetics system established here for PSaV highlighted the key role of the innate immune response in the restriction of PSaV infection and should greatly facilitate further molecular studies on sapovirus hostnndash;cell interactions.
MicroRNAs (miRNAs) are a class of short endogenous RNA molecules with the ability to control development, autophagy, apoptosis and the stress response in eukaryotes by pairing with partially complementary sites in the 3' UTRs of targeted genes. Recent studies have demonstrated that miRNAs serve as critical effectors in intricate networks of hostnndash;pathogen interactions. Notably, we found that Bos taurus bta-miR-29b (referred to as miR-29b herein) was significantly upregulated ggt;2.3-fold in bovine viral diarrhoea virus (BVDV) strain NADL-infected Madinnndash;Darby bovine kidney (MDBK) cells 6 h post-infection compared with normal MDBK cells. However, the roles of miR-29b in BVDV infection and pathogenesis remain unclear. Here, we report the inhibitory effects of miR-29b on BVDV NADL replication and viral infection-related autophagy. miR-29b overexpression mediated by miRNA precursor-expressing lentivirus resulted in the attenuation of BVDV NADL infection-related autophagy by directly downregulating the intracellular expression levels of two key autophagy-associated proteins, ATG14 and ATG9A. Moreover, ATG14 and ATG9A overexpression rescue not only reversed miR-29b-inhibited autophagy, but also increased BVDV NADL replication. In previous studies, we found that the early stages of autophagy contributed to BVDV NADL replication in MDBK cells and that the inhibition of autophagy repressed BVDV NADL replication, which was also proved in the present study. Collectively, our results establish a novel link between miR-29b and viral replication, and also provide a new pathway for the intimate interaction between host cells and pathogens.
Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) genome segment S4 showed that it consists of 3410 nt with a single ORF of 1110 aa which could encode a protein of ~127 kDa (p127). Bioinformatics analysis showed the presence of a 5' RNA triphosphatase (RTPase) domain (LRDR), a S-adenosyl-
Group A rotaviruses (RVAs) are enteric pathogens with well-documented zoonotic transmissions to humans. The segmented genome of the virus enables reassortment events which might alter host susceptibility and/or disease course. Genetic analysis of rotavirus in dogs has so far only revealed RVAs with the VP7 and VP4 genome constellation G3P. RVA G3P have also been found in cats, humans, monkeys and bats. In the present study, we described an unusual RVA of genotype G8P which was isolated from an asymptomatically infected young dog. The dog did not show signs of diarrhoea. Analysis of full-length segments of VP2, VP6 and VP7 as well as NSP1nndash;NSP5 revealed a typical bovine-like genotype constellation G8nndash;Pnndash;I2nndash;Rxnndash;C2nndash;Mxnndash;A3nndash;N2nndash;T6nndash;E2nndash;H3. Phylogenetic analysis supported the hypothesis of an interspecies transmission from a bovine/artiodactyl species or from humans to the young dog. The isolate was likely to represent a multiple reassortant virus.
Torque teno virus (TTV) is increasingly considered a universal marker of global immune function. The virus is supposed to replicate in lymphocytes, but poor information is available about fluctuations of viraemia after administration of anti-lymphocyte agents. We studied TTV kinetics in a cohort of 70 kidneypplusmn;pancreas recipients receiving one of two different anti-T-cell induction immunosuppressants. During the first 30 days after anti-T-cell antibody administration, we report kinetics of TTV viraemia compatible with replication in T lymphocytes, and highly dependent on the potency of the anti-T-cell drug administered.
Equine herpesvirus type 1 (EHV-1) replicates in the epithelial cells of the upper respiratory tract and disseminates through the body via a cell-associated viraemia in monocytic cells, despite the presence of neutralizing antibodies. However, the mechanism by which EHV-1 hijacks immune cells and uses them as llsquo;Trojan horsesrrsquo; in order to disseminate inside its host is still unclear. Here, we hypothesize that EHV-1 delays its replication in monocytic cells in order to avoid recognition by the immune system. We compared replication kinetics in vitro of EHV-1 in RK-13, a cell line fully susceptible to EHV-1 infection, and primary horse cells from the myeloid lineage (CD172a+). We found that EHV-1 replication was restricted to 4 % of CD172a+ cells compared with 100 % in RK-13 cells. In susceptible CD172a+ cells, the expression of immediate-early (IEP) and early (EICP22) proteins was delayed in the cell nuclei by 2nndash;3 h post-infection (p.i.) compared with RK-13, and the formation of replicative compartments by 15 h p.i. Virus production in CD172a+ cells was significantly lower (from 101.7 to 103.1 TCID50 per 105 inoculated cells) than in RK-13 (from 105 to 105.7 TCID50 per 105 inoculated cells). Less than 0.02 % of inoculated CD172a+ cells produced and transmitted infectious virus to neighbouring cells. Pre-treatment of CD172a+ cells with inhibitors of histone deacetylase activity increased and accelerated viral protein expression at very early times of infection and induced productive infection in CD172a+ cells. Our results demonstrated that the restriction and delay of EHV-1 replication in CD172a+ cells are part of an immune evasive strategy and involve silencing of EHV-1 gene expression associated with histone deacetylases.
Graft rejection in transplant patients is managed clinically by suppressing T-cell function with immunosuppressive drugs such as prednisolone and methylprednisolone. In such immunocompromised hosts, human cytomegalovirus (HCMV) is an important opportunistic pathogen and can cause severe morbidity and mortality. Currently, the effect of glucocorticosteroids (GCSs) on the HCMV life cycle remains unclear. Previous reports showed enhanced lytic replication of HCMV in vitro in the presence of GCSs. In the present study, we explored the implications of steroid exposure on latency and reactivation. We observed a direct effect of several GCSs used in the clinic on the activation of a quiescent viral major immediate-early promoter in stably transfected THP-1 monocytic cells. This activation was prevented by the glucocorticoid receptor (GR) antagonist Ru486 and by shRNA-mediated knockdown of the GR. Consistent with this observation, prednisolone treatment of latently infected primary monocytes resulted in HCMV reactivation. Analysis of the phenotype of these cells showed that treatment with GCSs was correlated with differentiation to an anti-inflammatory macrophage-like cell type. On the basis that these observations may be pertinent to HCMV reactivation in post-transplant settings, we retrospectively evaluated the incidence, viral kinetics and viral load of HCMV in liver transplant patients in the presence or absence of GCS treatment. We observed that combination therapy of baseline prednisolone and augmented methylprednisolone, upon organ rejection, significantly increased the incidence of HCMV infection in the intermediate risk group where donor and recipient are both HCMV seropositive (D+R+) to levels comparable with the high risk D+Rnndash; group.
Infection of astrocytes by the neuropathogenic mutant of Moloney murine leukemia virus, ts1, exhibits increased levels of reactive oxygen species (ROS) and signs of oxidative stress compared with uninfected astrocytes. Previously, we have demonstrated that ts1 infection caused two separate events of ROS upregulation. The first upregulation occurs during early viral establishment in host cells and the second during the virus-mediated apoptotic process. In this study, we show that virus-mediated ROS upregulation activates the protein kinase, ataxia telangiectasia mutated, which in turn phosphorylates serine 15 on p53. This activation of p53 however, is unlikely associated with ts1-induced cell death. Rather p53 appears to be involved in suppressing intracellular ROS levels in astrocytes under oxidative stress. The activated p53 appears to delay retroviral gene expression by suppressing NADPH oxidase, a superoxide-producing enzyme. These results suggest that p53 plays a role as a retrovirus-mediated oxidative stress modulator.
Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-producing chickens that had developed myeloid leukosis. However, ALV-J infections associated with hemangiomas have occurred in egg-producing (layer) flocks in China. In this study, we identified an ALV-J layer isolate (HLJ13SH01) as a recombinant of ALV-J and a Rous sarcoma virus Schmidt-Ruppin B strain (RSV-SRB), which contained the RSV-SRB 5'-LTR and the other genes of ALV-J. Replication kinetic testing indicated that the HLJ13SH01 strain replicated faster than other ALV-J layer isolates in vitro. Sequence analysis indicated that the main difference between the two isolates was the 5'-LTR sequences, particularly the U3 sequences. A 19 nt insertion was uniquely found in the U3 region of the HLJ13SH01 strain. The results of a Dual-Glo luciferase assay revealed that the 19 nt insertion in the HLJ13SH01 strain increased the enhancer activity of the U3 region. Moreover, an additional CCAAT/enhancer element was found in the 19 nt insertion and the luciferase assay indicated that this element played a key role in increasing the enhancer activity of the 5'-U3 region. To confirm the potentiation effect of the 19 nt insertion and the CCAAT/enhancer element on virus replication, three infectious clones with 5'-U3 region variations were constructed and rescued. Replication kinetic testing of the rescued viruses demonstrated that the CCAAT/enhancer element in the 19 nt insertion enhanced the replication capacity of the ALV-J recombinant in vitro.
Human T-cell leukaemia virus type-1 (HTLV-1) infection causes adult T-cell leukaemia (ATL). The viral protein HTLV-1 bZIP factor (HBZ) is constitutively expressed in ATL cells, suggesting that HBZ plays a major role in the pathogenesis of HTLV-1-associated disease. Here, we identified centromere protein B (CENP-B) as a novel interacting partner of HBZ. HBZ and CENP-B associate with their central regions in cells. Furthermore, overexpression of HBZ abrogated the DNA-binding activity of CENP-B to the aalpha;-satellite DNA region containing the CENP-B box motif, which in turn inhibited the CENP-B-mediated trimethylation of histone H3K9 in T-cells.
Grapevine vein clearing virus (GVCV) is a recently discovered DNA virus in grapevine that is closely associated with the grapevine vein clearing syndrome observed in vineyards in Missouri and surrounding states. The genome sequence of GVCV indicates that it belongs to the genus Badnavirus in the family Caulimoviridae. To identify the GVCV promoter, we cloned portions of the GVCV large intergenic region in front of a GFP gene present in an Agrobacterium tumefaciens binary vector. GFP expression was assessed by ELISA 3 days after agroinfiltration of Nicotiana benthamiana leaves. We found that the GVCV DNA segment between nts 7332 and 7672 directed expression of GFP and this expression was stronger than expression using the Cauliflower mosaic virus 35S promoter. It was revealed by 5' and 3' RACE that transcription was initiated predominantly at nt 7571 and terminated at nt 7676.
The IFN immune system plays an essential role in protecting the host against most viral infections. In order to explore the interactions between the IFN pathway and human respiratory syncytial virus (RSV) infection, and to identify potential IFN-stimulated genes (ISGs) that may be involved in suppressing the replication of RSV, we utilized an IFN pathway-specific microarray to study the effects of RSV infection on the IFN pathway in HeLa cells. We showed that RSV infection enhanced the expression of a series of ISGs, including oligoadenylate synthetase 2, IFITM1 (IFN-induced transmembrane protein 1) and myxovirus resistance 2. Our results also showed that the IFITM proteins potently inhibited RSV infection mainly by interfering with both virus entry and the subsequent replication steps, but not the attachment process. The antiviral effect of IFITM3 was not affected by ubiquitination modification. Furthermore, knocking down the endogenous and IFN-induced expression of IFITM1 and 3 facilitated RSV infection. Expression of the IFITM proteins was found to delay the phosphorylation of IFN regulatory factor 3 through interfering with the detection of viral RNA by MDA5 (melanoma differentiation-associated gene 5) and RIG-I (retinoic acid-inducible gene I). These results demonstrated that the restriction of RSV infection by the IFITM proteins was achieved through the inhibition of virus entry and replication, and they provided further insight for exploring the mechanism of IFITM-protein-mediated virus restriction.
