|Journal of General Virology current issue|
Laboratory animal models have provided valuable insight into foot-and-mouth disease virus (FMDV) pathogenesis in epidemiologically important target species. While not perfect, these models have delivered an accelerated time frame to characterize the immune responses in natural hosts and a platform to evaluate therapeutics and vaccine candidates at a reduced cost. Further expansion of these models in mice has allowed access to genetic mutations not available for target species, providing a powerful and versatile experimental system to interrogate the immune response to FMDV and to target more expensive studies in natural hosts. The purpose of this review is to describe commonly used FMDV infection models in laboratory animals and to cite examples of when these models have failed or successfully provided insight relevant for target species, with an emphasis on natural and vaccine-induced immunity.
A major research priority for HIV eradication is the elucidation of the events involved in HIV reservoir establishment and persistence. Cell-to-cell transmission of HIV represents an important area of study as it allows for the infection of cell types which are not easily infected by HIV, leading to the establishment of long-lived viral reservoirs. This phenomenon enables HIV to escape elimination by the immune system. This process may also enable HIV to escape suppressive effects of anti-retroviral drugs. During cell-to-cell transmission of HIV, a dynamic series of events ensues at the virological synapse that promotes viral dissemination. Cell-to-cell transmission involves various types of cells of the immune system and this mode of transmission has been shown to have an important role in sexual and mother-to-child transmission of HIV and spread of HIV within the central nervous system and gut-associated lymphoid tissues. There is also evidence that cell-to-cell transmission of HIV occurs between thymocytes and renal tubular cells. Herein, following a brief review of the processes involved at the virological synapse, evidence supporting the role for cell-to-cell transmission of HIV in the maintenance of the HIV reservoir will be highlighted. Therapeutic considerations and future directions for this area of research will also be discussed.
Hantaviruses are zoonotic viruses that cause life-threatening diseases when transmitted to humans. Severe hantavirus infection is manifested by impairment of renal function, pulmonary oedema and capillary leakage. Both innate and adaptive immune responses contribute to the pathogenesis, but the underlying mechanisms are not fully understood. Here, we showed that galectin-3-binding protein (Gal-3BP) was upregulated as a result of hantavirus infection both in vitro and in vivo. Gal-3BP is a secreted glycoprotein found in human serum, and increased Gal-3BP levels have been reported in chronic viral infections and in several types of cancer. Our in vitro experiments showed that, whilst Vero E6 cells (an African green monkey kidney cell line) constitutively expressed and secreted Gal-3BP, this protein was detected in primary human cells only as a result of hantavirus infection. Analysis of Gal-3BP levels in serum samples of cynomolgus macaques infected experimentally with hantavirus indicated that hantavirus infection induced Gal-3BP also in vivo. Finally, analysis of plasma samples collected from patients hospitalized because of acute hantavirus infection showed higher Gal-3BP levels during the acute than the convalescent phase. Furthermore, the Gal-3BP levels in patients with haemorrhagic fever with renal syndrome correlated with increased complement activation and with clinical variables reflecting the severity of acute hantavirus infection.
Whole-virus (WV) vaccines from influenza A/duck/Hokkaido/77 (H3N2), and its reassortant strains H3N4, H3N5 and H3N7, which have the same haemagglutinin (HA) gene but different neuraminidase (NA) genes, were prepared from our influenza virus library. Mice were intranasally immunized with equivalent doses of each vaccine (1nndash;0.01 mmicro;g per mouse). All of the mice that received the highest dose of each vaccine (1 mmicro;g per mouse) showed equivalent high HA-inhibiting (HI) antibody titres and survived the H3N2 challenge viruses. However, mice that received lower doses of vaccine (0.1 or 0.01 mmicro;g per mouse) containing a heterologous NA had lower survival rates than those given the H3N2-based vaccine. The lungs of mice challenged with H3N2 virus showed a significantly higher virus clearance rate when the vaccine contained the homologous NA (N2) versus a heterologous NA, suggesting that NA contributed to the protection, especially when the HI antibody level was low. These results suggested that, even if vaccines prepared for a possible upcoming pandemic do not induce sufficient HI antibodies, WV vaccines can still be effective through other matched proteins such as NA.
The avian-like swine influenza viruses emerged in 1979 in Belgium and Germany. Thereafter, they spread through many European swine-producing countries, replaced the circulating classical swine H1N1 influenza viruses, and became endemic. Serological and subsequent molecular data indicated an avian source, but details remained obscure due to a lack of relevant avian influenza virus sequence data. Here, the origin of the European avian-like swine influenza viruses was analysed using a collection of 16 European swine H1N1 influenza viruses sampled in 1979nndash;1981 in Germany, the Netherlands, Belgium, Italy and France, as well as several contemporaneous avian influenza viruses of various serotypes. The phylogenetic trees suggested a triple reassortant with a unique genotype constellation. Time-resolved maximum clade credibility trees indicated times to the most recent common ancestors of 34nndash;46 years (before 2008) depending on the RNA segment and the method of tree inference.
mAbs constitute an important biological tool for influenza virus haemagglutinin (HA) epitope mapping through the generation of escape mutants, which could provide insights into immune evasion mechanisms and may benefit the future development of vaccines. Several influenza A (H1N1) pandemic 2009 (pdm09) HA escape mutants have been recently described. However, the HA antigenic sites of the previous seasonal A/Brisbane/59/2007 (H1N1) (Bris07) virus remain poorly documented. Here, we produced mAbs against pdm09 and Bris07 HA proteins expressed in human HEK293 cells. Escape mutants were generated using mAbs that exhibited HA inhibition and neutralizing activities. The resulting epitope mapping of the pdm09 HA protein revealed 11 escape mutations including three that were previously described (G172E, N173D and K256E) and eight novel ones (T89R, F128L, G157E, K180E, A212E, R269K, N311T and G478E). Among the six HA mutations that were part of predicted antigenic sites (Ca1, Ca2, Cb, Sa or Sb), three (G172E, N173D and K180E) were within the Sa site. Eight escape mutations (H54N, N55D, N55K, L60H, N203D, A231T, V314I and K464E) were obtained for Bris07 HA, and all but one (N203D, Sb site) were outside the predicted antigenic sites. Our results suggest that the Sa antigenic site is immunodominant in pdm09 HA, whereas the N203D mutation (Sb site), present in three different Bris07 escape mutants, appears as the immunodominant epitope in that strain. The fact that some mutations were not part of predicted antigenic sites reinforces the necessity of further characterizing the HA of additional H1N1 strains.
Wild freshwater eel populations have dramatically declined in recent past decades in Europe and America, partially through the impact of several factors including the wide spread of infectious diseases. The anguillid rhabdoviruses eel virus European X (EVEX) and eel virus American (EVA) potentially play a role in this decline, even if their real contribution is still unclear. In this study, we investigate the evolutionary dynamics and genetic diversity of anguiillid rhabdoviruses by analysing sequences from the glycoprotein, nucleoprotein and phosphoprotein (P) genes of 57 viral strains collected from seven countries over 40 years using maximum-likelihood and Bayesian approaches. Phylogenetic trees from the three genes are congruent and allow two monophyletic groups, European and American, to be clearly distinguished. Results of nucleotide substitution rates per site per year indicate that the P gene is expected to evolve most rapidly. The nucleotide diversity observed is low (2nndash;3 %) for the three genes, with a significantly higher variability within the P gene, which encodes multiple proteins from a single genomic RNA sequence, particularly a small C protein. This putative C protein is a potential molecular marker suitable for characterization of distinct genotypes within anguillid rhabdoviruses. This study provides, to our knowledge, the first molecular characterization of EVA, brings new insights to the evolutionary dynamics of two genotypes of Anguillid rhabdovirus, and is a baseline for further investigations on the tracking of its spread.
