- Volume 94, Issue 5, 2013

Human immunodeficiency virus type 1 (HIV-1) persistently infected cell lines are characterized by the continuous viral production without cytopathic effect. However, it is not completely clear if this production is contributed only by viral transcription or also by new cycles of viral replication. We studied an HIV-1 persistently infected cell line, designated H61-D, providing evidence of new replication cycles as sustained by: (i) a decrease in viral production, measured by p24 protein, after treatment of the culture with 3′-azydo-3′-deoxythymydine; (ii) detection of new integration events in the course of cell culture, and (iii) finding of two-long-terminal repeat circles in the cells. H61-D cells were not infected by cell-free virus, but infection was possible by co-culture with another productive-infected cell line. In conclusion, ongoing viral replication is taking place in H61-D persistent cells and new infections are mediated by a cell-to-cell spread mechanism.

An SRV-like virus was isolated from a colony-born Japanese monkey. To identify this SRV-like virus, we designed universal primers at regions that were conserved among the reported SRV sequences in the 5′-LTR and the short ORF and we obtained plasmid clones containing the complete gag, prt, pol and env genes. The full-length sequences of the isolate were determined from the plasmids and by direct sequencing. Sequence comparisons and phylogenetic analyses indicated that this SRV-like virus had a sequence identical to the reported 626 bp of SRV-5. In this study, we isolated SRV5/JPN/2005/V1 from a Japanese monkey and characterized the full-length SRV-5 sequence.

Endogenous retroviruses (ERVs) are remnants of retroviral germ line infections and have been identified in all mammals investigated so far. Although the majority of ERVs are degenerated, some mammalian species, such as mice and pigs, carry replication-competent ERVs capable of forming infectious viral particles. In mice, ERVs are silenced by DNA methylation and histone modifications and some exogenous retroviruses were shown to be transcriptionally repressed after integration by a primer-binding site (PBS) targeting mechanism. However, epigenetic repression of porcine ERVs (PERVs) has remained largely unexplored so far. In this study, we screened the pig genome for PERVs using LTRharvest, a tool for de novo detection of ERVs, and investigated various aspects of epigenetic repression of three unrelated PERV families. We found that these PERV families are differentially up- or downregulated upon chemical inhibition of DNA methylation and histone deacetylation in cultured porcine cells. Furthermore, chromatin immunoprecipitation analysis revealed repressive histone methylation marks at PERV loci in primary porcine embryonic germ cells and immortalized embryonic kidney cells. PERV elements belonging to the PERV-γ1 family, which is the only known PERV family that has remained active up to the present, were marked by significantly higher levels of histone methylations than PERV-γ2 and PERV-β3 proviruses. Finally, we tested three PERV-associated PBS sequences for repression activity in murine and porcine cells using retroviral transduction experiments and showed that none of these PBS sequences induced immediate transcriptional silencing in the tested primary porcine cells.

The epithelium of conducting airways represents the main target for influenza virus in mammals. However, the peculiarities of virus interactions with differentiated airway epithelial cells remain largely unknown. Here, influenza virus budding was studied in differentiated cultures of human tracheobronchial epithelial cells using transmission electron microscopy. Budding of spherical and filamentous virions was observed on the apical surfaces of cells with no association with cilia and secretory granules. Quantitative analysis of the distribution of viral buds on the cell surface indicated that the tips of the microvilli represented a prominent site of influenza virus budding in the human airway epithelium. As the microvilli of differentiated cells are involved in many fundamental cell functions, these data will prompt further studies on the biological significance of microvilli-associated budding for virus replication, transmission and pathogenicity.

Influenza A virus vRNP nuclear export is CRM1-dependent. Ran-binding protein 3 (RanBP3) is a Ran-interacting protein that is best known for its role as a cofactor of CRM1-mediated cargo nuclear export. In this study, we investigated the role of RanBP3 during the influenza A virus life cycle. We found that RanBP3 was phosphorylated at Ser58 in the early and late phases of infection. Knockdown of RanBP3 expression led to vRNP nuclear retention, suggesting that RanBP3 is involved in vRNP nuclear export. Moreover, we demonstrated that the function of RanBP3 during vRNP nuclear export is regulated by phosphorylation at Ser58, and that RanBP3 phosphorylation is modulated by both PI3K/Akt and Ras/ERK/RSK pathways in the late phase of viral infection.