Biopsies from patients show that hepadnaviral core proteins and capsids nndash; collectively called core nndash; are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that , an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.
Invertebrate iridescent virus 6 (IIV-6) is a nucleocytoplasmic virus with a ~212 kb linear dsDNA genome that encodes 215 putative ORFs. The IIV-6 virion-associated proteins consist of at least 54 virally encoded proteins. One of our previous findings showed that most of these proteins are encoded by genes from the early transcriptional class. This indicated that these structural proteins may not only function in the formation of the virion, but also in the initial stage of viral infection. In the current study, we followed the protein expression profile of IIV-6 over time in Drosophila S2 cells by label-free quantification using a proteomic approach. A total of 95 virally encoded proteins were detected in infected cells, of which 37 were virion proteins. The expressed IIV-6 virion proteins could be categorized into three main clusters based on their expression profiles: proteins with stably low expression levels during infection, proteins with exponentially increasing expression levels during infection and proteins that were initially highly abundant, but showed slightly reduced levels after 48 h post-infection. We thus provided novel information on the kinetics of virion and infected cell-specific protein levels that assists in our understanding of gene regulation in this lesser-known DNA virus model.
Human papillomavirus (HPV) has a well-recognized aetiological role in the development of cervical cancer and other anogenital tumours. Recently, an association between colorectal cancer and HPV infection has been suggested, although this is still controversial. This study aimed at detecting and characterizing HPV infection in 57 paired biopsies from colorectal cancers and adjacent intact tissues using a degenerate PCR approach. All amplified fragments were genotyped by means of sequencing. Overall, HPV prevalence was 12.3 %. In particular, 15.8 % of tumour tissues and 8.8 % of non-cancerous tissue samples were HPV DNA-positive. Of these samples, 85.7 % were genotyped successfully, with 41.7 % of sequences identifying four genotypes of the HR (high oncogenic risk) clade Group 1; the remaining 58.3 % of HPV-genotyped specimens had an unclassified bbeta;-HPV. Examining additional cases and analysing whole genomes will help to outline the significance of these findings.
Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving the templated conversion of the normal cellular prion protein (PrPC) to a pathogenic misfolded conformation. Templated conversion has been modelled in several in vitro assays, including serial protein misfolding amplification, amyloid seeding and real-time quaking-induced conversion (RT-QuIC). As RT-QuIC measures formation of amyloid fibrils in real-time, it can be used to estimate the rate of seeded conversion. Here, we used samples from deer infected with chronic wasting disease (CWD) in RT-QuIC to show that serial dilution of prion seed was linearly related to the rate of amyloid formation over a range of 10nndash;3 to 10nndash;8 mmicro;g. We then used an amyloid formation rate standard curve derived from a bioassayed reference sample (CWD+ brain homogenate) to estimate the prion seed concentration and infectivity in tissues, body fluids and excreta. Using these methods, we estimated that urine and saliva from CWD-infected deer both contained 1nndash;5 LD50 per 10 ml. Thus, over the 1nndash;2 year course of an infection, a substantial environmental reservoir of CWD prion contamination accumulates.
|Journal of General Virology Publish Ahead of Print|
Infection with hepatitis C virus (HCV) is characterized by systemic oxidative stress that is caused by either viral Core or chronic inflammation. It is well recognized that reactive oxygen species (ROS) like H2O2 can induce apoptotic cell death and can therefore function as anti-tumorigenic species. However, the detailed mechanisms by which ROS induces apoptotic cell death and HCV copes with the oxidative condition are largely unknown. In the present study, we found that H2O2 induced apoptotic cell death in the p53-positive human hepatocytes but not in the p53-negative ones. For this effect, H2O2 up-regulated levels of p14, increased ubiquitin-dependent degradation of mouse double minute 2 (MDM2), and reduced the interaction between MDM2 and p53 to prevent p53 degradation, resulting in accumulation of p53 and subsequent activation of the p53-dependent apoptotic pathways. Interestingly, HCV Core repressed p14 expression via promoter hypermethylation to abolish the potential of H2O2 to activate the p14-MDM2-p53 pathway. As a consequence, the HCV Core-expressing cells could overcome the p53-mediated apoptosis provoked by H2O2. Taken together, HCV Core can contribute to hepatocellular carcinoma formation by removing deleterious roles of ROS inducing cell death.
During the last decades, metagenomic studies expanded the numbers of newly described, often unclassified, viruses within the Circoviridae family. Using broad-spectrum circo-/cyclovirus PCRs, we characterized a novel circo-like virus in Aedes vexans mosquitoes from Germany whose main putative open reading frames (ORFs) shared very low amino acid identity with those of previously characterized circo-/cycloviruses. Phylogenetic and genetic distance analysis revealed that this new virus species defines, with previously described mosquito- and bat feces-derived circo-like viruses, a different genus, tentatively called "krikovirus", within Circoviridae. We further demonstrated that viruses of the putative krikovirus genus all share a genomic organization which is unique among Circoviridae. Further investigations are needed to determine the host range, tissue tropism and transmission route(s). This report increases the current knowledge of the genetic diversity and evolution of the members of the Circoviridae family.
Analysing the evolution of FIV on the intra-host level is important, in order to address whether the diversity and composition of viral quasispecies affects disease progression.
We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at the rate of 1.16 x 10-3 substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3-V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3-V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation.
Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximal evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared to HIV might explain why many naturally infected cats do not progress to AIDS rapidly.
Adeno-associated virus (AAV) type 5 represents the genetically most distant AAV serotype and the only one isolated directly from human tissue. Seroepidemiological evidence suggests HSV as helper virus for human AAV5 infections underlining the in vivo relevance of the AAV-herpesvirus relationship. In this study we analyzed for the first time herpes simplex virus (HSV) helper functions for productive AAV5 replication and compared these to AAV2. Using a combination of HSV strains and plasmids for individual genes, the previously defined HSV helper functions for AAV2 replication were shown to induce AAV5 gene expression, DNA replication and production of infectious progeny. The helper functions comprise the replication genes for ICP8 (UL29), helicase-primase (UL5/8/52), and DNA polymerase (UL30/42). HSV immediate-early genes for ICP0 and ICP4 further enhanced AAV5 replication, mainly by induction of rep gene expression. In the presence of HSV helper functions AAV5 Rep colocalized with ICP8 in nuclear replication compartments and HSV alkaline exonuclease (UL12) enhanced AAV5 replication, similar to AAV2. UL12 in combination with ICP8 was shown to induce DNA strand exchange on partially double-stranded templates to resolve and repair concatemeric HSV replication intermediates. Similarly, concatemeric AAV replication intermediates appeared to be processed to yield AAV unit-length molecules, ready for AAV packaging. Taken together, our findings show that productive AAV5 replication is promoted by the same combination of HSV helper functions as AAV2.
Various herpesviruses have been discovered in marine mammals, associated with a wide spectrum of disease. In the present study we describe the detection and phylogenetic analysis of a novel gammaherpesvirus, tentatively called phocine herpesvirus 7 (PhHV-7), that was detected in samples collected during an outbreak of ulcerative gingivitis and glossitis from juvenile harbor seals (Phoca vitulina) at the Seal Rehabilitation and Research Centre, the Netherlands. The presence of this novel gammaherpesvirus was confirmed by viral metagenomics, while no other viruses except four novel anelloviruses were detected. However, PhHV-7 DNA was also detected in harbor and grey seals (Halichoerus grypus) without gingivitis or glossitis. Genetic analysis of the partial polymerase gene of PhHV-7 detected in both species revealed limited sequence variation. Additional studies are needed to elucidate whether the discovered viruses played a role in the observed disease.
A granulovirus (GV) producing occlusion bodies (OBs) with an unusual appearance was isolated from Adoxophyes spp. larvae in a field. Ultrastructural observations revealed that its OBs were significantly larger and cuboidal in shape, rather than the standard ovo-cylindrical shape typical of GVs. N-terminal amino acid sequence analysis of the OB matrix protein from this virus suggested that it was a variant of Adoxophyes orana granulovirus (AdorGV). Bioassays of this GV (termed AdorGV-M) and an English isolate of AdorGV (termed AdorGV-E) indicated that the two isolates were equally pathogenic against larvae of A. honmai. However, AdorGV-M retained more infectivity towards larvae after irradiation with UV light than AdorGV-E. Sequencing and analysis of the AdorGV-M genome revealed little sequence divergence between this isolate and AdorGV-E. Comparison of selected genes among the two AdorGVs isolates and other Japanese AdorGV isolates revealed differences that may account for the unusual OB morphology of the AdorGV-M.
African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens responsible for protection in the pig. Available data from vaccination/challenge experiments in pigs indicate ASF protective immunity is hemadsorption inhibition (HAI) serotype-specific. A better understanding of ASFV HAI serologic groups and their diversity in nature, as well as improved methods to serotype ASFV isolates, is needed. Here, we demonstrate that the genetic locus encoding ASFV CD2v and C-type lectin proteins mediates HAI serologic specificity and that CD2v/C-type lectin genotyping provides a simple method to group ASF viruses by serotype, thus facilitating study of ASFV strain diversity in nature and providing information necessary for eventual vaccine design, development and efficacious use.
Hepatitis B virus X protein (HBx) is involved in the development of hepatocellular carcinoma (HCC). The HBx sequence is a preferential site of integration into the human genome, leading to the formation of C-terminal truncated HBx proteins (Ct-HBx). We previously reported that Ct-HBx proteins were able to potentiate cell transformation in vitro. Our present goal was to compare the ability of Ct-HBx and full-length HBx (FL-HBx) proteins to develop or enhance HCC in transgenic mice. In the absence of treatment, neither Ct-HBx nor FL-HBx transgenic mice developed HCC. In young mice treated with diethylnitrosamine (DEN), at 8 months of age, a significantly higher incidence and number of liver lesions were observed in Ct-HBx mice than in FL-HBx and control mice. The earlier development of tumours in Ct-HBx transgenic mice was associated with increased liver inflammation. At 10 months, macroscopic and microscopic analyses showed that statistically, FL-HBx mice developed more liver lesions with a larger surface area compared to control mice. Following the DEN-induced initiation of HCC, Ct-HBx and FL-HBx transgenic mice displayed an increase in the inflammatory response, evidenced by IL-6, TNF-aalpha; and IL-1bbeta; expression and the activation of STAT3 ERK and JNK. This was associated with an increase in cell apoptosis following DEN treatment. In conclusion, in DEN-treated transgenic mice, the expression of Ct-HBx protein causes a more rapid onset of HCC than that of FL-HBx protein. HBV genome integration leading to the expression of a truncated form of HBx protein may therefore facilitate HCC development in chronically infected patients.
The HMPV fusion protein (F) is the most immunodominant protein, yet subunit vaccines containing only this protein do not confer complete protection. The HMPV matrix protein (M) induces the maturation of antigen presenting cells in vitro. The inclusion of the M protein into an F protein subunit vaccine might therefore provide an adjuvant effect. We administered the F protein twice intramuscularly, adjuvanted with alum, the M protein or both, to BALB/c mice at 3-week intervals. Three weeks after the boost, mice were infected with HMPV and monitored for 14 days. At day 5 post challenge, pulmonary viral titers, histopathology and cytokine levels were analyzed. Mice immunized with F+alum and F+M+alum generated significantly more neutralizing antibodies than mice immunized with F only (titers of 47pplusmn;7 (pllt;0.01) and 147pplusmn;13 (pllt;0.001) vs 17pplusmn;2). Unlike F only (1.6pplusmn;0.5x103 TCID50/g lung), pulmonary viral titers in mice immunized with F+M and F+M+alum were undetectable. Mice immunized with F+M presented the most important reduction in pulmonary inflammation and the lowest Th2/Th1 cytokine ratio. In conclusion, addition of the HMPV M protein to an F protein-based vaccine modulates both humoral and cellular immune responses to subsequent infection, thereby increasing the protection conferred by the vaccine.