The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the virus-encoded 3C protease to VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210E) within the VP1 protein and close to the VP1/2A cleavage site, inhibited cleavage of this junction and produced llsquo;self-taggedrrsquo; virus particles. A second site substitution (E83K) within VP1 was also observed within the rescued virus [Gullberg et al. (2013). J Virol 87, 11591nndash;11603]. It was shown here that introduction of this E83K change alone into a serotype O virus resulted in the rapid accumulation of a second site substitution within the 2A sequence (L2P), which also blocked VP1/2A cleavage. This suggests a linkage between the E83K change in VP1 and cleavage of the VP1/2A junction. Cells infected with viruses containing the VP1 K210E or the 2A L2P substitutions contained the uncleaved VP1-2A protein. The 2A L2P substitution resulted in the VP1/2A junction being highly resistant to cleavage by the 3C protease, hence it may be a preferred route for llsquo;taggingrrsquo; virus particles.
Tick-borne encephalitis (TBE), a disease caused by tick-borne encephalitis virus (TBEV), represents the most important flaviviral neural infection in Europe and north-eastern Asia. In the central nervous system (CNS), neurons are the primary target for TBEV infection; however, infection of non-neuronal CNS cells, such as astrocytes, is not well understood. In this study, we investigated the interaction between TBEV and primary human astrocytes. We report for the first time, to the best of our knowledge, that primary human astrocytes are sensitive to TBEV infection, although the infection did not affect their viability. The infection induced a marked increase in the expression of glial fibrillary acidic protein, a marker of astrocyte activation. In addition, expression of matrix metalloproteinase 9 and several key pro-inflammatory cytokines/chemokines (e.g. tumour necrosis factor aalpha;, interferon aalpha;, interleukin (IL)-1bbeta;, IL-6, IL-8, interferon -induced protein 10, macrophage inflammatory protein, but not monocyte chemotactic protein 1) was upregulated. Moreover, we present a detailed description of morphological changes in TBEV-infected cells, as investigated using three-dimensional electron tomography. Several novel ultrastructural changes were observed, including the formation of unique tubule-like structures of 17.9 pplusmn;0.15 nm diameter with associated viral particles and/or virus-induced vesicles and located in the rough endoplasmic reticulum of the TBEV-infected cells. This is the first demonstration that TBEV infection activates primary human astrocytes. The infected astrocytes might be a potential source of pro-inflammatory cytokines in the TBEV-infected brain, and might contribute to the TBEV-induced neurotoxicity and bloodnndash;brain barrier breakdown that occurs during TBE. The neuropathological significance of our observations is also discussed.
Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1-based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated subgenomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late-acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted, chimeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.
Astroviruses infect humans and many animal species and cause gastroenteritis. To extensively understand the distribution and genetic diversity of astrovirus in small mammals, we tested 968 anal swabs from 39 animal species, most of which were bats and rodents. We detected diverse astroviruses in 10 bat species, including known bat astroviruses and a large number of novel viruses. Meanwhile, novel groups of astroviruses were identified in three wild rodent species and a remarkably high genetic diversity of astrovirus was revealed in Eothenomys cachinus. We detected astroviruses in captive-bred porcupines and a nearly full-length genome sequence was determined for one strain. Phylogenetic analysis of the complete ORF2 sequence suggested that this strain may share a common ancestor with porcine astrovirus type 2. Moreover, to our knowledge, this study reports the first discovery of astroviruses in shrews and pikas. Our results provide new insights for understanding these small mammals as natural reservoirs of astroviruses.
Bone marrow stromal antigen 2 (BST-2; also known as tetherin or CD317) is an IFN-inducible gene that functions to block the release of a range of nascent enveloped virions from infected host cells. However, the role of BST-2 in viral pathogenesis remains poorly understood. BST-2 plays a multifaceted role in innate immunity, as it hinders retroviral infection and possibly promotes infection with some rhabdo- and orthomyxoviruses. This paradoxical role has probably hindered exploration of BST-2 antiviral function in vivo. We reported previously that BST-2 tethers Chikungunya virus (CHIKV)-like particles on the cell plasma membrane. To explore the role of BST-2 in CHIKV replication and host protection, we utilized CHIKV strain 181/25 to examine early events during CHIKV infection in a BST-2nndash;/nndash; mouse model. We observed an interesting dichotomy between WT and BST-2nndash;/nndash; mice. BST-2 deficiency increased inoculation site viral load, culminating in higher systemic viraemia and increased lymphoid tissues tropism. A suppressed inflammatory innate response demonstrated by impaired expression of IFN-aalpha;, IFN- and CD40 ligand was observed in BST-2nndash;/nndash; mice compared with the WT controls. These findings suggested that, in part, BST-2 protects lymphoid tissues from CHIKV infection and regulates CHIKV-induced inflammatory response by the host.
Reverse genetics is a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but methods based on the preparation of plasmid-based complete viral genomes are laborious and unpredictable. Here, both wild-type and genetically modified infectious RNA viruses were generated in days using the newly described ISA (infectious-subgenomic-amplicons) method. This new versatile and simple procedure may enhance our capacity to obtain infectious RNA viruses from PCR-amplified genetic material.
Direct-acting antivirals have significantly improved treatment outcomes in chronic hepatitis C (CHC), but side effects, drug resistance and cost mean that better treatments are still needed. Lipid metabolism is closely linked with hepatitis C virus (HCV) replication, and endocannabinoids are major regulators of lipid homeostasis. The cannabinoid 1 (CB1) receptor mediates these effects in the liver. We have previously shown upregulation of CB1 receptors in the livers of patients with CHC, and in a HCV cell-culture model. Here, we investigated whether CB1 blockade inhibited HCV replication. The antiviral effect of a CB1 antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), was examined in HCV strain JFH1 cell-culture and subgenomic replicon models. The effects on the expression of genes involved in lipid metabolism were also measured. CB1 short hairpin RNA (shRNA) was used to confirm that the effects were specific for the cannabinoid receptor. Treatment with AM251 strongly inhibited HCV RNA (~70 %), viral protein (~80 %), the production of new virus particles (~70 %) and virus infectivity (~90 %). As expected, AM251 reduced the expression of pro-lipogenic genes (SREBP-1c, FASN, SCD1 and ACC1) and stimulated genes promoting lipid oxidation (CPT1 and PPARaalpha;). This effect was mediated by AMP-activated protein kinase (AMPK). Stable CB1 knockdown of cells infected with HCV showed reduced levels of HCV RNA compared with controls. Thus, reduced CB1 signalling inhibits HCV replication using either pharmacological inhibitors or CB1 shRNA. This may be due, at least in part, to reduced lipogenesis, mediated by AMPK activation. We suggest that CB1 antagonists may represent an entirely new class of drug with activity against HCV.
The order Nidovirales contains large, enveloped viruses with a non-segmented positive-stranded RNA genome. Nidoviruses have been detected in man and various animal species, but, to date, there have been no reports of nidovirus in reptiles. In the present study, we describe the detection, characterization, phylogenetic analyses and disease association of a novel divergent nidovirus in the lung of an Indian python (Python molurus) with necrotizing pneumonia. Characterization of the partial genome (ggt;33 000 nt) of this virus revealed several genetic features that are distinct from other nidoviruses, including a very large polyprotein 1a, a putative ribosomal frameshift signal that was identical to the frameshift signal of astroviruses and retroviruses and an accessory ORF that showed some similarity with the haemagglutininnndash;neuraminidase of paramyxoviruses. Analysis of genome organization and phylogenetic analysis of polyprotein 1ab suggests that this virus belongs to the subfamily Torovirinae. Results of this study provide novel insights into the genetic diversity within the order Nidovirales.
The objective of this study was to compare the virulence and pathogenicity of a combination of concurrent infections of two genotypes of porcine circovirus type 2 (PCV2) and two genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) in terms of PCV2 viraemia, and PCV2-associated lesions and antigens in co-infected pigs. Pigs with PCV2a (or 2b)/type 1 (or type 2) PRRSV had significantly (Pllt;0.05) higher mean clinical respiratory scores and lower average daily weight gain compared with pigs with PCV2a (or 2b). Co-infection induced significantly lower levels of anti-PCV2 and anti-PRRSV IgG antibodies than infection with one genotype alone, regardless of the genotype of the two viruses. Pigs with PCV2a (or 2b)/type 2 PRRSV had significantly (Pllt;0.05) higher levels of PCV2 viraemia, more severe PCV2-associated lesions, and more PCV2 DNA within the lesions compared with pigs with PCV2a (or 2b)/type 1 PRRSV. However, there was no significant difference in these parameters in pigs with PCV2a/type 2 PRRSV or PCV2b/type 2 PRRSV. The results of this study demonstrate significant differences in the virulence and pathogenicity of type 1 and type 2 PRRSV but no significant differences in the virulence and pathogenicity of PCV2a and PCV2b with respect to the production of PCV2-associated lesions.