While post-transcriptional regulation of gene expression by microRNAs (miRNAs) has been shown to be involved in influenza virus replication cycle, only a few studies have further investigated this aspect in a human cellular model infected with human influenza viruses. In this study, we performed miRNA global profiling in human lung epithelial cells (A549) infected by two different subtypes of human influenza A viruses (H1N1 and H3N2). We identified a common miRNA signature in response to infection by the two different strains, highlighting a pool of five miRNAs commonly deregulated, which are known to be involved in the innate immune response or apoptosis. Among the five miRNA hits, the only upregulated miRNA in response to influenza infection corresponded to miR-146a. Based on a previously published gene expression dataset, we extracted inversely correlated miR-146a target genes and determined their first-level interactants. This functional analysis revealed eight distinct biological processes strongly associated with these interactants: Toll-like receptor pathway, innate immune response, cytokine production and apoptosis. To better understand the biological significance of miR-146a upregulation, using a reporter assay and a specific anti-miR-146a inhibitor, we confirmed that infection increased the endogenous miR-146a promoter activity and that inhibition of miR-146a significantly increased viral propagation. Altogether, our results suggest a functional role of miR-146a in the outcome of influenza infection, at the crossroads of several biological processes.

A trans-packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression in trans of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression in trans of core or core, p7 and NS2 with envelope proteins did not enhance trans-encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of trans-encapsidation and infection were performed. Four cycles resulted in a 100-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located at the NS5A-B cleavage site did not affect viral replication, but enhanced the single-round infectious particles assembly only in trans-encapsidation model. In conclusion, our trans-encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote trans-encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines.

The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and the assembly of infectious virus particles. NS5A comprises three domains, separated by low-complexity sequences. Whilst the function of domain I appears to be predominantly involved with genome replication, the roles of domains II and III are less well defined. It has been reported previously that a deletion spanning the majority of domain II but retaining the C-terminal 35 residues had no effect on virus production; however, deletion of the entire domain II eliminated genome replication, pointing to a key role for the C terminus of this domain. Recent work has also highlighted this region as the potential binding site of the host factor cyclophilin A (CypA). To define this requirement for replication in more detail, and to investigate the involvement of CypA, we conducted a mutagenic study of the C-terminal 30 residues of domain II within the context of both the infectious JFH-1 virus and a JFH-1-derived subgenomic replicon. We showed that 12 of these residues were absolutely required for virus genome replication, whilst mutations of the remainder either had no phenotype or exhibited a partial reduction in genome replication. There was an absolute correlation between the datasets for virus and subgenomic replicon, indicating that this region is involved solely in the process of genome replication. Comparison of our data with a previously published analysis of the same region in genotype 1b revealed some important differences between the two genotypes of HCV.

Enterovirus 71 (EV71) infection has induced fatal encephalitis in thousands of young children in the Asia–Pacific region over the last decade. EV71 infection continues to cause serious problems in areas with outbreaks, because vaccines and antiviral therapies are not available. Lymphocytes are present in the brains of infected patients and mice, and they protect mice from infection by decreasing the viral burden. The chemokines responsible for recruiting lymphocytes to infected organs are yet to be identified. Among the lymphocyte chemokines detected, high levels of interferon-gamma-inducible protein-10 (IP-10) are found in the plasma and cerebral spinal fluid of patients with brainstem encephalitis as compared with the levels of a monokine induced by gamma interferon (Mig). Using a murine model to investigate the induction of IP-10 by EV71 infection, we observed that EV71 infection significantly enhanced IP-10 protein expression in the serum and brain, with kinetics similar to viral titres in the blood and brain. Brain neurons of infected mice expressed IP-10. Using wild-type mice and IP-10 gene knockout mice to investigate the role of IP-10 in EV71 infection, we found that IP-10 deficiency significantly reduced levels of Mig in serum, and levels of gamma interferon and the number of CD8 T cells in the mouse brain. Absence of IP-10 significantly increased the mortality of infected mice by 45 %, with slow virus clearance in several vital tissues. Our observations are consistent with a model where EV71 infection boosts IP-10 expression to increase gamma interferon and Mig levels, infiltration of CD8 T cells, virus clearance in tissues and the survival of mice.