The segment-specific non-coding regions (NCRs) of influenza A virus RNA genome play important roles in controlling viral RNA transcription, replication and genome packaging. In this report, we present, for the first time to our knowledge, a full view of the segment-specific (NCRs) of all influenza A viruses by bioinformatics analysis. Our systematic functional analysis reveal that the eight segment-specific NCRs could differently regulate viral RNA synthesis and protein expression at both transcription and translation levels. Interestingly, a highly conserved suboptimal nucleotide at -3 position of Kozak sequence, that could down-regulate protein expression at translation level, is only present in the segment-specific NCR of PB1. By reverse genetics, we demonstrate that recombinant viruses with an optimized Kozak sequence at -3 position in PB1 results in a significant multiple-cycle replication reduction that is independent of PB1-F2 expression. Our detailed dynamic analysis of the virus infection reveals that, the mutant virus displays slightly altered dynamics from the wild type virus on both viral RNA synthesis and protein production. Furthermore, we demonstrate that the level of PB1 expression is involved in regulating type I interferon production. Together, these data reveal a novel strategy exploited by influenza A virus to fine-tune virus replication dynamics and host anti-viral response through regulating PB1 protein expression.
Bats are important reservoirs of many viruses with zoonotic potential worldwide, including Europe. Among bat viruses, members of the Picornaviridae family remain a neglected group. We performed viral metagenomic analyses on Miniopterus schreibersii bat fecal samples, collected in Hungary in 2013. In the present study we report the first molecular data and genomic characterization of a novel picornavirus from the bat species M. schreibersii in Europe. Based on phylogenetic analyses, the novel bat picornaviruses unambiguously belong to the Mischivirus genus and were highly divergent from other bat-derived picornaviruses of the Sapelovirus genus. Although the Hungarian viruses were most closely related to Mischivirus A, they formed a separate monophyletic branch within the genus.
Yellow fever virus (YFV)-17D is an empirically developed, highly effective live-attenuated vaccine that has been administered to human beings for almost a century. YFV-17D has stood as a paradigm for a successful viral vaccine, and has been exploited as a potential virus vector for the development of recombinant vaccines against other diseases. In this study, a DNA-launched YFV-17D construct (pBeloBAC-FLYF) was explored as a new modality to the standard vaccine to combine the commendable features of both DNA vaccine and live attenuated viral vaccine. The DNA-launched YFV-17D construct was characterized extensively both in cell culture and in mice. High titers of YFV-17D were generated upon transfection of the DNA into cells, whereas a mutant with deletion in the capsid coding region (pBeloBAC-YF/C) was restricted to a single round of infection, with no release of progeny virus. Homologous prime-boost immunization of mice with both pBeloBAC-FLYF and pBeloBAC-YF/C elicited specific dose-dependent cellular immune response against YFV-17D. Vaccination of A129 mice with pBeloBAC-FLYF resulted in induction YFV-specific neutralizing antibodies in all vaccinated subjects. These promising results underlined the potential of the DNA-launched YFV both as an alternative to standard YFV-17D vaccination but also as a vaccine platform for the development of DNA-based recombinant YFV vaccines.
Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cellular DNA deaminases that restrict a broad spectrum of lentiviruses. This process is counteracted by the viral infectivity factor (Vif) of lentiviruses, which binds APOBEC3s and promotes their degradation. Core binding factor subunit bbeta; (CBF-bbeta;) is an essential co-factor for the function of human immunodeficiency virus type 1 Vif to degrade human APOBEC3s. However, the requirement for CBF-bbeta; in Vif-mediated degradation of other mammalian APOBEC3 proteins is less clear. Here we determine the sequence of feline CBFB and perform phylogenetic analyses. These analyses reveal that mammalian CBFB is under purifying selection. Moreover, we demonstrate that CBF-bbeta; is dispensable for feline immunodeficiency virus Vif-mediated degradation of APOBEC3s of its host. These findings suggest that primate lentiviruses have adapted to use CBF-bbeta;, an evolutionary stable protein, to counteract APOBEC3 proteins of their hosts after diverging from other lentiviruses.
Many insect-transmissible pathogens are transmitted by specific insect species and not by others, even if the insect species are closely related. The molecular mechanisms underlying such strict pathogen-insect specificity are poorly understood. Rice dwarf virus (RDV), a plant reovirus, is mainly transmitted by the leafhopper species Nephotettix cincticeps, but is ineffectively transmitted by the leafhopper Recilia dorsalis. Here, we demonstrated that virus-containing tubules composed of viral nonstructural protein Pns10 of RDV, associated with the intestinal microvilli of N. cincticeps, but not with those of R. dorsalis. Furthermore, Pns10 of RDV specifically interacted with cytoplasmic actin, the main component of microvilli of N. cincticeps, but not with that of R. dorsalis, suggesting that the interaction of Pns10 with insect cytoplasmic actin was consistent with the transmissibility of RDV by leafhoppers. All these results suggested that the interaction of Pns10 of RDV with insect cytoplasmic actin may determine pathogen-vector specificity.
We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here we show that the same mutations improve the growth kinetics of a virus containing the A/VietNam/1203/2004 (H5N1) HA and NA. Total protein yield and neuraminidase activity were increased when a chimeric NA was included. These increases indicate that the synergistic effect is due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.
During productive infection with Epstein-Barr virus (EBV), a dramatic suppression of cellular protein expression is caused by the viral alkaline exonuclease BGLF5. Among the proteins downregulated by BGLF5 are multiple immune components. Here, we show that shutoff reduces expression of the innate EBV-sensing Toll-like receptor-2 and the lipid antigen-presenting CD1d molecule, thereby identifying these proteins as novel targets of BGLF5.
To silence BGLF5 expression in B cells undergoing productive EBV infection, we employed an shRNA approach. Viral replication still occurred in these cells, albeit with reduced late gene expression. Surface levels of a group of proteins, including immunologically relevant molecules such as CD1d and HLA class I and class II, were only partly rescued by depletion of BGLF5, suggesting that additional viral gene products interfere with their expression. Our combined approach thus provides a means to unmask novel EBV (innate) immune evasion strategies that may operate in productively infected B cells.
Human noroviruses are one of the major causes of acute gastroenteritis worldwide. Due to the lack of an efficient human norovirus cell culture system coupled with an animal model, human norovirus research mainly relies on human volunteer studies and surrogate models. Current models either utilize human norovirus infected animals including the gnotobiotic pig or calf and the chimpanzee models, or employ other members of the Caliciviridae family including cell culture propagable surrogate caliciviruses such as the feline calicivirus, murine norovirus and most recently the Tulane virus. One of the major features of human noroviruses is their extreme biological diversity, including genetic, antigenic and histo-blood group antigen binding diversity, and possible differences of virulence and environmental stability. This extreme biological diversity and its effect on intervention/prevention strategies cannot be modeled by uniform groups of surrogates much less by single isolates. Tulane virus, the prototype recovirus strain, was discovered in 2008. Since then, several other novel recoviruses have been described and cell culture adapted. Recent studies indicate that the epidemiology, the biological features and diversity of recoviruses and the course of infection and clinical disease in recovirus infected macaques more closely reflect those properties of human noroviruses than any of the current surrogates. This review aims to summarize what is currently known about recoviruses, highlight their biological similarities to human noroviruses and discuss applications of the model in addressing questions relevant for human norovirus research.
Marek's disease virus (MDV) is an important oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts. The Meq-clustered miRNAs encoded by MDV have been suggested to play potentially critical roles in the induction of lymphomas. Using the technique of bacterial artificial chromosome mutagenesis, we have presently constructed a series of specific miRNA-deleted mutants and demonstrate that these miRNAs are not essential for replication of MDV and have no effects on the early cytolytic or latent phases of the developing disease. However, compared to the parental GX0101, mortality of birds infected with the mutants GXmiR-M2, GXmiR-M3, GXmiR-M5, GXmiR-M9 and GXmiR-M12 was reduced from 100% to 18%, 30%, 48%, 24% and 14%, coupled with the gross tumour incidence reduction from 28% to 8%, 4%, 12%, 8% and 0%, respectively. Our data confirms that except for mdv1-miR-M4, the other Meq-clustered miRNAs also play critical roles in MDV oncogenesis. Further work will be needed to elucidate the miRNA-mediated regulatory mechanisms that trigger the development of MD lymphomas.
Cassava brown streak disease (CBSD) has emerged as the most important viral disease of cassava (Manihot esculenta) in Africa and is a major threat to food security. CBSD is caused by two distinct species of ipomoviruses, Cassava brown streak virus and Ugandan cassava brown streak virus, belonging to the family Potyviridae. Previously CBSD was reported only from the coastal lowlands of East Africa, but recently it has begun to spread as an epidemic throughout the Great Lakes region of East Africa and Central Africa. This new spread represents a major threat to the cassava-growing regions of West Africa. CBSD resistant cassava cultivars are being developed through breeding and transgenic RNAi-derived field resistance to CBSD has also been demonstrated. This review aims to provide a summary of the most important studies on aetiology, epidemiology and control of CBSD and highlight key research areas that need prioritization.
CRISPR/Cas9 system is a highly efficient and powerful tool for RNA-guided editing of cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here we reported on CRISPR/Cas9-mediated editing of EBV genome in human cells. Two guide RNAs were used to direct a targeted deletion of 558 bp in the promoter region of BART transcript which encodes viral microRNAs. Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and guide RNAs. No off-target cleavage was found by deep sequencing. The loss of BART microRNA expression and activity was verified, supporting that the BART promoter is the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses can be recovered and introduced into other cells at low multiplicity of infection. Recombinant viruses with an edited genome can be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provides not only the first genetic evidence that the BART promoter drives the expression of BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.
As a member of the newly established Betaflexiviridae family, Grapevine rupestris stem pitting-associated virus (GRSPaV) has a RNA genome containing five open reading frames (ORFs). ORF1 encodes a putative replicase polyprotein typical of the Alphavirus superfamily of positive-strand, ssRNA viruses. Several viruses of this superfamily have been demonstrated to replicate in structures designated as viral replication complexes associated with intracellular membranes. However, structure and cellular localization of the replicase complex have not been studied for members of Betaflexiviridae, a family of mostly woody plant viruses. As a first step toward the elucidation of the replication complex of GRSPaV, we investigated the subcellular localization of full-length and truncated versions of its replicase polyprotein via fluorescent tagging, followed by fluorescence microscopy. We found that the replicase polyprotein formed distinctive punctate bodies in both Nicotiana benthamiana leaf cells and tobacco protoplasts. We further mapped a region of 76 amino acid in the methyl-transferase domain responsible for the formation of these punctate structures. These punctate structures are distributed in close proximity to the endoplasmic reticulum network. Membrane flotation and biochemical analyses demonstrate that the N-terminal region responsible for punctate structure formation associated with cellular membrane likely through an amphipathic a helix serving as an in-plane anchor. The identity of this membrane is yet to be determined. This is, to our knowledge, the first report on the localization and membrane association of the replicase proteins of a member of the family Betaflexiviridae.