In 2012, a mutant porcine circovirus type 2 (mPCV2) strain was identified in cases of PCV-associated disease (PCVAD) in the USA. The mPCV2 had an additional amino acid, lysine (K), in the capsid at position 234. The objectives of this study were to compare the pathogenicity of mPCV2, PCV2a and PCV2b in pigs using biologically pure infectious virus stocks derived from respective infectious DNA clones, and to investigate the importance of genotype-specific ORF2 and the presence of lysine at position 234 of the capsid. A total of 47, 2-week-old, caesarean-derived, colostrum-deprived (CDCD) pigs were assigned to one of seven groups. At 3 weeks of age, the pigs were experimentally inoculated with saline, PCV2a, PCV2b, mPCV2, PCV2b-234-K (lysine addition in ORF2), chimeric PCV2b-ORF1/mPCV2-ORF2 or reciprocal chimeric mPCV2-ORF1/PCV2b-ORF2. All pigs were necropsied 21 days post-infection (p.i.). Gross lesions were limited to visible icterus and loss of body condition in a portion of the mPCV2 pigs. The amount of PCV2 DNA was significantly higher in pigs inoculated with mPCV2 compared with PCV2b in sera at 7 days p.i. and faecal swabs at 14 days p.i. Based on lymphoid lesions, a higher prevalence of PCVAD was seen in pigs infected with PCV2s containing the additional 234-K (64.3 %) compared with those infected with a PCV2 with the regular 233 bp ORF2 (40 %). Results indicated that all PCV2 isolates were capable of inducing severe lesions and disease in the CDCD pig model, and there was no significant difference in virulence.
We used SILAC (stable isotope labelling of amino acids in cell culture) and high-throughput quantitative MS mass spectrometry to analyse the protein composition of highly purified WT wild type adenoviruses, mutant adenoviruses lacking an internal protein component (protein V) and recombinant adenoviruses of the type commonly used in gene therapy, including one virus that had been used in a clinical trial. We found that the viral protein abundance and composition were consistent across all types of virus examined except for the virus lacking protein V, which also had reduced amounts of another viral core protein, protein VII. In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins. We believe this technique is a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus-like particles. The raw data have been deposited at proteomexchange, identifer PXD001120.
The strain JS11C1, a member of a putative new subgroup of avian leukosis virus (ALV) that is different from all six known subgroups from chickens based on Gp85 amino acid sequence comparison, was isolated from Chinese native chicken breeds in 2012. In order to further study the genome structure, biological characteristics, and the evolutionary relationship of the virus with others of known subgroups from infected chickens, we determined the complete genome sequence, constructed an infectious clone of ALV strain JS11C1, and performed comparative analysis using the whole genome sequence or elements with that of other ALVs available in GenBank. The results showed that the full-length sequence of the JS11C1 DNA provirus genome was 7707 bp, which is consistent with a genetic organization typical of a replication-competent type C retrovirus lacking viral oncogenes. The rescued infectious clone of JS11C1 showed similar growth rate and biological characteristics to its original virus. All the comparison analyses based on whole genomes support the opinion that the new isolates are relatively distantly related to any known subgroups of ALVs and might be classified as a new subgroup.
A high prevalence of the rtI187V polymerase substitution of hepatitis B virus (HBV) was detected in nucleoside/nucleotide-analogue-naive and -treated chronic hepatitis B (CHB) patients. We aimed at assessing the replicative capacity and susceptibility to lamivudine (LAM) and adefovir (ADV) in vitro of HBV harbouring rtI187V alone or in conjunction with LAM- or ADV-resistant mutations. The reverse transcriptase region of HBV isolates was directly sequenced from a cohort of 300 CHB patients from China. Replication-competent HBV constructs containing rtI187V and combined with LAM-resistant (rtM204I, rtL180M/rtM204V) mutations were generated, and compared with WT, LAM-resistant single (rtM204I) or double (rtL180M/rtM204V) and ADV-resistant (rtN236T) clones. In a Chinese cohort of 300 CHB patients, 8.7 % (26/300) showed substitution of rtI187 with V. Of note, the rtI187V prevalence in HBV genotype B was significantly higher than that in HBV genotype C (95.2 vs 4.8 %). In vitro phenotypic assays showed that the viruses bearing the rtI187V substitution had impaired replication efficacy when compared with the WT and the virus carrying rtI187V combined with LAM-resistant single or double mutations showed even more significantly impaired replicative capacities. Furthermore, rtI187V HBV remained susceptible towards treatment with LAM or ADV in vitro whereas the combination of the rtI187V substitution with LAM-resistant mutations rendered HBV resistant to LAM but still sensitive to ADV. Our study revealed that the rtI187V substitution in the HBV polymerase frequently occurred in CHB patients, particularly those with HBV genotype B. However, the emergence of the rtI187V substitution significantly impaired viral replication but without affecting drug sensitivity in vitro.
The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host-range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with EGFP fused to the nucleocapsid to monitor virus entry into primary midgut epithelium cells ex vivo using confocal fluorescence microscopy. The EGFP-labelled virus showed similar BV virulence and ODV infectivity as WT virus. The ability to bind and enter host cells was then visualized for WT AcMNPV and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, whilst PIF1 and PIF2 play important roles in the entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.
Begomoviruses are whitefly-transmitted, ssDNA plant viruses and are among the most damaging pathogens causing epidemics in economically important crops worldwide. Wild/non-cultivated plants play a crucial epidemiological role, acting as begomovirus reservoirs and as llsquo;mixing vessels' where recombination can occur. Previous work suggests a higher degree of genetic variability in begomovirus populations from non-cultivated hosts compared with cultivated hosts. To assess this supposed host effect on the genetic variability of begomovirus populations, cultivated (common bean, Phaseolus vulgaris, and lima bean, Phaseolus lunatus) and non-cultivated (Macroptilium lathyroides) legume hosts were sampled from two regions of Brazil. A total of 212 full-length DNA-A genome segments were sequenced from samples collected between 2005 and 2012, and populations of the begomoviruses Bean golden mosaic virus (BGMV) and Macroptilium yellow spot virus (MaYSV) were obtained. We found, for each begomovirus species, similar genetic variation between populations infecting cultivated and non-cultivated hosts, indicating that the presumed genetic variability of the host did not a priori affect viral variability. We observed a higher degree of genetic variation in isolates from MaYSV populations than BGMV populations, which was explained by numerous recombination events in MaYSV. MaYSV and BGMV showed distinct distributions of genetic variation, with the BGMV population (but not MaYSV) being structured by both host and geography.
We describe the metagenomics-derived feline enteric virome in the faeces of 25 cats from a single shelter in California. More than 90 % of the recognizable viral reads were related to mammalian viruses and the rest to bacterial viruses. Eight viral families were detected: Astroviridae, Coronaviridae, Parvoviridae, Circoviridae, Herpesviridae, Anelloviridae, Caliciviridae and Picobirnaviridae. Six previously known viruses were also identified: feline coronavirus type 1, felid herpes 1, feline calicivirus, feline norovirus, feline panleukopenia virus and picobirnavirus. Novel species of astroviruses and bocaviruses, and the first genome of a cyclovirus in a feline were characterized. The RNA-dependent RNA polymerase region from four highly divergent partial viral genomes in the order Picornavirales were sequenced. The detection of such a diverse collection of viruses shed within a single shelter suggested that such animals experience robust viral exposures. This study increases our understanding of the viral diversity in cats, facilitating future evaluation of their pathogenic and zoonotic potentials.