Bats are reservoirs for a wide range of human pathogens including Nipah, Hendra, rabies, Ebola, Marburg and severe acute respiratory syndrome coronavirus (CoV). The recent implication of a novel beta (β)-CoV as the cause of fatal respiratory disease in the Middle East emphasizes the importance of surveillance for CoVs that have potential to move from bats into the human population. In a screen of 606 bats from 42 different species in Campeche, Chiapas and Mexico City we identified 13 distinct CoVs. Nine were alpha (α)-CoVs; four were β-CoVs. Twelve were novel. Analyses of these viruses in the context of their hosts and ecological habitat indicated that host species is a strong selective driver in CoV evolution, even in allopatric populations separated by significant geographical distance; and that a single species/genus of bat can contain multiple CoVs. A β-CoV with 96.5 % amino acid identity to the β-CoV associated with human disease in the Middle East was found in a Nyctinomops laticaudatus bat, suggesting that efforts to identify the viral reservoir should include surveillance of the bat families Molossidae/Vespertilionidae, or the closely related Nycteridae/Emballonuridae. While it is important to investigate unknown viral diversity in bats, it is also important to remember that the majority of viruses they carry will not pose any clinical risk, and bats should not be stigmatized ubiquitously as significant threats to public health.

Piscine reovirus (PRV) is a tentative new member of the family Reoviridae and has been linked to heart and skeletal muscle inflammation in farmed Atlantic salmon (Salmo salar L.). Recent sequence-based evidence suggests that PRV is about equally related to members of the genera Orthoreovirus and Aquareovirus. Sequence similarities have also suggested that PRV might encode a fusion-associated small transmembrane (FAST) protein, which in turn suggests that PRV might be the prototype of a new genus with syncytium-inducing potential. In previous support of this designation has been the absence of identifiable PRV-encoded homologues of either the virion outer-clamp protein of ortho- and aquareoviruses or the virion outer-fibre protein of most orthoreoviruses. In the current report, we have provided experimental evidence that the putative p13 FAST protein of PRV lacks the defining feature of the FAST protein family – the ability to induce syncytium formation. Instead, p13 is the first example of a cytosolic, integral membrane protein encoded by ortho- or aquareoviruses, and induces cytotoxicity in the absence of cell–cell fusion. Sequence analysis also identified signature motifs of the outer-clamp and outer-fibre proteins of other reoviruses in two of the predicted PRV gene products. Based on these findings, we conclude that PRV does not encode a FAST protein and is therefore unlikely to be a new fusogenic reovirus. The presence of a novel integral membrane protein and two previously unrecognized, essential outer-capsid proteins has important implications for the biology, evolution and taxonomic classification of this virus.

The genus Orbivirus of the family Reoviridae includes a genetically diverse group of dsRNA arthropod-borne viruses that infect a wide variety of animal species. Here, we report the complete genome and phylogenetic analysis of a novel orbivirus (IAn-66411 or Sathuvachari virus, SVIV) isolated in 1963 from starlings (Brahminy myna) collected in Vellore, Tamil Nadu, India. Comparative genetic analysis of the SVIV polymerase (VP1 protein), core protein (VP3) and outer core protein (VP7) confirmed that SVIV is most closely related to the mosquito-borne orbiviruses, but that it is equally divergent from all known species. Therefore, SVIV should be tentatively considered as the prototype of a novel mosquito-associated Orbivirus species. These findings will aid in the development of molecular reagents that can identify genetically similar orbiviruses and help elucidate their geographical distribution, epidemiology, species tropism and possible disease association.

Triatoma virus (TrV) is a member of the insect virus family Dicistroviridae and consists of a small, non-enveloped capsid that encloses its positive-sense ssRNA genome. Using cryo-transmission electron microscopy and three-dimensional reconstruction techniques combined with fitting of the available crystallographic models, this study analysed the capsids corresponding to mature and several RNA-empty TrV particles. After genome release, the resulting reconstruction of the empty capsids displayed no prominent conformational changes with respect to the full virion capsid. The results showed that RNA delivery led to empty capsids with an apparent overall intact protein shell and suggested that, in a subsequent step, empty capsids disassemble into small symmetrical particles. Contrary to what is observed upon genome release in mammalian picornaviruses, the empty TrV capsid maintained a protein shell thickness and size identical to that in full virions.

Previous mutational analyses of naturally occurring West Nile virus (WNV) strains and engineered mutant WNV strains have identified locations in the viral genome that can have profound phenotypic effect on viral infectivity, temperature sensitivity and neuroinvasiveness. We chose six mutant WNV strains to evaluate for vector competence in the natural WNV vector Culex tarsalis, two of which contain multiple ablations of glycosylation sites in the envelope and NS1 proteins; three of which contain mutations in the NS4B protein and an attenuated natural bird isolate (Bird 1153) harbouring an NS4B mutation. Despite vertebrate attenuation, all NS4B mutant viruses displayed enhanced vector competence by Cx. tarsalis. Non-glycosylated mutant viruses displayed decreased vector competence in Cx. tarsalis mosquitoes, particularly when all three NS1 glycosylation sites were abolished. These results indicate the importance of both the NS4B protein and NS1 glycosylation in the transmission of WNV by a significant mosquito vector.