Interferon-induced restriction factors can significantly affect the replicative capacity of retroviruses in mammals. Tripartite motif protein 5, isoform aalpha; (TRIM5aalpha;) is a restriction factor that acts at early stages of the virus life cycle by intercepting and destabilizing incoming retroviral cores. Sensitivity to TRIM5aalpha; maps to the N-terminal domain of the retroviral capsid (CA) proteins. In several New World and Old World monkey species, independent events of retrotransposon-mediated insertion of the cyclophilin A (CypA) coding sequence in the trim5 gene have given rise to TRIMCyp (also called TRIM5-CypA), a hybrid protein that is active against some lentiviruses in a species-specific fashion. In particular, TRIMCyp from the owl monkey (omkTRIMCyp) very efficiently inhibits HIV-1. Previously, we showed that disrupting the integrity of microtubules (MTs) and of cytoplasmic dynein complexes partially rescued replication of retroviruses, including HIV-1, from restriction mediated by TRIM5aalpha;. Here we show that efficient restriction of HIV-1 by omkTRIMCyp is similarly dependent on the MT network and on dynein complexes, but in a context-dependent fashion. When omkTRIMCyp was expressed in human HeLa cells, restriction was partially counteracted by pharmacological agents targeting MTs or by siRNA-mediated inhibition of dynein. The same drugs (nocodazole and paclitaxel) also rescued HIV-1 from restriction in cat CRFK cells, although to a lesser extent. Strikingly, neither nocodazole, paclitaxel nor depletion of the dynein heavy chain (DHC) had a significant effect on the restriction of HIV-1 in an owl monkey cell line. These results suggest the existence of cell-specific functional interactions between MTs/dynein and TRIMCyp.
The division of viruses into orders, families, genera and species provides a classification framework that seeks to organise and make sense of the diversity of viruses infecting animals, plants and bacteria. Classifications are based on similarities in genome structure and organisation, the presence of homologous genes and sequence motifs, and, at lower levels such as species, host range, nucleotide and antigenic relatedness and epidemiology. Classification below the level of family must also be consistent with phylogeny and virus evolutionary histories. Recently developed methods such as PASC, DEMaRC and NVR offer alternative strategies for genus and species assignments that are based purely on degrees of divergence between genome sequences. They offer the possibility of automating classification of the vast number of novel virus sequences being generated by next generation metagenomic sequencing. However, distance-based methods struggle to deal with the complex evolutionary history of virus genomes which are shuffled by recombination and re-assortment and where taxonomic lineages evolve at different rates. In biological terms, classifications based on sequences distances alone are also arbitrary whereas the current system of virus taxonomy is of utility precisely because it is primarily based upon phenotypic characteristics. However, a separate system is clearly needed by which virus variants that lack biological information might be incorporated in to the ICTV classification even if based solely on sequence relationships to existing taxa. For these, simplified taxonomic proposals and naming conventions represent a practical way to expand the existing virus classification and catalogue our rapidly increasing knowledge of virus diversity.
The human genome is comprised of 8% endogenous retroviruses (ERVs), the majority of which are defective due to deleterious mutations. Nonetheless, transcripts of ERVs are found in most tissues and these transcripts could either be reverse transcribed to generate single-stranded DNA or expressed to generate proteins. Thus, the expression of ERVs could produce nucleic acids or proteins with viral signatures, much like the pathogen-associated molecular patterns (PAMPs) of exogenous viruses, which would enable them to be detected by the innate immune system. The activation of some pattern recognition receptors (PRRs) in response to ERVs has been described in mice and in the context of human autoimmune diseases. Here, we review the evidence for detection of ERVs by PRRs and the resultant activation of innate immune signalling. This is an emerging area of research within the field of innate antiviral immunity, showing how ERVs could possibly initiate immune signalling pathways and might have implications for numerous inflammatory diseases.
Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally transmitted by mosquitoes. During the infection, JEV generally enters host cells via receptor-mediated clathrin-dependent endocytosis that requires the involvement of the 70 kDa heat shock protein (Hsp70). Heat shock cognate protein 70 (Hsc70) is one member of the Hsp70 family and is mainly constitutive; thus, it may be expressed at physiological condition. In C6/36 cells, Hsc70 is up-regulated in response to JEV infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface, it probably does not serve as the receptor according to our results in the present study. In contrast, Hsc70 is evidently associated with virus penetration into the cell and resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly involved in clathrin-mediated endocytosis, particularly at a late stage of viral entry into host cells. Furthermore, we found that Hsc70 is composed of at least three isoforms, including B, C, and D. Of them, the isoform D is the one that helps JEV to penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant evidence that sheds light on the regulatory mechanisms of JEV infection in host cells, especially on the process of clathrin-mediated endocytosis.
Human herpesvirus-6A (HHV-6A) is rarer than HHV-6B in many infant populations. However, they are similarly prevalent as germline chromosomally integrated genomes (ciHHV-6A/B). This integrated form affects 0.1-1% human populations, where potentially virus gene expression could be in every cell, although virus relationships and health effects are not clear. In a Czech/German patient cohort ciHHV-6A was more common and diverse than ciHHV-6B. Quantitative PCR, nucleotide sequencing and telomeric integration site amplification characterised ciHHV-6 in 44 German myocarditis/cardiomyopathy and Czech malignancy/inflammatory disease (MI) patients plus donors. Comparisons were made to sequences from global virus reference strains, and blood DNA from childhood-infections from Zambia (HHV-6A mainly) and Japan (HHV-6B). The MI cohort were 95%(19/21) ciHHV-6A, the cardiac cohort 65%(13/20) ciHHV-6B, suggesting different disease links. Reactivation was supported by findings of 1) recombination between ciHHV-6A and HHV-6B genes in 20%(4/21) of the MI cohort; 2) expression in a subset, of early/late transcripts tested from encoded inflammatory mediator genes chemokine receptor U51 and chemokine U83, both identical to ciHHV-6A DNA sequences; and 3) superinfection shown by deep sequencing identifying minor virus-variants only in ciHHV-6A which expressed transcripts, indicating virus infection reactivates latent ciHHV-6A. Half the MI cohort had more than two copies/cell, median 5.2, indicative of reactivation. Remarkably, the integrated genomes encoded the secreted-active form of virus chemokines, rare in virus from childhood-infections. This shows integrated virus genomes can contribute new human genes with links to inflammatory pathology and supports ciHHV-6A reactivation as a source for emergent infection.
Chronic hepatitis B (CHB) is treated with nucleos(t)ide analogs (NA). The reverse transcriptase (RT) region in the Hepatitis B virus (HBV) genome mutates to resist NA treatment, yet the RT mutations have not been well characterized. Further, the HBV genotype might influence RT sequence evolution, NA resistance (NAr) mutation patterns, and drug resistance development. We examined 42 NAr mutation sites in 169 untreated and 131 NA-treated CHB patient samples. Patients were identified with genotype B (HBV-B) and HBV-C infections, with a higher prevalence and mutation frequency of HBV-C than HBV-B. Seventeen reported NAr mutation sites and 13 novel mutations were detected. NAr-related mutation prevalence was significantly higher in NA-treated versus untreated patients. Primary antiviral-resistant mutants only existed in NA-treated patients. Sequencing data revealed 7 HBV-C-specific mutations and 3 HBV-B-specific mutations. In conclusion, NA treatment and HBV genotype might constitute the selection basis and promote NA-resistant HBV strain evolution under antiviral therapy.
Pathogenesis of viral hemorrhagic fevers (VHF) is associated with alteration of vascular barrier function and hemorrhage. To date, the specific mechanism behind this is unknown. Programmed cell death and regulation of apoptosis in response to viral infection is an important factor for host or virus survival but this has not been well-studied in the case of Crimean-Congo hemorrhagic fever virus (CCHFV).
In this study, we demonstrated that CCHFV infection suppresses cleavage of poly (ADP-ribose) polymerase (PARP), triggered by staurosporine at early post infection. We also demonstrated that CCHFV infection suppresses activation of caspase-3 and caspase-9. Most interestingly, we found that CCHFV N can suppress induction of apoptosis by Bax and inhibit the release of cytochrome-c from the inner membrane of mitochondria to cytosol. However, CCHFV infection induces activation of Bid at late post infection, suggesting the activation of extrinsic apoptotic signaling. Consistently, supernatant from late post-infected cells stimulated was found to induce PARP cleavage, most probably through the TNF-aalpha; dead receptor pathway. In summary, we found that CCHFV has strategies to interplay with apoptosis pathways and thereby regulate caspase cascade. We suggest that CCHFV suppresses caspase activation at early stages of the CCHFV replication cycle, which perhaps benefits the establishment of infection. Furthermore, we suggest that the host cellular response at late post infection induces host cellular pro-apoptotic molecules through the death receptor pathway. External host-derived stimuli most probably initiate the apoptotic process, and the route continues either by crosstalk between the death receptor and mitochondria routes, or separately.
Baculoviruses are arthropod-specific pathogens and find extensive applications in pest control strategies and recombinant protein expression. Spodoptera litura nucleopolyhedrovirus infects the Tropical armyworm, Spodoptera litura which is an important polyphagous crop pest widely distributed in regions of Asia and Oceania. Using next generation sequencing, we report stage specific profiling of SpltNPV encoded miRNAs at different time intervals post infection of Sf21 cell lines. Sequence length distribution analysis of the small RNA libraries revealed a significant increase in 20nt reads and reduction of other size fractions during late phases of infection. In-silico miRNA prediction tools identified 48 novel SpltNPV encoded miRNAs, out of which ten were experimentally validated in Sf21 cell lines using northern blot analysis and Taqman qPCR. The viral miRNAs were found to express in fat body and mid gut tissues of infected fifth instar Spodoptera litura larva as well. Real time PCR analysis confirmed that expression of most viral miRNAs was triggered post 12 hours of infection and continued thereafter. Gene Ontology and KEGG pathway annotation of computationally predicted targets of the reported miRNAs suggests a major impact of these miRNAs on cell signalling, protein translation and metabolic processes.
Porcine reproductive and respiratory syndrome virus (PRRSV) is now prevalent throughout the world and has caused major economic losses to the pig industry. Arterivirus nonstructural protein 10 (nsp10) is a superfamily 1 helicase participating in multiple processes of virus replication. Nsp10 of PRRSV, however, has not yet been well characterized. In this study, a series of nsp10 mutants were constructed and analyzed for functional sites of different enzymatic activities. We found that nsp10 could bind both ssDNA and dsDNA, and this binding activity could be inactivated by mutations at Cys25 and His32. These two mutations also abolished unwinding activity without affecting ATPase activity. In addition, substitution of Ala227 by serine eliminated helicase activity, while substitution by valine enhanced unwinding activity. Taken together, our results showed that Cys25 and His32 in PRRSV nsp10 were critical for nucleic acid binding and unwinding, and that Ala227 played an important role in helicase activity.
Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for epidemics of debilitating arthritic disease. The recent outbreak (2004-2014) resulted in an estimated 1.4-6.5 million cases, with imported cases reported in nearly 40 countries. The development of CHIKV-specific diagnostics and research tools is thus highly desirable. Herein we describe the generation and characterization of the first monoclonal antibodies (mAbs) specific for the capsid protein (CP) of CHIKV. The antibodies recognized isolates representing the major genotypes of CHIKV, as well as several other alphaviruses, and were reactive in a range of assays including ELISA, Western blot, immunofluorescence (IFA) and immunohistochemistry (IHC). We have also used the anti-CP mAb, 5.5G9, in IHC studies to show that capsid antigen is persistently expressed 30 days post-infection in cells with macrophage morphology in a mouse model of chronic CHIKV disease. These antibodies may thus represent useful tools for further research including investigations on the structure and function of CHIKV CP, and as valuable reagents for CHIKV detection in a range of settings.