We isolated and characterized a novel virulent bacteriophage, IME-EFm1, specifically infecting multidrug-resistant Enterococcus faecium. IME-EFm1 is morphologically similar to members of the family Siphoviridae. It was found capable of lysing a wide range of our E. faecium collections, including two strains resistant to vancomycin. One-step growth tests revealed the host lysis activity of phage IME-EFm1, with a latent time of 30 min and a large burst size of 116 p.f.u. per cell. These biological characteristics suggested that IME-EFm1 has the potential to be used as a therapeutic agent. The complete genome of IME-EFm1 was 42 597 bp, and was linear, with terminally non-redundant dsDNA and a G+C content of 35.2 mol%. The termini of the phage genome were determined with next-generation sequencing and were further confirmed by nuclease digestion analysis. To our knowledge, this is the first report of a complete genome sequence of a bacteriophage infecting E. faecium. IME-EFm1 exhibited a low similarity to other phages in terms of genome organization and structural protein amino acid sequences. The coding region corresponded to 90.7 % of the genome; 70 putative ORFs were deduced and, of these, 29 could be functionally identified based on their homology to previously characterized proteins. A predicted metallo-bbeta;-lactamase gene was detected in the genome sequence. The identification of an antibiotic resistance gene emphasizes the necessity for complete genome sequencing of a phage to ensure it is free of any undesirable genes before use as a therapeutic agent against bacterial pathogens.
Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrPSc) in the central nervous system. The pathological features and biochemical properties of PrPSc in macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldtnndash;Jakob disease (vCJD). Non-human primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method for in vitro amplification of cynomolgus macaque BSE PrPSc. This method involves amplifying PrPSc by protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrPC substrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSc contained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrPSc in the CSF by serial PMCA, and the CSF levels of PrPSc tended to increase with disease progression. In addition, PrPSc was detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrPSc in non-human primate models of CJD.
|Journal of General Virology Publish Ahead of Print|
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.
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 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 reactivation of latent virus by 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 methylprednisone, 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+R- group.
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 open reading frames encoded by 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 etiological 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 Caliciviridae, is the only member of the Sapovirus genus 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 the reduction in STAT1-mediated signaling 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 interferon because treatment of cells with recombinant porcine IFN-bbeta; resulted in significantly reduced viral replication. Moreover, IFN-mediated signaling pathways (IFN, STAT1 and OAS1) were activated during PSaV infection. Characterization of PSaV growth in cell lines deficient in their ability to induce or respond to interferon, resulted in a 100-150 fold increase in infectious virus production, indicating that the primary role of bile acids is 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 highlights the key role of the innate immune response in the restriction of PSaV infection and should greatly facilitate further molecular studies on sapovirus host-cell interactions.
Torquetenovirus (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 viremia 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 viremia compatible with replication in T-lymphocytes, and highly dependent on the potency of the anti-T cell drug administered.
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 modeled in several in vitro assays, including serial protein misfolding amplification (sPMCA), amyloid seeding, and real time quaking induced conversion (RT-QuIC). Because RT-QuIC measures formation of amyloid fibrils in real time, it can be used to estimate the rate of seeded conversion. Here we use samples from deer infected with chronic wasting disease (CWD) in RT-QuIC to show that serial dilution of prion seed is linearly related to the rate of amyloid formation over a range of 10-3 to 10-8 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 estimate that urine and saliva from CWD-infected deer contain between 1 and 5 LD50 per 10 ml, respectively. Thus, over the 1 to 2 year course of infection, a substantial environmental reservoir of CWD prion contamination accumulates.
Rotaviruses of group A (RVA) 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 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 an unusual RVA of genotype G8P is described 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 NSP1 to NSP5 revealed a typical bovine-like genotype constellation G8-P-I2-Rx-C2-Mx-A3-N2-T6-E2-H3. Phylogenetic analysis supports the hypothesis of an interspecies transmission from a bovine/artiodactyl species or from humans to the young dog. The isolate is likely to represent a multiple reassortant virus.
Human papillomavirus (HPV) has a well-recognized etiological role in the development of cervical cancer and other anogenital tumors. Recently, an association between colorectal cancer and HPV infection has been suggested, although still controversial. This study aimed at detecting and characterizing HPV infection in fifty-seven 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 tumor tissues and 8.8% of noncancerous tissue samples were HPV DNA positive. 85.7% of these samples were successfully genotyped, with 41.7% of sequences identifying four genotypes comprised in the HR-clade Group 1; the remaining 58.3% of HPV-genotyped specimens had an unclassified beta-HPV. Examining additional cases and analyzing whole genomes will help to outline the significance of this finding.
A common critical cellular event 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 hemagglutinin precursor (HA0) subtypes is critical for yielding fusion-competent infectious virions. In this study, we hypothesize 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 lifecycle. We explore the biological role in this process of a newly identified furin-directed human microRNA, miR-24, 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 demonstrate a robust decrease in both furin mRNA level and intracellular furin activity in A549 cells. Importantly, pretreatment of A549 cells with miR-24 mimics results in a robust decrease in the production 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 down-regulation of furin-directed microRNAs such as miR-24 during the lifecycle of HP infA viruses may represent a novel regulatory mechanism that governs furin-mediated proteolytic activation of HA0 glycoproteins and production of infectious virions.
Human T-cell leukemia virus type-1 (HTLV-1) infection causes adult T-cell leukemia (ATL). The viral protein HTLV-1 bZIP factor (HBZ) is constitutively expressed in ATL cells, suggesting that HBZ plays a major role in HTLV-1 pathogenesis. Here, we identified centromere protein B (CENP-B) as a novel interacting partner with 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 newly discovered DNA virus in grapevine that is closely associated with 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 three days after agroinfiltration of Nicotiana benthamiana leaves. We found that the GVCV DNA segment between nucleotides 7,332 and 7,672 directed expression of GFP and it was stronger than the Cauliflower mosaic virus 35S promoter. 5rrsquo; and 3rrsquo; RACE revealed that transcription was initiated predominantly at nucleotide 7,571 and terminated at nucleotide 7,676.
Invertebrate iridescent virus 6 (IIV-6) is a nucleocytoplasmic virus with a 212 kb-long linear double-stranded DNA genome that encodes 215 putative open reading frames. 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 indicates 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 quantitation using a proteomic approach. A total of 95 viral encoded proteins were detected in infected cells, of which 37 are virion proteins. The expressed IIV-6 virion proteins could be categorized into three main clusters based on their expression profiles. These clusters were: 1) proteins with stably low or 2) exponentially increasing expression levels during infection, and 3) proteins that were initially highly abundant, but showed slightly reduced levels after 48 hours (h) post infection (p.i.). Here, we provide novel information on the kinetics of virion and infected cell-specific protein levels that assists in understanding gene regulation in this lesser known DNA virus model.
Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-type chickens that had developed myeloid leukosis. However, ALV-J infections associated with hemangiomas have occurred in layer flock 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 5rrsquo;-LTR region 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 5rrsquo;-LTR sequences, particularly the U3 sequences. A 19-nt insertion was uniquely found in the U3 region of the HLJ13SH01 strain. The results of the Dual-Glo Luciferase 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 5rrsquo;-U3 region. To confirm the potentiation effect of the 19-nt insertion and the CCAAT/enhancer element on virus replication, three infectious clones with 5rrsquo;-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.
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 titer 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.
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 JFH1 envelope glycoprotein to profile antibody response in four HCV chronically infected individuals. 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 (80.9-99.8%) while that against E1 was relatively small (0.3-19.0%). A total of five major antigenic domains (D1-D5) were identified; one was within the E1 and additional four within the 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 the E1 contained additional neutralizing epitopes. Our results have provided additional insghts into HCV-specific antibody response in human and should assist our better understanding of protective antibody immunity and to guide development of effective vaccines and therapeutics against HCV infection.
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 3rrsquo; end and gradually decreased as the gene was inserted closer to the 5rrsquo; end. Measurement of the GFP fluorescence intensity in recombinant virus-infected cells demonstrated that the noncoding region between the P and M genes is the optimal insertion site for foreign gene expression in the VG/GA vaccine vector.
Patients' biopsies show that hepadnaviral core proteins and capsids - collectively called core - are found in the nucleus and in the cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viremia while nuclear core is associated with high viral load. In order to better understand the molecular interactions leading to core localization, we analyzed 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 viral context resulted in a predominantly cytoplasmic stain. Analyzing which viral partner was responsible for the cytoplasmic retention we found the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that not only the polymerase but also , an RNA structure to which the viral polymerase binds, was essential. Furthermore we showed that core protein phosphorylation at serine 164 was essential for the cytoplasmic phenotype of core, which likely explains differences between individual cells.