Providence virus (PrV) is the sole member of the family Carmotetraviridae (formerly Tetraviridae) sharing the characteristic T = 4 capsid architecture with other tetravirus families. Despite significant structural similarities, PrV differs from other tetraviruses in terms of genome organization, non-structural protein sequence and regulation of gene expression. In addition, it is the only tetravirus that infects tissue culture cells. Previous studies showed that in persistently infected Helicoverpa zea MG8 cells, the PrV replicase associates with detergent-resistant membranes in punctate cytosolic structures, which is similar to the distribution of an alpha-like tetravirus replicase (Helicoverpa armigera stunt virus). Here, we demonstrate that the site of PrV vRNA replication coincides with the presence of PrV p40/p104 proteins in infected cells and that these replication proteins associate with the Golgi apparatus and secretory vesicles in transfected cells.

The human bocavirus 1 (HBoV1) parvovirus causes respiratory disease and primarily affects children. Despite its worldwide prevalence, the mechanisms of HBoV1 replication and pathogenesis remain largely undefined. In this study of 846 children hospitalized at the Children’s Hospital of Chongqing Medical University in China for respiratory tract infection between June 2009 and May 2011, HBoV1 was detected in 112 (13.2 %) by real-time quantitative PCR. The median age of HBoV1-positive patients was 10 months old. Forty-five (40.2 %) of the HBoV1 cases were monoinfections, and 67 (59.8 %) were viral co-infections. Genotyping of all 112 HBoV1-positive cases yielded 27 full HBoV1 sequences, as well as two NS1 gene sequences, 15 NP1 gene sequences and 10 VP1/VP2 gene sequences harbouring 24, 10 and 43 mutations, respectively. Statistical analysis revealed no relationship between genetic mutations and clinical manifestations of HBoV1-positive patients. However, the viral loads were significantly lower in samples with mutations G236A or A447G in NP1, or G1461A in VP1/VP2, than in samples with wild-type HBoV1. Future studies should investigate whether these mutations in the HBoV1 gene may represent useful markers of disease pathogenesis.

Beak and feather disease virus (BFDV) causes the highly contagious, in some cases fatal, psittacine beak and feather disease in parrots. The European continent has no native parrots, yet in the past has been one of the world’s biggest importers of wild-caught exotic parrot species. Following the banning of this practice in 2007, the demand for exotic pet parrots has largely been met by established European breeding facilities, which can also supply buyers outside Europe. However, the years of unregulated importation have provided numerous opportunities for BFDV to enter Europe, meaning the likelihood of birds within captive breeding facilities being BFDV positive is high. This study examined the BFDV status of such facilities in Poland, a country previously shown to have BFDV among captive birds. A total of 209 birds from over 50 captive breeding facilities across Poland were tested, and 43 birds from 18 different facilities tested positive for BFDV. The full BFDV genomes from these 43 positive birds were determined, and phylogenetic analysis revealed that these samples harboured a relatively high degree of diversity and that they were highly recombinant. It is evident that there have been multiple introductions of BFDV into Poland over a long period of time, and the close association of different species of birds in the captive environment has probably facilitated the evolution of new BFDV strains through recombination.

Whereas the antibody response to the anogenital human papillomaviruses (HPVs) is known to be mainly type-specific, correlated with the presence of viral DNA and mainly directed to conformational epitopes of the virion, it is not known if this applies also to the antibody response to cutaneous HPVs. For 434 non-immunosuppressed patients with skin lesions (squamous cell carcinoma and basal cell carcinoma of the skin, actinic keratosis and benign skin lesions), we compared HPV DNA status with seroreactivity to HPV pseudovirions (PsV) and to GST-L1 fusion proteins from HPV types -5, -6, -15, -16, -32 and -38. Biopsies from the skin lesions were tested for the presence of HPV DNA using three different PCR methods, with typing by sequencing. Serum samples from subjects with HPV DNA-positive biopsies and randomly selected serum samples from subjects with HPV DNA-negative biopsies were also tested with neutralization assays with HPV5, -38 and -76 PsV. Agreement of the three serological methods varied from poor to moderate. Type-specific seroprevalences among patients positive for the same type of HPV DNA (sensitivity of serology) was improved with the PsV-based method (mean of 40 %, maximum 63 %) compared with the GST-L1 method (mean of 20 %, maximum of 25 %). Neutralization was the most sensitive assay for HPV38 (50 %). In summary, cutaneous HPVs also appear to induce a type-specific antibody response that correlates with the presence of HPV DNA and that can be detected with improved sensitivity using PsV-based serology.