Since one of us co-authored a review on NS5A a decade ago the hepatitis C virus (HCV) field has changed dramatically, primarily due to the advent of the JFH-1 cell culture infectious clone which allowed the study of all aspects of the virus lifecycle from entry to exit. This review will describe advances in our understanding of NS5A biology over the past decade, highlighting how the JFH-1 system has allowed us to determine that NS5A is essential not only in genome replication but also in the assembly of infectious virions. We will review the recent structural insights - NS5A is predicted to comprise 3 domains, X-ray crystallography has revealed the structure of domain I but there is a lack of detailed structural information about the other two domains which are predicted to be largely unstructured. Recent insights into the phosphorylation of NS5A will be discussed, and we will highlight a few pertinent examples from the ever expanding list of NS5A binding partners identified over the past decade. Lastly, we will review the literature showing that NS5A is a potential target for a new class of highly potent small molecules that function to inhibit virus replication. These direct acting antivirals (DAAs) are now either licenced, or in the late stages of approval, for clinical use both in the USA and UK/Europe. In combination with other DAAs targeting the viral protease (NS3) and polymerase (NS5B), they are revolutionising treatment for HCV infection.
Turnip mosaic virus (TuMV) is a potyvirus that is transmitted by aphids and infects a wide range of plant species. We investigated the evolution of this pathogen by collecting 32 isolates of TuMV, mostly from Brassicaceae plants, in Australia and in New Zealand. We performed a variety of sequence-based phylogenetic and population genetic analyses of the complete genomic sequences and of three non-recombinogenic regions of those sequences. The substitution rates, divergence times, and phylogeographic patterns of the virus populations were estimated. Six inter- and five intralineage recombination type patterns were found in the genomes of the Australian and New Zealand isolates, and all were novel. Only one recombination type pattern has been found in both countries. Australian and New Zealand populations were genetically different, and were different from the European and Asian populations. Our Bayesian coalescent analyses, based on a combination of novel and published sequence data from three non-recombinogenic protein-encoding regions, showed that TuMV probably started to migrate from Europe to Australia and New Zealand more than 80 years ago, and that distinct populations arose as a result of evolutionary drivers such as recombination. The basal-B2 subpopulation in Australia and New Zealand seems to be older than those of the world-B2 and B3 populations. To our knowledge, our study presents the first population genetic analysis of TuMV in Australia and New Zealand. We have shown that the time of migration of TuMV correlates well with the establishment of agriculture and migration of Europeans to these countries.
Mouse mammary tumour virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse tranferrin receptor 1 for cell entry. Several MMTV strains have been shown to productively infect, in addition to murine cells, various heterologous cell lines including those of human origin, albeit less efficiently than murine cells. Here, we analysed whether MMTV(C3H), previously reported to be incapable of infecting human cells, can productively infect human cells. Using a recently described high titer MMTV-based vector carrying the MMTV(C3H)Env we successfully transduced cells of human origin. Furthermore, wild-type MMTV(C3H) was able to infect human cells, albeit less efficiently than mouse cells. The established infection was, however, sufficient to enable virus spread to every cell in culture. Infectivity of wild-type MMTV(C3H) and MMTV-based vectors carrying MMTV(C3H)Env was blocked by heat-inactivation, an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine and pre-incubation with neutralizing anti-MMTV antibodies that did not neutralize vectors pseudotyped with amphotropic MLV Env, providing evidence for an authentic, receptor-mediated and reverse transcriptase-dependent infection process. Persistently infected human Hs578T cells produced infectious virions capable of infecting naive human breast cells in culture, the infectivity of which could also be blocked by neutralizing anti-MMTV antibodies, demonstrating that virus particles released by the persistently infected Hs578T cells are antigenically related to the virus produced from murine cells. Taken together, our results show that MMTV(C3H), like MMTV(GR) and MMTV(RIII), is able to not only infect, but also to replicate in cultured human breast cells.
Oropouche virus (OROV) is a medically important orthobunyavirus, which causes frequent outbreaks of a febrile illness in the Northern parts of Brazil. However, despite being the cause for an estimated half a million human infections since its first isolation in Trinidad, 1955, details of the molecular biology of this tripartite, negative-sense RNA virus remain limited. We have determined the complete nucleotide sequence of the Brazilian prototype strain of OROV, BeAn 19991, and found a number of differences compared to sequences in the database. Most notable were that the S segment contains an additional 204 nucleotides at the 3' end and that there is a critical nucleotide mismatch at position 9 within the base-paired terminal panhandle structure of each genome segment. In addition, we obtained the complete sequence of the Trinidadian prototype strain TRVL 9760 that showed similar characteristics to the BeAn 19991 strain. By using a T7 RNA polymerase-driven minigenome system, we demonstrated that cDNA clones of the BeAn 19991 L and S segments expressed functional proteins and also that the newly determined terminal untranslated sequences acted as functional promoters in the minigenome assay. By co-transfecting a cDNA to the viral glycoproteins, virus-like particles (VLP) were generated that packaged a minigenome and were capable of infecting naive cells.
Analysis of virus-derived small RNAs with high-throughput sequencing has been successful for detecting novel viruses in plants and invertebrates. However, the applicability of this method has not been demonstrated in fungi, although fungi were among the first organisms reported to utilize RNA silencing. Here, we used virus-infected isolates of the fungal species complex Heterobasidion annosum sensu lato as a model system to test whether mycovirus genome segments can be detected with small RNA deep sequencing. Species of Heterobasidion are some of the most devastating forest pathogens in boreal forests. These fungi cause wood decay and are commonly infected with species of Partitiviridae and the yet unassigned virus species Heterobasidion RNA virus 6 (HetRV6). Small RNA deep sequencing allowed the simultaneous detection of all eight double-stranded RNA virus strains known to be present in the tested samples and one putative mitovirus species (family Narnaviridae) with a single-stranded RNA genome, designated here as Heterobasidion mitovirus 1. Prior to this study, no members of the family Narnaviridae had been described as infecting species of Heterobasidion. Quantification of viral double- and single-stranded RNA with quantitative PCR indicated that co-infecting viral species and viruses with segmented genomes can be detected with small RNA deep sequencing despite vast differences in the amount of RNA. This is the first study demonstrating the usefulness of this method for detecting fungal viruses. Moreover, the results suggest that viral genomes are processed into small RNAs by different species of Heterobasidion.
Rotavirus A is a very common cause of acute diarrhea in infants and young children worldwide. Most of human strains are classified into 2 major Wa-like and DS-1-like genotype constellations, while a minor third strain, AU-1, was described in 1989 among human rotavirus isolates from Japan. AU-1 demonstrates a high degree of homology to a feline rotavirus, FRV-1, which suggests interspecies transmission of feline rotavirus. However, there has been no subsequent report of RVs possessing AU-1 genotype throughout all the 11 genes. Between March 1997 and December 1999, 157 rotavirus positive stool samples were collected from Brazilian children, and 16 of 157 (10.2%) were P genotype. We analyzed 8 strains by nearly full genome sequencing. These 8 strains were divided into 2 groups, that is, 5 AU-1-like and 3 Wa-like strains. Four of 5 AU-1-like strains had AU-1-like genotype constellation throughout the 11 genes. The remaining AU-1-like strain was considered to be a reassortant strain comprised of 9, 2 and 1 genes from AU-1-like, Wa-like and G9 strains, respectively. Three Wa-like strains were considered to be reassortant comprised of 7 to 8 genes and 3 to 4 genes from Wa-like and non-Wa-like strains, respectively. This report of human G3P rotavirus strains possessing AU-1 genotype constellation throughout all genes demonstrates stability and infectivity of AU-1-like strain with its original genotype beyond space and time.
A fundamental aspect of herpesviruses is their ability to establish a life long latent infection within the host. Current research would suggest that small regulatory RNAs called microRNAs play a central role in all three aspects of virus latency and persistence. In this review we will discuss the role of miRNAs in virus persistence and latency, specifically focusing on herpesviruses. We will cover the current knowledge on the role of miRNAs in establishing and maintaining virus latency and promoting survival of infected cells through targeting of both viral and cellular transcripts, highlighting key publications in the field. We will also discuss potential areas of future research and how novel technologies may aid in determining how miRNAs shape virus latency in the context of herpesvirus infections.
The stability and conservation of the sequences of RNA viruses in the field and the high error rates measured in vitro are paradoxical. The field stability indicates that there are very strong selective constraints on sequence diversity. The nature of these constraints is discussed. Apart from constraints on variation in cis-acting RNA and the amino acid sequences of viral proteins, there are other ones relating to the presence of specific dinucleotides such CpG and UpA as well as the importance of RNA secondary structures and RNA degradation rates. Recent other constraints identified in other RNA viruses such as effects of secondary RNA structure on protein folding or modification of cellular tRNA complements are also discussed. Using the family Paramyxoviridae I show that the codon usage pattern (CUP) is (i) specific for each virus species and (ii) that it is markedly different from the host - it does not vary even in vaccine viruses that have been derived by passage in a number of inappropriate host cells. The CUP might thus be an additional constraint on variation and I propose the concept of codon constellation to indicate the informational content of the sequences of RNA molecules relating not only to stability and structure but also to the efficiency of translation of a viral mRNA resulting from the CUP and the numbers and position of rare codons.
Two strains of African swine fever virus (ASFV), the high virulence Lisboa60 (L60) and the low virulence NH/P68 (NHV), which have been used previously in effective immunization/protection studies, were sequenced. Both were isolated in Portugal in the 11-year period after the introduction of ASFV into the European continent in 1957. The predicted proteins coded by both strains were compared, and where differences were found, these were also compared to other strains of known virulence. This highlighted several genes with significant alterations in low virulence strains of ASFV that may constitute virulence factors, several of which still lacking knowledge regarding their function. Phylogenetic analysis grouped L60 and NHV closest with other P72 genotype I ASFV strains from Europe and West Africa, consistent with the assumed West-African origin of all European strains. Interestingly, a relatively lower genomic identity exists between L60 and NHV, both isolated in a similar geographical location 8-years apart, than with other European and West-African strains isolated later in time and in more distant locations. This may reflect the intensive passage in tissue culture during the early 60rrsquo;s of a Portuguese isolate to obtain an attenuated vaccine, which may have led to NHV. This study contributes to a better understanding of the evolution of ASFV and defines additional potential virulence genes for future studies of pathogenesis towards the development of effective vaccines.
Human Papillomaviruses (HPVs) have been detected in urban wastewaters, demonstrating that epitheliotropic viruses can find their way into sewage through the washing of skin and mucous membranes. Papillomavirus shedding through feces is still an unexplored issue. The objective of the present study was to investigate the presence of HPVs in stool samples. We analyzed 103 fecal specimens collected from hospitalized patients with diarrhea using validated primers able to detect aalpha;, bbeta; and HPVs. PCR products underwent sequencing analysis and sequences were aligned to reference genomes from the Papillomavirus Episteme database. A total of 15 sequences were characterized from the fecal samples. Thirteen samples (12.6%) were positive for 9 genotypes belonging to aalpha; and bbeta; genus: HPV32 (LR, aalpha;1), HPV39 (HR, aalpha;7), HPV44 (LR, aalpha;10), HPV8 (bbeta;1), HPV9, HPV23, HPV37, HPV38, and HPV120 (bbeta;2). Two putative novel genotypes of the bbeta; genus, species 1 and 2 were also detected. The tissue (s) of origin is unknown, since feces can collect HPVs originating from or passing through the entire digestive system. To our knowledge, this is the first investigation on the occurrence and diversity of HPVs in fecal samples. Results from this study demonstrate that HPVs can find their way into sewage as a result of shedding in the feces. This highlights the need of further studies aimed at understanding the prevalence of HPV in different water environments and the potential for waterborne transmission.