Infection of astrocytes by the neuropathogenic mutant of Moloney murine leukemia virus (MuLV), ts1, exhibits increased levels of reactive oxygen species (ROS) and signs of oxidative stress compared to 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 ATM kinase, which in turn phosphorylates the serine 15 site 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.
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.
In December 1983 a seminal paper appeared on the overexpression of human interferon-bbeta; in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produce massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes are dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years major improvements have been 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 baculovirus expression vectors are used to deliver and express heterologous genes in mammalian cells and may find applications for gene therapy and cancer treatment in the future. The purpose of this paper is to highlight the 30-years 'anniversary' of this expression system by summarizing the fundamental research that allowed the development of this expression system and by indicating the major technological advances since 1983. Finally, attention will be paid to the future challenges to further optimize this amazing technology.
Chikungunya virus (CHIKV) 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 geographic range primarily restricted to Sub-Saharan Africa, the Indian subcontinent, and Southeast 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.
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 viremia 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 hypothesized 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+ compared to 100% in RK-13. In susceptible CD172a+, the expression of immediate-early (IEP) and early (EICP22) proteins was delayed in the cell nuclei by 2-3 hpi compared to RK-13, and the formation of replicative compartments by 15 hpi. The viral production in CD172a+ was significantly lower (from 101.7 to 3.1 TCID50/105 inoculated cells) than in RK-13 (from 105 to 5.7 TCID50/105 inoculated cells). Less than 0.02% of inoculated CD172a+ produced and transmitted infectious virus to neighbor cells. Pretreatment of CD172a+ with inhibitors of HDAC activity increased and accelerated viral protein expression at very early time of infection and induced productive infection in CD172a+. Our results demonstrated that the restriction and delay of EHV-1 replication in CD172a+ is part of an immune evasive strategy and involves silencing of EHV-1 gene expression associated with histone deacetylases.
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' untranslated regions (UTRs) of targeted genes. Recent studies have demonstrated that miRNAs serve as critical effectors in intricate networks of host-pathogen interactions. Notably, we found that Bos taurus bta-miR-29b (referred to as miR-29b herein) was significantly upregulated more than 2.3-fold in bovine viral diarrhea virus (BVDV) NADL-infected Madin-Darby bovine kidney (MDBK) cells 6 h post-infection (pi) compared to normal MDBK cells. However, the roles of miR-29b in BVDV infection and pathogenesis remain unclear. Here, we discover 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 contribute to BVDV NADL replication in MDBK cells and that the inhibition of autophagy represses 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 they provide a new avenue for the intimate interaction between host cells and pathogens.
Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) genome segment 4 (S4) showed that it consists of 3410 nucleotides with a single ORF of 1110 amino acids which could encode a protein of ~ 127 kDa (p127). Bioinformatics analysis showed the presence of 5rrsquo; RNA triphosphatase (RTPase) domain (LRDR), S-adenosyl-L-methionine (SAM) binding (GxGxG) motif and K-D-K-E tetrad of 2rrsquo;-O-methyltransferase (MTase), which suggests that S4 may encode RTPase and MTase. The ORF of S4 was expressed in E. coli as His-tagged fusion protein and purified by Ni-NTA affinity chromatography. Biochemical analysis of recombinant p127 showed its 5rrsquo; RTPase as well as SAM dependent guanine N-7 and ribose 2rrsquo;-O-MTase activity. MTase assay using in vitro transcribed AmCPV S2 RNA having 5rrsquo; G*pppG end showed that guanine N-7- methylation occurs prior to the ribose 2rrsquo;-O-methylation to yield m7GpppG/ m7GpppGm RNA cap.. Mutagenesis of the SAM binding (GxGxG) motif (G831A) completely abolished N-7 and 2rrsquo;-O-MTase activity indicating importance of these residues for capping. From the kinetic analysis the Km of N-7-MTase for SAM and RNA was calculated as 4.41 and 0.39 mmu;M, respectively. These results suggest that AmCPV S4 encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5rrsquo; end of viral RNA.
Bats have been found to harbor 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 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 2009-2011.
The interferon (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 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 (OAS2), interferon-induced transmembrane protein 1 (IFITM1) and myxovirus-resistance 2 (Mx2). 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 anti-viral effect of IFITM3 protein was not affected by ubiquitination modification. Furthermore, knocking down the endogenous and IFN-induced expressions of IFITM1 and IFITM3 proteins facilitated RSV infection. Expression of the IFITM proteins was found to delay the phosphorylation of interferon regulatory factor-3 (IRF3) through interfering with the detection of viral RNA by the melanoma differentiation-associated gene 5 (MDA5) and the retinoic acid-inducible gene I (RIG-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 provide further insight for exploring the mechanism of IFITM proteins-mediated virus restriction.
Evidence is accumulating that one or more beta-retrovirus is associated with human breast cancer. Retroviruses can exist as an infectious (exogenous) virus or as a part of the genetic information of cells due to germline integration (endogenous). An exogenous virus with a genome that is highly homologous to mouse mammary tumor virus (MMTV) is gaining acceptance as possibly being associated with human breast cancer and recently furnished evidence is discussed in this article, as is the evidence for involvement of an endogenous human beta-retrovirus, HERV-K. Modes of interaction are also reviewed and linkage to APOBEC3 suggested.
Previous studies of duck hepatitis A virus infection have focused only on the pathogenicity and host response of one strain. Here, we showed that the virulent SH strain and the attenuated FC64 strain induced varied pathogenicity, apoptosis, and immune responses in the liver of 1-day-old ducklings. SH infection caused apoptosis and visible lesions in the liver, and serum AST, ALT, ALP, GGT, and T-Bil activities were greatly up-regulated, and ducklings died at 36 hours post-infection (h.p.i.). However, FC64 infection did not induce significant symptoms or impair liver function, and all of the infected ducklings remained healthy. In addition, both virus strains replicated well in the liver, spleen, and intestine, while the SH strain replicated more efficiently than FC64. IFN-, IL-2, iNOS, and NO were strongly induced by SH infection, and may be associated with the pathogenicity of the SH strain. IFN-aalpha;, IFN-bbeta;, IFITM1, ISG12, OASL, and IL-6 were moderately induced by SH infection at 24 h.p.i., and dramatically induced by FC64 infection at 36 h.p.i. The intensive induction of cytokines by FC64 may be involved in restriction of virus replication and stimulation of adaptive immune responses. Ducklings inoculated with FC64 produced high levels of anti-viral antibodies within 45 days post-infection. The low virulence and strong immune response of FC64 rendered this strain a good vaccine candidate, as confirmed by a protective assay in this study.
Human herpesvirus 6 (HHV-6) glycoprotein M (gM) is an envelope glycoprotein that associates with glycoprotein N (gN), forming the gM/gN protein complex, as similar manner to the other herpesviruses. LC-MS/MS analysis showed that the HHV-6 gM/gN complex interacts with the v-SNARE protein, VAMP3. VAMP3 colocalized with the gM/gN complex at the trans-Golgi network and the other compartments, possibly late endosome in HHV-6-infected cells, and its expression gradually increased during the late phase of virus infection. Finally, VAMP3 was incorporated into mature virions, maybe being transported with the gM/gN complex.
The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the remaining N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified. The domain was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain was not reacted with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis.
Replicon particle-based vaccines combine the efficacy of live-attenuated vaccines with the safety of inactivated or subunit vaccines. Recently, we developed Rift Valley fever virus (RVFV) replicon particles, also known as nonspreading RVFV (NSR) and demonstrated that a single vaccination with these particles can confer sterile immunity in target animals. NSR particles can be produced by transfection of replicon cells, which stably maintain replicating RVFV S and L genome segments, with an expression plasmid encoding the RVFV glycoproteins, Gn and Gc, normally encoded by the M-genome segment. Here, we explored the possibility to produce NSR with the use of a helper virus. We show that replicon cells infected with a Newcastle disease virus expressing Gn and Gc (NDV-GnGc) are able to produce high levels of NSR particles. In addition, using reverse-genetics and site-directed mutagenesis, we were able to create an NDV-GnGc variant that lacks the NDV fusion protein and contains 2 amino acid substitutions in respectively Gn and HN. The resulting virus uses a unique entry pathway that facilitates the efficient production of NSR in a one-component system. The novel system provides a promising alternative for transfection-based NSR production.
Despite the success of combined antiretroviral therapy in controlling viral replication in HIV-infected individuals, HIV-associated neurocognitive disorders (Manganaro et al., 2014), commonly referred to as neuroAIDS, remain a frequent and poorly understood complication. Infection of CD8+ lymphocyte-depleted rhesus macaques with SIVmac251 viral swarm is a well-established rapid disease model of neuroAIDS that has provided critical insight into HAND onset and progression. However, no studies so far have characterized in depth the relationship between intra-host viral evolution and pathogenesis in this model. SIV env gp120 sequences were obtained from six infected animals. Sequences were sampled longitudinally from several lymphoid and non-lymphoid tissues, including individual lobes within the brain at necropsy, for four macaques; two animals were sacrificed at 21 days post-infection (dpi) to evaluate early viral seeding of the brain. Bayesian phylodynamic and phylogeographic analyses of the sequence data were used to ascertain viral population dynamics and gene flow between peripheral and brain tissues, respectively. A steady increase in viral effective population size, with a peak occurring at approximately 50-80 dpi, was observed across all longitudinally monitored macaques. Phylogeographic analysis indicated continual viral seeding of the brain from several peripheral tissues throughout infection, with the last migration event before terminal illness occurring in all macaques from cells within the bone marrow. The results strongly support the role of infected bone marrow cells in HIV/SIV neuropathogenesis. In addition, our work demonstrates the applicability of Bayesian phylogeography to intra-host studies in order to assess the interplay between viral evolution and pathogenesis.
A new nodavirus (named covert mortality nodavirus, CMNV) is associated with covert mortality disease of shrimp which caused serious loss in China since 2009. Histopathological examination of shrimp suffering the disease revealed coagulative necrosis of striated muscle similar to typical histopathology features of infectious myonecrosis virus (IMNV), Penaeus vannamei nodavirus (PvNV) and Macrobrachium rosenbergii nodavirus (MrNV). However, shrimp suffering this disease tested negative for IMNV, MrNV and PvNV by RT-PCR. Additionally, eosinophilic inclusions were found in epithelium of the tubules in hepatopancreas and lymphoid organ, and mass karyopyknotic nuclei existed in muscle and lymphoid organ. The tubular epithelium of hepatopancreas show significant atrophy. A cDNA library was constructed from total RNA of infected shrimp. Sequencing and alignment analysis showed that one clone with an 1185 bp insert (designated CMNV-7) shares 54%, 53% and 39% identities with the amino acid sequences of RNA-dependent RNA polymerase from flock house virus, black beetle virus, and MrNV. The results of fluorescence in situ hybridization (FISH) showed that the hepatopancreas, striated muscle, and lymphoid organ were positively reacting tissues. The average size of negative stained virus particles was 32 nm. In addition, a nested RT-PCR assay was developed for CMNV and the RT-PCR detection results revealed that Fenneropenaeus chinensis, Litopenaeus vannamei and Marsupenaeus japonicus suffering from this disease were CMNV positive.
Papillomaviruses are a family of slowly evolving DNA viruses and their evolution is commonly linked to that of their host species. However, while Bovine Papillomavirus-1 (BPV-1) primarily causes warts in its natural host the cow, it can also cause locally aggressive and invasive skin tumours in equids known as sarcoids, and thus provides a rare contemporary example of cross-species transmission of a papillomavirus. Here we describe the first phylogenetic analysis of BPV-1 in equine sarcoids, to our knowledge, allowing us to explore the evolutionary history of BPV-1 and investigate its cross-species association with equids. A phylogenetic analysis of the BPV-1 transcriptional promoter region (the long control region or LCR) was conducted on 15 bovine and 116 equine samples from four continents. Incorporating previous estimates for evolutionary rates in papillomavirus implies that the genetic diversity in the LCR variants is ancient and predates domestication of both equids and cattle. The phylogeny demonstrates geographic segregation into an ancestral group (African, South American and Australian samples), and a more recently derived, largely European clade. While our data are consistent with BPV-1 originating in cattle, we find evidence of multiple, likely relatively recent, cross-species transmission events into horses. We also demonstrate the high prevalence of one particular sequence variant (variant 20), and suggest this may indicate this variant shows a fitness advantage in equids. Our results add to the accumulating evidence that host specificity in papillomaviruses is more flexible than previously assumed.
Most viruses express one or several proteins that counter the anti-viral defences of the host-cell. This is the task of non-structural protein NS1 in influenza viruses. Absent from the viral particle but highly expressed in the infected cell, NS1 dramatically inhibits the cellular gene expression and prevents the activation of key players in the interferon system. In addition, NS1 selectively enhances the translation of viral mRNAs and may regulate the synthesis of viral RNAs. Our knowledge of the virus and of its protein NS1 has dramatically increased during the last fifteen years. The atomic structure of NS1 has been determined, many cellular partners were identified and its multiple activities have been studied in depth. This review presents our current knowledge and attempts to establish relationships between the RNA sequence, the structure of the protein, its ligands, its activities and the pathogenicity of the virus. A better knowledge of NS1 could help in elaborating novel antiviral strategies, based on either live vaccines with altered NS1, or on small-compounds inhibitors of NS1.
Aphid lethal paralysis virus (ALPV; Dicistroviridae) was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi. ALPV-like virus sequences have been reported from many insects and insect predators. We identified a new strain of ALPV (ALPV-AP) from the pea aphid, Acyrthosiphon pisum, and a new isolate (ALPV-DvV) from western corn rootworm, Diabrotica virgifera virgifera. ALPV-AP has a single-stranded RNA genome of 9,940 nucleotides. Based on phylogenetic analysis, ALPV-AP is closely related to ALPV-AM, an ALPV strain isolated from honeybees, Apis mellifera in Spain and Brookings, South Dakota, USA. The distinct evolutionary branches suggest the existence of two lineages of the ALPV virus. One consists of ALPV-AP and ALPV-AM while all other strains of ALPV group into the other lineage. The similarity of ALPV-AP and ALPV-AM is up to 88% at the RNA level, compared to 78-79% between ALPV-AP and other ALPV isolates. The sequence identity of proteins between ALPV-AP and ALPV-AM is 98-99% for both ORF 1 and ORF2, while only 85-87% for ORF1 and 91-92% for ORF2 between ALPV-AP and other ALPV isolates. Sequencing of RACE products and cDNA clones of the virus genome revealed sequence variation in the 5rrsquo; untranslated regions (5rrsquo;UTR) and in ORF1, indicating that ALPV may be under strong selection pressure, which could have important biological implications for ALPV host range and infectivity. Our results indicate that ALPV-like viruses infect insects in the order Coleoptera, in addition to the orders Hemiptera and Hymenoptera, and propose that ALPV isolates be classified as two separate viral species.
In the past decade there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated "Nhumirim virus"; NHUV) (Pauvolid-Correa et al., 2014) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential, 3rrsquo; UTR sequence homology with alternative flaviviruses, and evaluated the virusrrsquo;s capacity to suppress replication of representative Culex spp. vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell lines such as C6/36, C710, and Culex quinquefasciatus cells, were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3rrsquo; UTR indicate NHUV to be most similar to viruses within the yellow fever serogroup, Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. Interestingly, NHUV was found to share the fewest conserved sequence elements when compared to traditional insect-specific flaviviruses. This suggests that, despite being apparently insect-specific, this virus likely diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in significant reduction in viral production of West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Japanese encephalitis virus (JEV). The inhibitory effect was most effective against WNV and SLEV with over a million-fold and 10,000-fold reduction in peak titers, respectively. These data indicate the potential modulatory effect of flaviviral mosquito co-infections in the field and serve to identify a potential target for blocking mosquito infection with medically important flaviviruses.