The unfolded protein response (UPR) is a cellular defense mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1aalpha; become activated. PERK phosphorylates eIF2aalpha; leading to a general inhibition of cellular translation, while the expression of transcription factor ATF4 is upregulated. Active IRE1aalpha; splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Herein we demonstrate that transient expression of chikungunya virus (CHIKV) (Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2aalpha; and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIK infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV infected cells. Individual expression of CHIKV non-structural proteins (nsP) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that render nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER, but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.
T-lymphotropic feline leukemia virus (FeLV-T), a highly pathogenic variant of FeLV, induces severe immunosuppression in cats. FeLV-T is fusion-defective because in its PHQ motif, a gammaretroviral consensus motif in the N-terminal of an envelope protein, histidine is replaced with aspartate. Infection by FeLV-T requires FeLIX, a truncated envelope protein encoded by an endogenous FeLV, for transactivation of infectivity and Pit1 for binding FeLIX. Although Pit1 is present in most tissues in cats, the expression of FeLIX is limited to certain cells in lymphoid organs. Therefore, the host cell range of FeLV-T was thought to be restricted to cells expressing FeLIX. However, because FeLIX is a soluble factor and expressed constitutively in lymphoid organs, we presumed it to be present in blood and evaluated its activities in sera of various mammalian species using a pseudotype assay. We demonstrated that cat serum has FeLIX activity at a functional level, suggesting that FeLIX is present in cats and FeLV-T may be able to infect cells expressing Pit1 regardless of the expression of FeLIX in vivo. In addition, FeLIX activities in sera were detected only in domestic cats but not in other feline species tested. To our knowledge, this is the first report to prove that a large amount of truncated envelope protein of endogenous retrovirus is circulating in the blood to facilitate the infection of a pathogenic exogenous retrovirus.
Neutralising antibodies (NAbs) are believed to comprise an essential component of the protective immune response induced by vaccines against FIV and HIV infections. However, relatively little is known about the role of NAbs in controlling FIV infection and subsequent disease progression. Here we present studies examining the neutralisation of HIV-luciferase pseudotypes bearing homologous and heterologous FIV Envs (n=278) by sequential plasma samples collected at 6 month intervals from naturally infected cats (n=38) over a period of 18 months. We evaluated the breadth of the NAb response against non-recombinant homologous and heterologous clade A and clade B viral variants as well as recombinants and assessed the results, testing for evidence of an association between the potency of the NAb response and the duration of infection, CD4 T lymphocyte numbers, health status and survival times of the infected cats. Neutralisation profiles varied significantly between FIV infected cats and strong autologous neutralisation, assessed using luciferase based in vitro assays, did not correlate with the clinical outcome. No association was observed between strong NAb responses and either improved health status or increased survival time of infected animals, implying that other protective mechanisms are likely to be involved. Similarly, no correlation was observed between the development of autologous NAbs and the duration of infection. Furthermore, cross-neutralising antibodies were evident in only a small proportion (13%) of cats.
Pleconaril is a capsid-inhibitor previously used to treat enterovirus infections. A pleconaril resistant Echovirus (E) 11 strain was identified before pleconaril treatment was given in an immune-compromised patient. The patient was also treated with immune-globulins (IVIG) for a long period, but remained unresponsive. The pleconaril resistant strains could not be neutralized in vitro confirming IVIG treatment failure. To identify the basis of pleconaril resistance genetic and structural analyses were conducted. Analysis of a modelled viral capsid indicated conformational changes in the hydrophobic pocket that could prevent pleconaril docking. Substitutions (V117I, V119M, and I188L) in the pleconaril resistant viruses were found in the pocket region of VP1. Modelling suggests V119M could confer resistance, most probably due to the protruding sulphate side chain of methionine. Although pleconaril resistance induced in vitro in a susceptible E11 clinical isolate was characterised by a different substitution (I183M), resistance is suggested to also results by a similar mechanism i.e. due to a protruding sulphate side chain of methionine. Our results show resistant strains that arise in vivo display different markers than those identified in vitro and suggest that multiple factors may play a role in pleconaril resistance in patient strains. Based on IVIG treatment failure, we predict one of these factors could be immune-related. Thus both IVIG and capsid-inhibitors target the viral capsid and can induce mutations that can be cross-reactive enabling escape from both IVIG and drug. This could limit treatment options and should be investigated further.
Salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared to terrestrial alphavirus replicons (Olsen et al., 2013a). In this study, we have compared several modifications to a SAV replicon construct and analyzed their influence on foreign gene expression. We found that with an insertion of a translational enhancer consisting of the N-terminal 102 nucleotides of the capsid gene together with a nucleotide sequence encoding the FMDV 2A peptide caused a significant increase of the EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 5rrsquo; end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at 3rrsquo; end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production which can be applied for immunization of salmonid fish in the future.
Crimean-Congo hemorrhagic fever virus (CCHFV) is a Nairovirus of the family Bunyaviridae, which can cause severe hemorrhagic fever in humans, with mortality rates above 30%. CCHFV is the most widespread of the tick-borne human viruses and it is endemic in areas of central Asia, the Middle East, Africa and southern Europe. Its viral genome consists of three negative sense RNA segments. The large segment (L) encodes a viral RNA-dependent RNA polymerase (L protein), the small segment (S) encodes the nucleocapsid protein (N) and the medium segment (M), encodes for the envelope proteins. The nucleocapsid protein of bunyaviruses binds genomic RNA forming the viral RiboNucleoProtein (RNP) complex. The L protein interacts with these RNP structures allowing the initiation of viral replication. The nucleocapsid protein also interacts with actin, although the regions and specific residues involved in these interactions have not been yet described. Here, by means of immunoprecipitation and immunofluorescence assays we identify the regions within the CCHFV nucleocapsid protein implicated in homo-oligomerization and actin binding. We describe the interaction of N with the CCHFV L protein, and identify the amino- and carboxy-terminal regions within the L protein that might be necessary for the formation of these N-L protein complexes. These results may guide the development of potent inhibitors of these complexes that could potentially block CCHFV replication.
Development of numerous advanced techniques in recent years have allowed detection of the pathological prion protein (PrPTSE), the unique marker of transmissible spongiform encephalopathies (TSEs, or prion diseases), in the blood of animals and humans; however, an ante-mortem screening test that can be used for the routine diagnosis of human prion diseases remains unavailable. A critical, analytical review of all the diagnostic assays developed to date will allow an evaluation of progress in this field and may facilitate the identification of the possible reason/s for this delay. Thus, in this review, I provide a detailed overview of the techniques currently available for detecting PrPTSE and other markers of the disease in blood, as well as an analysis of the significance, feasibility, reliability, and the application spectrum for these methods. I highlight that factors intrinsic and extrinsic to blood may interfere with the detection of PrPTSE/prions, and that this is not yet taken into account in current tests. This may inspire researchers in this field to not only aspire to increase test sensitivity but also to adopt other strategies in order to identify and overcome the limitations that hamper the development of a successful routine blood test for prion diseases.
In simian virus 40 (SV40) and several other polyomaviruses, the TATA box of the early promoter is embedded in an AT tract that is also an essential part of the replication origin. We generated an "AT trap", an SV40 genome lacking the AT tract and unable to grow in CV-1 monkey cells. Co-transfection of the AT trap with oligonucleotides containing AT tracts of human polyomaviruses, a poly (A:T) tract, or variants of the SV40 wild type sequence all restored infectious virus. In a transfection of the AT trap without suitable oligonucleotide, an AT-rich segment was incorporated, stemming either from bovine (calf serum) or monkey (host cell) DNA. Similarly, when cells were grown with human serum, a human DNA segment was captured by SV40 to substitute for the missing AT stretch. We conclude that the virus is quite opportunistic in accepting heterologous substitutes, and that even low-abundance DNA from serum can be incorporated into the viral genome.
In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2), consistently gained positively charged amino acids in the putative heparan sulfate-binding pocket (VP2 bbeta;E-bbeta;F loop, VP1 C terminus and VP3 bbeta;-B knob), surrounding the five-fold symmetry axis (VP1 bbeta;F-bbeta;G loop) and at other discrete sites on the capsid (VP3 bbeta;G-bbeta;H loop, VP1 C terminus, VP2 bbeta;C strand and VP1 bbeta;G-bbeta;H loop). A lysine insertion in the VP1 bbeta;F-bbeta;G loop of two of the BHK-21 adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line suggesting their possible role in ionic interaction with ubiquitous negatively charged cell surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared to the lower passage viruses with wild-type genotypes. Substitutions fixed in the VP1 bbeta;G-bbeta;H loop (-3, -2 and +2 'RGD' positions) or in the structural element known to be juxtaposed against that loop (VP1 bbeta;B-bbeta;C loop) suggest their possible role in modulating the efficiency and specificity of interaction of 'RGD' motif with aalpha;V-integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production.
Hepatitis E virus (HEV), the causative agent of hepatitis E, is a single-stranded positive-sense RNA virus belonging to the family Hepeviridae. At least four genotypes of the family infect humans: genotypes 1 and 2 are transmitted to humans through contaminated water, while genotypes 3 and 4 are zoonotic and have animal reservoirs. A novel strain of HEV recently identified in rabbits is a distant member of genotype 3, and thus poses a potential risk of zoonotic transmission to humans. The objective of this study was to construct and characterize an infectious cDNA clone of the rabbit HEV. Two full-length cDNA clones of rabbit HEV, pT7g-rabHEV and pT7-rabHEV, were constructed and their infectivity was tested by in vitro transfection of Huh7 human liver cells and by direct intrahepatic inoculation of rabbits with capped RNA transcripts. Results showed that positive signal for rabbit HEV protein was detected by an immunofluorescence assay with a HEV-specific antibody in Huh7 human liver cells transfected with capped RNA transcripts from the two full-length cDNA clones. Rabbits intrahepatically inoculated with capped RNA transcripts from each of the two clones developed active HEV infection as evidenced by seroconversion to anti-HEV antibodies, and detection of rabbit HEV RNA in sera and feces of inoculated animals. The availability of a rabbit HEV infectious cDNA clone now affords us the ability to delineate the mechanism of HEV replication and cross-species infection in a small animal model.
Shrews are small insectivorous mammals that are distributed worldwide. Similar to rodents, shrews live on the ground and are commonly found near human residences. In this study, we investigated the enteric virome of wild shrews in the genus Crocidurinae using a sequence-independent viral metagenomics approach. A large portion of the shrew enteric virome was composed of insect viruses, while novel viruses including cyclovirus, picornavirus and picorna-like virus were also identified. Several cycloviruses, including variants of human cycloviruses detected in cerebrospinal fluid (CSF) and stool, were detected in wild shrews at a high prevalence rate. The identified picornavirus is distantly related to human parechovirus, inferring the presence of a new genus in this family. The identified picorna-like viruses were characterized as different species of calhevirus 1, which was previously discovered in human stool. Complete or nearly complete genome sequences of these novel viruses were determined in this study and then were subjected to further genetic characterization. Our study provides an initial view of the diversity and distinctiveness of the shrew enteric virome and highlights unique novel viruses related to human stool-associated viruses.
Coronaviruses are enveloped RNA viruses that have evolved complex relationship with their host cells and modulate their lipid composition, lipid synthesis and signaling of host cell. Lipid rafts, enriched in sphingolipids, cholesterol and associated proteins, are special plasma membrane microdomains involved in several processes of viruses infections. The extraction of cholesterol leads to disorganization of lipid microdomains and to dissociation of proteins bound to the lipid rafts. Because of cholesterol-rich microdomains appear to be a general feature of the entry mechanism of noneneveloped viruses and of several coronaviruses, the purpose of this study was to analyze the contribution of lipids in the infectivity of canine coronavirus (CCoV). CCoV lifecycle is closely connected to plasma membrane cholesterol, from cell entry to viral particle production. The methyl-bbeta;-cyclodextrin (Mbbeta;CD) was employed to remove cholesterol and to disrupt the lipid rafts. Cholesterol depletion from cellular membrane resulted in a dose-dependent reduction but not in the abolishment of virus infectivity and at a concentration of 15 mM, the reduction of the infection rate was about 68%. Mbbeta;CD treatment to verify if cholesterol in the envelope was required for CCoV infection, resulted in a dose-dependent inhibitory effect and at a concentration of 9 mM Mbbeta;CD infectivity was reduced by about 73%. Since viral entry would constitute a target for antiviral strategies, inhibitory molecules interacting with viral and/or cellular membranes or interfering with the function of lipid metabolism, could offer strong antiviral potential. It will be interesting in future to analyze the membrane microdomains in CCoV envelope.
Several members of the Circoviridae family have been shown to encode proteins with apoptotic activity. For example, both Porcine Circovirus Type 2 (PCV2) and Chicken Anemia Virus (CAV) encode a third viral protein (VP3) that has been shown to be cytotoxic. Interestingly, in the case of the CAV protein (deemed Apoptin), apoptosis is specific to transformed cell types. Similarities in genome structure and organization suggest that PCV Type 1 (PCV1) may also contain a third open reading frame that codes for a protein with homologous activity. To investigate this, ORF prediction followed by gene expression analyses were conducted on a gene found to be homologous to CAV and PCV2 VP3. Our data presented herein elucidate a putative ORF3 that codes for a viral protein with functional similarity to that of Apoptin and PCV2 VP3. Unlike its homologs, sequence analysis revealed a highly hydrophobic, extended C terminal domain in PCV1 VP3, which harbors a strong nuclear export signal. Subcellular localization analysis demonstrated divergent PCV1 VP3 localization patterns compared to that of CAV VP3. Interestingly, cytotoxicity studies revealed evidence that apoptosis may be selective to transformed cell types, similar to Apoptin. These results indicate that nuclear localization of PCV1 VP3 is neither necessary for induction of apoptosis nor for transformed cell selectivity unlike CAV VP3, suggesting an alternate mechanism of action.
Coxsackievirus B4 (CV-B4) belongs to the genus Enterovirus within family Picornaviridae. To investigate target proteins recognised by T-cells in human enterovirus B infections, viral-encoded structural (VP0 [VP4 and VP2], VP1, VP3) and non-structural (2A, 2B, 2C, 3C and 3D) proteins were expressed and purified in E. coli. Peripheral blood of 19 healthy adult donors was used to create enterovirus-specific T-cell lines by repeated stimulation with CV-B4 cell lysate antigen. T-cell lines responded in individual patterns, and responses to all purified proteins were observed. The most often recognised enteroviral protein was VP0, which is the fusion between the most conserved structural proteins, VP4 and VP2. T-cell responses to VP0 were detected in 15 of 19 (79%) donor lines. Non-structural 2C protein was recognised in 11 of 19 (58%) lines, and 11 of 19 (58%) lines also had a response to 3D protein. Furthermore, responses to other non-structural proteins (2A, 2B and 3C) were also detected. T-cell responses did not correlate clearly to the individual HLA-DR-DQ phenotype or the history of past coxsackie B virus infections of the donors.
Feline rotaviruses, members of Rotavirus A, are an infrequent source of zoonotic infections, and were previously shown by RNA-RNA hybridization assays to possess two distinct genomic RNA constellations each of which was represented by strains FRV-1 and FRV64. Due to the lack of whole genome sequence information of FRV-1, human rotavirus strain AU-1 has been used as a surrogate for the genotype constellation of feline rotaviruses. The aim of this study was to determine the whole genome sequence of FRV-1 and FRV64 to help understand the genetic relationships among existing feline rotaviruses from the evolutionary perspective. The genotype constellation of FRV-1 and FRV64 were G3-P-I3-R3-C3-M3-A3-N3-T3-E3-H3 and G3-P-I3-R3-C2-M3-A9-N2-T3-E3-H6, respectively. FRV-1 has a genotype constellation identical with that of the AU-1 strain. Although for individual genes they shared lineages with the exception of genes encoding VP2, VP6 and VP7, the sequence identity between FRV-1 and AU-1 was considered to be sufficiently high for the AU-1 to be regarded as an example of the direct transmission of a feline rotavirus to a child. On the other hand, the FRV64 strain was not only similar in all the 11 genome segments to another feline rotavirus strain, Cat97, but also to canine rotavirus strains (K9 and CU-1) and feline/canine-like human rotavirus strains (Ro1845 and HCR3). In conclusion, this study revealed intermingled sharing of genotypes and lineages among feline rotaviruses, suggesting the occurrence of frequent reassortment events over the course of evolution to emerge in four genotype constellations represented by FRV-1, FRV64/Cat97, Cat2 and BA222 strains.
Retinoic acid-induced gene I (RIG-I), plays a crucial role in sensing viral RNA and facilitating the production of IFN-bbeta;. It varies in length and sequence among different species. The present study assessed the functional differences among RIG-I proteins derived from mammals and birds. The transfection of duck caspase recruitment domains (CARDs) and RIG-I (dCARDs and dRIG-I) and goose CARDs and RIG-I (gCARDs and gRIG-I) into DF-1 cells increased the production of IFN-bbeta; mRNA and IFN-stimulated genes and decreased influenza A virus (IAV) replication, whereas, human CARDs and RIG-I (hCARDs and hRIG-I) and mouse CARDs and RIG-I (mCARDs and mRIG-I) had no effect. In 293T and A549 cells, hCARDs had the strongest IFN-inducing activity, followed by mCARDs, dCARDs, and gCARDs. The IFN-inducing activity of hRIG-I was stronger than that of mRIG-I, dRIG-I, and gRIG-I, in that order. The results also showed that although the ability dCARDs to activate IFN was stronger than that of gCARDs in DF-1, 293T, and A549 cells, dRIG-I had a weaker ability to activate IFN than gRIG-I in DF-1 with or without IAV infection. Taken together, these data suggest that RIG-I protein has different amino acid sequences and functions among species. This genetic and functional diversity renders it flexible, adaptable, and capable of recognizing many viruses in different species.
Bacteriophages and their derivatives are continuously gaining impetus as viable alternative therapeutic agents to control harmful multi-drug resistant bacterial pathogens, particularly in the food industry. The reduced efficacy of conventional antibiotics has resulted in a quest to find novel alternatives in the war against infectious disease. This study describes the full genome sequence of Cronobacter phage Ss1, with subsequent cloning and expression of its endolysin, capable of hydrolysing Gram-negative peptidoglycan. Cronobacter phage Ss1 is composed of 42,205 bp of double stranded DNA with a GC content of 46.1%. 57 ORFs were identified of which 18 could be assigned a putative function based on similarity to characterised proteins. The genome of Cronobacter phage Ss1 showed little similarity to the any other bacteriophage genomes available in the database and thus was considered unique. In addition, functional analysis on the predicted endolysin (LysSs1) was also investigated. Zymographic experiments demonstrated the hydrolytic activity of LysSs1 against Gram-negative peptidoglycan, thus highlighting the potential use of this enzyme as an antibacterial against Gram-negative pathogens.
Human cytomegalovirus (HCMV) infected cells a dramatic remodeling of the nuclear architecture linked to the creation, utilization and manipulation of sub-nuclear structures. This review outlines the involvement of several viral and cellular sub-nuclear structures in areas of HCMV replication and virus-host interaction that include viral transcription, viral DNA synthesis and the production of DNA filled viral capsids. The structures discussed include those that promote or impede HCMV replication (such as viral replication compartments and PML nuclear bodies, respectively) and those whose role in the infected cell is unclear (for example, nucleoli and nuclear speckles). Viral and cellular proteins associated with sub-nuclear structures are also discussed. The data reviewed here highlights advances in our understanding of HCMV biology and emphasizes the complexity of HCMV replication and virus-host interaction in the nucleus.
Part of the Togaviridae family, alphaviruses are arthropod-borne viruses that are widely distributed throughout the globe. Alphaviruses are able to infect a variety of vertebrate hosts, but in humans infection can result in extensive morbidity and mortality. Symptomatic infection can manifest as fever, an erythematous rash and/or significant inflammatory pathologies such as arthritis and encephalitis. Recent overwhelming outbreaks of alphaviral disease have highlighted the void in our understanding of alphavirus pathogenesis and the re-emergence of alphaviruses has given new impetus to anti-alphaviral drug design. In this review, the development of viable mouse models of Old Word and New World alphaviruses is examined. How mouse models that best replicate human disease have been used to elucidate the immunopathology of alphavirus pathogenesis and trial novel therapeutic discoveries is also discussed.
Summary: Chikungunya virus (CHIKV) has recently affected millions of people and with rare cases of encephalopathy and encephalitis essentially in neonates. We herein tested the capacity of mouse brain cells to control infection through innate immune antiviral responses. In vitro, CHIKV infected principally a subpopulation of mouse GFAP+ primary astrocytes. Oligodendrocytes and neurons could also be infected. An innate immune response was engaged by CHIKV-infected astrocytes and with elevated expression of mRNAs for IFN-aalpha;-bbeta;, inflammatory cytokines (e.g. IL-1bbeta;, IL-12, IL-10, IL-24), and proapoptotic factors (e.g. TNF-aalpha;, FasL, Lymphotoxin B). Programmed-cell death through the intrinsic caspase 9 pathway was observed by immunofluorescence in infected astrocytes, neurons but not in oligodendrocytes. Interestingly, microglia were not replicating CHIKV but responded as indicated by elevated MAPK activity. Intracerebroventricular injection of CHIKV in neonate mice led to the infection of astrocytes. The astrogliosis response was accompanied by a dendritic CD206+ cell mobilization restricted to the site of infection. Our results support the paradigm that a multifaceted innate immune response can be mobilized by both, professional immune and glial cells, to control CHIKV neuroinfection events in neonates.
Core plays a critical role during HCV assembly, not only as a structural component of the virion, but also as a regulator of the formation of assembly sites. In this study, we observed that core is expressed later than other HCV proteins in a single viral cycle assay, resulting in a relative increase of core expression during a late step of the viral life cycle. This delayed core expression results from an increase of core half-life, indicating that core is initially degraded and is stabilized at a late step of the HCV life cycle. A stabilization-mediated delayed kinetics of core expression was also observed using heterologous expression systems. Core stabilization did not depend on its interaction with non-structural proteins or lipid droplets but was correlated to its expression levels and its oligomerization status. Therefore in the course of a HCV infection, core stabilization likely occurs when the prior amplification of the viral genome during an initial replication step allows core to be synthesized at higher levels as a stable protein during the assembly step of the viral life cycle.
Vaccinia virus (VACV) is a large DNA virus that replicates in the cytoplasm and encodes about 200 proteins of which ~50% may be non-essential for virus replication. These proteins enable VACV to suppress transcription and translation of cellular genes, to inhibit the innate immune response, to exploit microtubule and actin based transport for virus entry and spread, and to subvert cellular metabolism for the benefit of the virus. VACV strain WR protein C16 induces stabilisation of the hypoxia inducible transcription factor (HIF)-1aalpha; by binding to the cellular oxygen sensor prolyl hydroxylase domain containing protein (PHD)2. Stabilisation of HIF-1aalpha; is induced by several virus groups, but the purpose and consequences are unclear. Here, 1H-NMR spectroscopy and liquid chromatography-mass spectrometry are used to investigate the metabolic alterations during VACV infection in HeLa and 2FTGH cells. The role of C16 in such alterations was examined by comparing infection with wild type VACV (strain WR) and a derivative virus lacking gene C16L (vC16). Compared to uninfected cells, VACV infection caused increased nucleotide and glutamine metabolism. In addition, there were increased concentrations of glutamine derivatives in cells infected with wild type VACV compared to vC16. This indicates that C16 contributes to enhanced glutamine metabolism, and this may help preserve tricarboxylic acid cycle activity. These data show that VACV infection reprograms cellular energy metabolism towards increased synthesis of the metabolic precursors utilised during viral replication, and that C16 contributes to this anabolic reprogramming of the cell, likely via the stabilisation of HIF-1aalpha;.