Vaccination is the best measure to prevent influenza pandemics. Here we studied the protective effect against heterologous influenza viruses, including A/reassortant/NYMC X-179A (pH1N1), A/chicken/Henan/12/2004 (H5N1), A/Chicken/Jiangsu/7/2002 (H9N2) and A/Guizhou/54/89xA/PR/8/34 (A/Guizhou-X) (H3N2), in mice first vaccinated with a DNA vaccine of hemagglutinin (HA) or neuraminidase (NA) of A/PR/8/34 (PR8) and then infected with the homologous virus. We showed that PR8 HA or NA vaccination both protected mice against a lethal dose of the homologous virus; PR8 HA or NA vaccination and then PR8 infection in mice respectively offered poor or excellent protection against a second, heterologous influenza virus challenge. In addition,before the second heterologous influenza infection, the highest antibody level against the NP, M1 and M2 was found in PR8 NA DNA vaccinated and PR8 infected group. The level of induced cellular immunity against NP and M1 showed a trend consistent with that seen in antibody levels. However,PR8HA+NA and then PR8 infection resulted in limited protection against heterologous influenza virus challenges. Results of the present study demonstrated that infection of the homologous influenza virus in mice already immunized with a NA vaccine could provide excellent protection against subsequent infection of a heterologous influenza virus. These findings suggest that NA, a major antigen of influenza virus, could be an important candidate antigen for universal influenza vaccines.
Classical swine fever virus (CSFV) has a tropism for vascular endothelial cells and immune system cells. The process and release of proinflammatory cytokines, including IL-1bbeta; and IL-18, is one of the fundamental reactions of the innate immune response to viral infection. In this study, we investigated the production of IL-1bbeta; from macrophages following CSFV infection. Our results showed that IL-1bbeta; was upregulated after CSFV infection through activating caspase-1. Subsequent studies demonstrated that reactive oxygen species (ROS) may not be involved in CSFV mediated IL-1bbeta; release. Recently, researchers indicate a novel mechanism by which inflammasomes are triggered through detection of activity of viroporin. We further demonstrated that CSFV viroporin p7 protein induces IL-1bbeta; secretion and also discovered that p7 protein is a short-lived protein degraded by the proteasome. Together, our observations provide an insight into the mechanism of CSFV-induced inflammatory response.
Viral suppressors of RNA silencing (VSRs) are critical for the success of virus infection and efficient accumulation of the virus progeny. The chrysanthemum virus B p12 protein acts as a transcription factor (TF) to regulate cell size and proliferation favorable for the virus infection. Here we show that the p12 protein suppresses RNA silencing and is able to complement a VSR-deficient unrelated virus. Moreover, p12 counter-silencing activity can be uncoupled from its function as a TF in the nucleus. The altered p12 protein, which lacks nuclear localization signal and is not imported into the nucleus, was able to suppress RNA silencing as efficiently as the native protein. These data reveal new aspects of p12 functioning and identify a novel role for this viral zinc-finger transcription factor. The results provide a general insight into one of the activities of the p12 protein, which appears to possess more than one function.
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay 32P-CTP was incorporated into CHIKV genome and subgenomic (sg) RNA, as well as into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5rrsquo;-end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15,000 x g. Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated RTCs. The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3rrsquo;dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the CHIKV RTC and mode of action studies on compounds with anti-CHIKV activity.
The 2009 pandemic H1N1 influenza A virus spread across the globe and caused the first influenza pandemic of the 21st century. Many of the molecular factors that contributed to the airborne transmission of this pandemic virus have been determined; however, the direct-contact transmission of this virus remains poorly understood. In this study, we report that a combination of two mutations (N159D and Q226R) in the hemagglutinin (HA) protein of the representative 2009 H1N1 influenza virus A/California/04/2009 (CA04) caused a switch in receptor binding preference from the aalpha;2,6-sialoglycan to the aalpha;2,3-sialoglycan receptor and decreased the binding intensities for both glycans. In conjunction with a significantly decreased replication efficiency in the nasal epithelium, this limited human receptor-binding affinity resulted in inefficient direct-contact transmission of CA04 between guinea pigs. Our findings highlight the role of the HA gene in the transmission of the influenza virus.
Interactions between the viral surface glycoprotein hemagglutinin (HA) and the corresponding receptors on host cells is one important aspect of influenza virus infection. Mutations in the HA have been described to affect pathogenicity, antigenicity and the transmission of influenza viruses. Here, we detected polymorphisms present in the HA genes of two 2009 pandemic H1N1 isolates, A/California/04/2009 (Ca/09) and A/Mexico/4108/2009 (Mx/09), that resulted in amino acid changes at position 186 (S to P) and 194 (L to I) of the mature HA1 protein. Although not reported in the published 2009 pandemic H1N1 (H1N1pdm09) consensus sequence, the P186 genotype was more readily detected in primary infected and contact naiiuml;ve pigs when inoculated with a heterogeneous mixed stock of Ca/09. By reverse genetics we engineered Ca/09 and Mx/09 genomes by introducing the Ca/09 HA with two naturally occurring variants expressing S186/I194 (HA-S/I) and P186/L194 (HA-P/L), respectively. The Ca/09 HA with the combination of P186/L194 with either Ca/09 or Mx/09 backbones resulted in higher and prolonged viral shedding in naiiuml;ve pigs. This efficiency appeared to be more likely through an advantage in cell surface attachment rather than replication efficiency. Although these mutations occur within the receptor binding pocket and the Sb antigenic site, they did not affect serologic cross-reactivity. Relative increases of P186 in publically available sequences from swine H1N1pdm09 viruses supports the experimental data indicating this amino acid substitution confers an advantage in swine.
Rats are known as reservoirs and vectors for several zoonotic pathogens. However, information on the viruses shed by urban wild rats that could pose zoonotic risk to human health is scare. Here, intestinal contents from 20 wild Norway rats (Rattus norvegicus) collected in the city of Berlin, Germany, were subjected to metagenomic analysis of viral nucleic acids. The determined faecal viromes of rats consisted of a variety of known and unknown viruses and were highly variable among the individuals. Members of the families Parvoviridae and Picobirnaviridae represented the most abundant species. Novel picorna-, boca-, sapo- and stool-associated circular ssDNA viruses (SCV) were identified, which showed only low sequence identities to known representatives of the corresponding taxa. In addition, noro- and rotaviruses were detected as potential zoonotic gastroenteritis viruses. However, partial genome sequence analyses indicated that the norovirus was closely related to the recently identified rat norovirus and the rotavirus B was closely related to the rat rotavirus strain IDIR; both viruses clustered separately from respective human virus strains in phylogenetic trees. In contrast, the rotavirus A sequences showed high identities to human and animal strains. Analysis of the nearly complete genome of this virus revealed the known genotypes G3, P and N2 for three of the genome segments, whereas the remaining eight genome segments represented the novel genotypes I20-R11-C11-M10-A22-T14-E18-H13. In conclusion, the results indicate a high heterogeneity of enteric viruses present in urban wild rats; their ability to be transmitted to humans remains to be assessed in future.
During a hospital-based diarrheal disease study conducted in Ho Chi Minh City, Vietnam from 2009nndash;2010, we identified four symptomatic children infected with G26P rotavirus, an atypical variant that has not previously been reported in human gastroenteritis. To determine the genetic structure and investigate the origin of this G26P strain, the whole genome of a representative example was characterized, revealing a novel mixed genotype constellation of G26-P-I5-R1-C1-M1-A8-N1-T1-E1-H1. The genome segments were most closely related to porcine (VP7, VP4, VP6 and NSP1) and Wa-like porcine rotaviruses (VP1-3, NSP2-5). We propose that this G26P strain was a result from a pig-human zoonotic transmission, followed by a limited onward transmission train in humans. The identification of such strains has potential implications for vaccine efficacy in Southeast Asia and outlines the utility of whole genome sequencing for studying rotavirus genetic diversity and zoonotic potential during disease surveillance.