Introductions of H7 Influenza A virus (IAV) from wild birds into poultry have been documented worldwide, resulting in varying degrees of morbidity and mortality. H7 IAV infection in domestic poultry has served as a source of human infection and disease. We report the detection of H7N9 subtype IAV in Minnesota turkey farms during 2009 and 2011. The full-genome was sequenced from eight isolates as well as the hemagglutinin (HA) and neuraminidase (NA) gene segments of H7 and N9 virus subtypes for 108 isolates from North American wild birds between 1986 and 2012. Through maximum likelihood and coalescent phylogenetic analyses, we identified the recent H7 and N9 IAV ancestors of the turkey-origin H7N9 IAV, estimated the time and geographic origin of the ancestral viruses, and determined the relatedness between the 2009 and the 2011 turkey-origin H7N9 IAV. Analyses supported that the 2009 and the 2011 viruses were distantly related genetically, suggesting that the two outbreaks arose from independent introduction events from wild birds. Our findings further support that the 2011 MN turkey-origin H7N9 virus was closely related to H7N9 IAV isolated in poultry in Nebraska during the same year. Although the precise origin of the wild-bird donor of the turkey-origin H7N9 IAV could not be determined, our findings suggest that, for both the NA and HA gene segments, the MN turkey-origin H7N9 viruses were related to viruses circulating in wild birds between 2006 and 2011 in the Mississippi flyway.
In this study, we identified a novel virus from gentian (Gentiana triflora) that caused ring-spots on ovaries. Pollen grains carrying the virus were used to infect host plants by hand-pollination. Furthermore, the virus causes unusual symptoms, ring-spots that appear specifically on the outer surface of the ovarian wall after pollination. RNA extracted from the purified virions indicated that the virus has two segments, RNA1 and RNA2. The full-length cDNA sequence indicated that RNA1 had two ORFs: ORF1 had methyltransferase and helicase motifs, and ORF2 had an RNA-dependent RNA polymerase motif. RNA2 had five ORFs encoding a coat protein, triple gene block proteins 1-3 and a cysteine-rich protein. The length of RNA1 was 5519 bases and that of RNA2 was 3810 bases not including a polyU/polyA region between the first and second ORFs. Viral RNA does not have a polyA tail at the 3' end. Sequence homology and phylogenetic analysis suggested that the virus is closely related to Pecluvirus and Hordeivirus but was distinct from them. These combined results suggest that the causal agent inducing ring-spot symptoms on gentian ovaries is a new virus belonging to the family Virgaviridae but not to any presently known genus. We tentatively name the virus Gentian ovary ring-spot virus.
The dengue virus (DENV) envelope protein domain 3 (ED3) is the target of potent virus neutralizing antibodies. The DENV-2 ED3 contains adjacent type-specific and DENV complex-reactive antigenic sites that are composed of a small number of residues that were previously demonstrated to be critical for antibody binding. Site-directed mutagenesis of a DENV-2 16681 infectious clone was used to mutate critical residues in the DENV-2 type-specific (K305A and P384A) and DENV complex-reactive (K310A) antigenic sites. The K305A mutant virus multiplied like the parent virus in mosquito and mammalian cells, as did the P384A mutant virus, which required a compensatory mutation (G330D) for viability. However, the K310A mutant virus could not be recovered. The DENV-2 type-specific critical residue mutations K305A and P384A+G330D reduced the ability of DENV-2 type-specific, but not DENV complex-reactive, monoclonal antibodies (mAbs) to neutralize virus infectivity and this was directly correlated with mAb binding affinity to the rED3 mutants.
H9N2 avian influenza viruses are enzootic around the world, and can infect many different avian and mammalian hosts, including humans. Unlike the H9N2 viruses, which mainly originated in other countries and possess an non-structural protein 1( NS1) of 230 amino acids (aa), 98% of the H9N2 viruses isolated in China lack the 13 aa at the C-terminus of NS1 (217 aa in total). The biological significance of NS1 elongation remains elusive. In the current study, reverse genetics was used to generate a wild-type avian influenza H9N2 virus containing a 217 aa NS1 (H9N2NS1217) and two mutant viruses with elongated NS1s of 230 aa and 237 aa (H9N2NS1230 and H9N2NS1237), in order to examine the effect NS1 C-terminal elongation in the influenza virus. The C-terminal elongation of NS1 did not have significant impact on virus replication in MDCK or DF-1 cells. Although the three variants exhibited similar replicability in mice, the H9N2NS1230 and H9N2NS1237 up-regulated the level of inflammatory cytokines. In addition, both the H9N2NS1230 and H9N2NS1237 viruses increased replication and induced a high level of inflammatory cytokines and transmission in chickens, compared to the wild-type virus. These findings suggest that the NS1 extension conferred a gain of fitness to some extent.
The pattern of flavivirus infection in mosquitoes belonging to the genus Aedes and Culex collected in two regions of north-eastern Italy (Trentino and Veneto) was assessed. Mosquitoes were collected during 2012 and screened for Flavivirus using a generic RT-nested-PCR targeted on a region of the nonstructural 5 (NS5) gene. The phylogenetic analysis was performed on a fragment of around 1000 bp. Virus isolation was attempted in C6/36 insect cell lines and the infected cell cultures were studied by electron microscopy. We detected a wide distribution of Aedes Flavivirus (AeFV) in Aedes (Ae.) albopictus, with higher infection prevalence in Trentino than in Veneto. In Culex (Cx.) pipiens collected in Veneto, we detected a new sequence of an insect-specific flavivirus and one of Usutu virus (USUV). Interestingly, we detected AeFV in Cx. Pipiens for the first time in both regions. Viral isolation in cell culture was successful for AeFV. AeFV sequences found in Veneto showed a high percentage of similarity to those detected in Trentino and to those previously reported in other areas of northern Italy. Co-infections with different flaviviruses were not detected.
Human monkeypox is a viral zoonosis caused by monkeypox virus, an orthopoxvirus (OPXV). The majority of human monkeypox cases have been reported in moist forested regions in West and Central Africa, particularly in the Democratic Republic of the Congo (DRC). In this study we investigated zoonotic OPXV infection among wild animals in Zambia, which shares a border with DRC, to assess geographical distribution of OPXV. We screened for OPXV antibodies in sera from non-human primates (NHPs), rodents, and shrews by ELISA, and performed real-time PCR to detect OPXV DNA in spleen samples. Serological analysis indicated that 38 of 259 (14.7%) rodents, 14 of 42 (33.3%) shrews, and four of 188 (2.1%) NHPs had antibodies against OPXV. The OPXV DNA could not be detected in spleens from any animals tested. Our results indicated that wild animals living in rural human habitation areas of Zambia have been infected with OPXV.
The comparative long-term kinetics of human cytomegalovirus (HCMV) load and HCMV-specific antibody responses in the immunocompetent and immunocompromised solid-organ transplanted host during primary HCMV infection was investigated. On the whole, 40 immunocompetent subjects and 17 transplanted patients were examined for viral load as well as for IgG antibody responses to HCMV glycoproteins gH/gL/pUL128L, gH/gL and gB, and neutralizing antibodies in ARPE-19 epithelial cells and human fibroblasts. In parallel, the CD4+ and CD8+ HCMV-specific T-cell responses were determined by cytokine flow cytometry. Transplanted patients reached significantly higher viral DNA peaks, which persisted longer than in immunocompetent subjects. The ELISA-IgG responses to the pentamer, gH/gL and gB were significantly higher in primary infections of the immunocompetent until six months after onset, then the two antibody levels overlapped from six to 12 months. Antibody levels neutralizing infection of epithelial cells were significantly higher in transplanted patients after six months, persisting up to a year after transplantation. This trend was not observed for antibodies neutralizing infection of human fibroblasts, which showed higher titers in the immunocompetent over the entire 1-year follow-up. In conclusion, in immunocompromised patients the viral load peak was much higher, while the neutralizing antibody response exceeded that detected in the immunocompetent host starting six months after onset of follow-up, often concomitantly with a lack of specific CD4+ T-cells. In these setting, the elevated antibody response occurred in the presence of differentiated follicular helper T-cells in blood, which decreased in number as did antibody titers upon reappearance of HCMV-specific CD4+ T-cells.
PRD1 is a gram-negative bacteria infecting complex tailless icosahedral virus with an inner membrane. This type virus of Tectiviridae family contains at least 18 structural protein species of which several are membrane associated. Vertices of PRD1 virion consist of complexes recognizing the host cell except one special vertex through which the genome is packaged. Despite the extensive knowledge of the overall structure of PRD1 virion and several individual proteins at the atomic details, the locations and interactions of various integral membrane proteins and membrane-associated proteins still remains a mystery. Here, we demonstrate that blue native polyacrylamide gel electrophoresis can be used to probe protein-protein interactions in complex membrane-containing viruses. Using this technique and PRD1 as a model, we identified the known PRD1 multiprotein vertex structure composed of penton protein P31, spike protein P5, receptor binding protein P2 and stabilizing protein P16 linking the vertex to the internal membrane. Our results also indicate that two transmembrane proteins P7 and P14, involved in the viral nucleic acid delivery, make a complex. In addition, we performed a zymogram analysis using mutant particles devoid of special vertex indicating that the lytic enzyme P15 of PRD1 is not part of the packaging vertex thus contradicting previously published results.
We investigated the infectivity and transmissibility of the human seasonal H3N2, pandemic (pdm) H1N1 (2009), and B influenza viruses in dogs. Dogs inoculated with human seasonal H3N2 and pdm H1N1 influenza viruses exhibited nasal shedding and were seroconverted against the viruses; this did not occur in the influenza B virus-inoculated dogs. Transmission of human H3N2 virus between dogs was demonstrated by observing nasal shedding and seroconversion in naiiuml;ve dogs after contact with inoculated dogs. The seroprevalence study offered evidence of human H3N2 infection in dogs since 2008. Further, serological evidence of pdm H1N1 influenza virus infection alone and in combination with canine H3N2 virus was found in the serum samples collected from field dogs during 2010 and 2011. Our results suggest that dogs may be hosts for human seasonal H3N2 and pdm H1N1 influenza viruses.
Coronaviruses have been studied for over 60 years, but have only recently gained notoriety as deadly human pathogens with the emergence of severe respiratory syndrome coronavirus and Middle East respiratory syndrome virus. The rapid emergence of these viruses has demonstrated the need for good models to study severe coronavirus respiratory infection and pathogenesis. There are, currently, different methods and models for the study of coronavirus disease. The available genetic methods for the study and evaluation of coronavirus genetics are reviewed here. There are several animal models, both mouse and alternative animals, for the study of severe coronavirus respiratory disease that have been examined, each with different pros and cons relative to the actual pathogenesis of the disease in humans. A current limitation of these models is that no animal model perfectly recapitulates the disease seen in humans. Through the review and analysis of the available disease models investigators can employ the most appropriate available model to study coronavirus various aspects of pathogenesis and evaluate potential antiviral treatments that may potentially be successful in future treatment and prevention of severe coronavirus respiratory infections.