The aim of this study was to investigate the infection and replication of swine-derived hepatitis E virus (HEV) in primary-cultured human hepatocytes (PHCs). Hepatocytes were cultured from the resected normal livers of patients with metastatic tumours. These cultured hepatocytes were infected with swine-derived genotype 3 or 4 HEV. Viral replication was monitored using reverse transcription-polymerase chain reaction (RT-PCR). The amount of HEV RNA increased in the culture media and cells following infection. Immunofluorescence staining implied that the spread of HEV infection in hepatocytes is mainly attributed to cell-to-cell transmission via the cell membrane. The sequences of the inoculated and propagated HEV were determined to examine whether sequence variation occurred during infection. Sequence analysis showed that there were no differences between inoculated and propagated HEV, demonstrating that in vitro infection and replication of swine HEV in PHCs occur without sequence variation.
The major envelope fusion protein F of budded virus (BV) of baculoviruses consists of two disulfide-linked subunits, an N-terminal F2 subunit and a C-terminal, membrane-anchored F1 subunit. There is one cysteine (C) in F2 and there are fifteen cysteines in F1, but their role in disulfide linking is largely unknown. In this study, the inter- and intra-subunit disulfide bonds of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) F protein was analyzed by site-directed mutagenesis. Results indicate that in a functional F protein an inter-subunit disulfide-bond exists between amino acids C108 (F2) and C241 (F1). When C241 was mutated, an alternative disulfide-bond was formed between C108 and C232, but rendering F non-functional. No inter-subunit bridge was observed in a double C232/C241 mutant of F1. C403 was not involved in the formation of inter-subunit disulfide bonding, but mutation of this amino acid decreased viral infectivity significantly, suggesting that it might be involved in intra-subunit disulfide bonds. The influence of reductant (TCEP) and free-thiol inhibitors (AMS and DTNB) on the infectivity of HearNPV was tested. The results indicated that TCEP greatly decreased the infection of HzAm1 cells by HearNPV. In contrast, AMS and DTNB had no inhibitory effect on viral infectivity. These data suggest that free thiol/disulfide isomerization was not likely to play roles in viral entry and infectivity.
Human immunodeficiency virus type 2 (HIV-2) infects about 2 million people worldwide. HIV-2 has fewer treatment options than HIV-1, yet may evolve drug resistance more quickly. We have analyzed several novel drugs for anti-HIV-2 activity. It was observed that 5-azacytidine, clofarabine, gemcitabine and resveratrol have potent anti-HIV-2 activity. The EC50 values for 5-azacytidine, clofarabine and resveratrol were found to be significantly lower with HIV-2 compared to that of HIV-1. A time-of-addition assay was used to analyze the ability of these drugs to interfere with HIV-2 replication. Reverse transcription was the likely target for antiretroviral activity. Taken together, several novel drugs have been discovered to have activity against HIV-2. Based upon their known activities, these drugs may elicit enhanced HIV-2 mutagenesis and therefore be useful for inducing HIV-2 lethal mutagenesis. In addition, the data are consistent with HIV-2 reverse transcriptase (RT) being more sensitive than HIV-1 RT to dNTP pool alterations.
Based on the hypothesis that interactions between virions and serum components may influence the outcome of Dengue virus (DENV) infections, we decided to use affinity chromatography with domain III from protein E of DENV2 (DIIIE2) as a ligand to isolate virus-binding proteins from human plasma. This approach yielded serum amyloid P (SAP) and alpha 2-macroglobulin (aalpha;2M) as novel viral interactors. After confirming the specific binding of both SAP and aalpha;2M to DIIIE2 by ELISA, the latter interaction was examined in greater detail. We obtain evidence suggesting that the binding species is actually the receptor-activated form of aalpha;2M (aalpha;2M*), that aalpha;2M* can bind monovalently to recombinant domain III from all four DENV serotypes with affinities in the micromolar range ranking as DENV4 ggt; DENV1 ~ DENV2 ggt; DENV3, and that this interaction exhibits a strong avidity effect when multivalent binding is favored (KD 8 x10-8 M for DIIIE2). We also show that aalpha;2M* binds to DENV virions of the four serotypes, protecting the virus from temperature-induced inactivation in the absence of serum and enhancing infectivity. The latter effect exhibits an ED50 of 2.9 x10-8 M, also suggesting an avidity effect due to multivalent binding. These results will further contribute to the characterization of the virus-host factors interaction network during human DENV infection.
Previous studies have shown that hepatitis C virus (HCV) enters human hepatic cells through interaction with a series of cellular receptors, followed by clathrin-mediated, pH-dependent endocytosis. Here, we investigated the mechanisms of HCV entry into multiple HCV-permissive human hepatocyte-derived cells using trans-complemented HCV particles (HCVtcp). Knockdown of CD81 and claudin-1, or treatment with bafilomycin A1, reduced infection in Huh-7 and Huh7.5.1 cells, suggesting that HCV enters both cell types via receptor-mediated, pH-dependent endocytosis. Interestingly, knockdown of the clathrin heavy chain or dynamin-2, as well as expression of the dominant-negative form of dynamin-2, reduced infection of Huh-7 cells with HCVtcp, whereas infectious entry of HCVtcp into Huh7.5.1 cells was not impaired. Infection of Huh7.5.1 cells with culture-derived HCV (HCVcc) via a clathrin-independent pathway was also observed. Knockdown of caveolin-1, ADP-ribosylation factor 6 (Arf6), flotillin, p21-activated kinase 1 (PAK1) and the PAK1 effector C-terminal binding protein 1 of E1A had no inhibitory effects on HCVtcp infection into Huh7.5.1 cells, thus suggesting that the infectious entry pathway of HCV into Huh7.5.1 cells is not caveolae-mediated, or Arf6- and flotillin-mediated endocytosis and macropinocytosis, but rather may occur via an undefined endocytic pathway. Further analysis revealed that HCV entry is clathrin- and dynamin-dependent in ORL8c and HepCD81/miR122 cells, but productive entry of HCV is clathrin- and dynamin-independent in Hep3B/miR122 cells. Collectively, these data indicate that HCV enters different target cells through different entry routes.
We have previously documented the inhibitory activity of RNA aptamers to the RNA-dependent RNA polymerase of foot-and-mouth disease virus (3Dpol). Here we report their modification and use with a sub-genomic replicon incorporating GFP (pGFP-PAC replicon), allowing replication to be monitored and quantified in real time. GFP expression in transfected BHK-21 cells reached a maximum approximately 8 hours post-transfection, at which time change in morphology of the cells was consistent with virus-induced cytopathic effect (cpe). However, transfection of replicon-bearing cells with a 3Dpol aptamer RNA resulted in inhibition of GFP expression and maintenance of normal cell morphology, whereas a control aptamer RNA had little effect. The inhibition was correlated with a reduction in 3Dpol (detected by immunoblotting) and shown to be dose-dependent. The 3Dpol aptamers appeared to be more effective than 2llsquo;-C-methylcytidine. Aptamers to components of the replication complex are therefore useful molecular tools for studying viral replication and also have potential as diagnostic molecules in the future.
Vaccinia virus (VACV) is a large dsDNA virus encoding about 200 proteins, several of which inhibit apoptosis. Here a comparative study of anti-apoptotic proteins N1, F1, B13 and Golgi anti-apoptotic protein (GAAP) within and outwith virus infection is presented. VACVs strains engineered to lack each gene separately still block apoptosis to some degree because of functional redundancy provided by the other anti-apoptotic proteins. To overcome this redundancy we inserted each gene separately into a VACV strain (vv811) that lacks all these anti-apoptotic proteins and that induces apoptosis efficiently during infection. Each protein was also expressed in cells using lentivirus vectors. In isolation, each VACV protein showed anti-apoptotic activity in response to specific stimuli, as measured by immunoblotting for cleaved PARP-1 and caspase-3 activation. Of the proteins tested, B13 was the most potent inhibitor, blocking both intrinsic and extrinsic stimuli, whilst the activity of the other proteins was largely restricted to inhibition of intrinsic stimuli. In addition, B13 and F1 were effective blockers of apoptosis induced by vv811 infection. Finally, whilst differences in induction of apoptosis were barely detectable during infection with VACV strain WR compared to derivative viruses lacking individual anti-apoptotic genes, several of these proteins reduced activation of caspase 3 during infection by vv811 strains expressing these proteins. These results illustrate that vv811 is a useful tool to determine the role of VACV proteins during infection and that whilst all of these proteins have some anti-apoptotic activity, B13 is most potent.