Recombination of hepatitis D virus RNA sequences and its implications.

Recombination between RNA sequences plays a role in the fast evolution of a few viruses. There has been no report on hepatitis D virus (HDV) recombination. In this study, we analyzed genetic recombination of HDV and its possible impact on evolution and clinical course. The aligned HDV sequences allowed us to construct a phylogenetic tree which supported the notion of distinct lineages of HDV. The tree was also used in the analysis of recombination using partial likelihoods assessed through optimization. Nine segments of the HDV genome with significant levels of genetic recombination were detected. Five segments were in the hypervariable region, and four were in the delta-antigen- coding region. None could be found in the well-conserved autocleavage region that is essential for replication. Recombination occurred both between and within types. The results of this study indicated that the remarkable variation in HDV genomic sequences, particularly in the hypervariable region, among different genotypes may at least partly result from multiple episodes of genetic recombination during evolution. Genetic recombination may play a significant role in increasing genetic diversity. Importantly, a genetic recombination (nt 1082-1093) occurred in one of the immunogenic domains of hepatitis delta virus antigen recognized by human and woodchuck antibodies (amino acids 174-195). Genetic recombination also occurred at another segment between nt 1517 and 1535, which was close to one of the predicted T-cell epitopes (amino acids 26-41). In longitudinal analysis of HDV genomes at different time points during chronic infection, novel dominant HDV strains with amino acid changes at these epitopes usually emerged after severe hepatitis attacks. In the comparison of HDV clones during or shortly after flare-up of liver disease, Ka/Ks ratios of > 1 were frequently found, suggesting Darwinian positive selection. Therefore, recombination in these two segments may play an important role for HDV in the evasion of immunity.     

Mapping of the hepatitis B virus pre-S1 domain involved in receptor recognition

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) are primate hepadnaviruses that display restricted tissue and host tropisms. Hepatitis D virus (HDV) particles pseudotyped with HBV and WMHBV envelopes (HBV-HDV and WM-HDV) preferentially infect human and spider monkey hepatocytes, respectively, thereby confirming host range bias in vitro. The analysis of chimeric HBV and WMHBV large (L) envelope proteins suggests that the pre-S1 domain may comprise two regions that affect infectivity: one within the amino-terminal 40 amino acids of pre-S1 and one downstream of this region. In the present study, we further characterized the role of the amino terminus of pre-S1 in infectivity by examining the ability of synthetic peptides to competitively block HDV infection of primary human and spider monkey hepatocytes. A synthetic peptide representing the first 45 residues of the pre-S1 domain of the HBV L protein blocked infectivity of HBV-HDV and WM-HDV, with a requirement for myristylation of the amino terminal residue. Competition studies with truncated peptides suggested that pre-S1 residues 5 to 20 represent the minimal domain for inhibition of HDV infection and, thus, presumably represent the residues involved in virus-host receptor interaction. Recombinant pre-S1 proteins expressed in insect cells blocked infection with HBV-HDV and WM-HDV at a concentration of 1 nanomolar. The ability of short pre-S1 peptides to efficiently inhibit HDV infection suggests that they represent suitable ligands for identification of the HBV receptor and that a pre-S1 mimetic may represent a rational therapy for the treatment of HBV infection.     

Sequence analysis of NS3 protease gene in clinical strains of hepatitis C virus

The amino terminal region of the non structural gene 3 (NS3) of hepatitis C virus (HCV) is a chymotripsinlike serine-protease responsible for cleavage of the non structural proteins of Hepatitis C virus (HCV). In order to investigate the genetic variation of this region, we developed a nested PCR to obtain NS3 protease sequences from 54 patients chronically infected with HCV genotypes 1a, 1b and 3, respectively. Comparison of nucleotide and amino acids sequences of NS3 protease domain with consensus sequence obtained within the same genotype, showed 3.73% nucleotide divergence and 1.64% amino acid divergence in isolates of genotype 3a, whereas isolates 1a exhibited 4.45% nucleotide and 4% amino acid change, respectively. Finally, NS3 sequence from 1b isolates revealed 6.47% nucleotide and 3.5 % aa changes. Comparison of consensus amino acid sequences derived from isolates 1a, 1b and 3, with the HCV prototypes showed a low amino acid sequence diversity. However, the consensus sequence of HCV genotype 3 isolates showed an amino acid changed from the prototype, that was located within a region important for enzyme structure and activity. These results indicated that the NS3 protease gene is highly conserved within the same HCV genotype. The domains involved in enzyme function were highly conserved in 1a and 1b strains, whereas consensus sequence of isolates 3a showed that the majority of these strains were not perfectly conserved in one of such regions. These findings altogether suggested that the NS3 protease enzyme of HCV may constitute an important target for antiviral therapy, but the NS3 protease variability of isolates 3 within a region that is a potential target for antiviral therapy could pose a problem for structure based drug development.     

Apparent helper-independent infection of woodchucks by hepatitis delta virus and subsequent rescue with woodchuck hepatitis virus.

Hepatitis delta virus (HDV) is a subviral agent of humans which is dependent upon hepatitis B virus as a helper for transmission. HDV can be experimentally transmitted to woodchucks by using woodchuck hepatitis virus (WHV) as the helper. We used this model system to study two types of HDV infections: those of animals already chronically infected with WHV and those of animals without any evidence of prior exposure to WHV. At 5 to 10 days after infection with HDV, liver biopsies of these two groups of animals indicated that around 1% of the hepatocytes were infected (HDV antigen positive). Moreover, similar amounts of replicative forms of HDV RNA were detected. In contrast, by 20 days postinfection, the two groups of animals were quite different in the extent of the HDV infection. The animals chronically infected with WHV showed spread of the infection within the liver and the release of high titers of HDV into the serum. In contrast, the animals not previously exposed to WHV showed a progressive reduction in liver involvement, and at no time up to 165 days postinfection could we detect HDV particles in the serum. However, if these animals were inoculated with a relatively high titer of WHV at either 7 or even 33 days after the HDV infection, HDV viremia was observed. Our data support the interpretation that in these animals, hepatocytes were initially infected in the absence of helper virus, HDV genome replication took place, and ultimately these replicating genomes were rescued by the secondary WHV infection. The observation that HDV can survive in the liver for at least 33 days in the absence of coinfecting helper virus may be relevant to the reemergence of HDV infection following liver transplantation.     

Genetic characterization of Chikungunya virus from New Delhi reveal emergence of a new molecular signature in Indian isolates

ABSTRACT: BACKGROUND: Chikungunya (CHIK) is currently endemic in South and Central India and exist as co-infections with dengue in Northern India. In 2010, New Delhi witnessed an outbreak of CHIK in the months October-December. This was the first incidence of a dominant CHIK outbreak in Delhi and prompted us to characterize the Delhi virus strains. We have also investigated the evolution of CHIK spread in India. FINDINGS: Clinical samples were subjected to RT-PCR to detect CHIK viral RNA. The PCR amplified products were sequenced and the resulting sequences were genetically analyzed. Phylogenetic analysis based on partial sequences of the structural proteins E1 and E2 revealed that the viruses in the latest outbreak exhibited ECSA lineage. Two novel mutations, E1 K211E and E2 V264A were observed in all Delhi isolates. In addition, CHIKV sequences from eight states in India were analyzed along with Delhi sequences to map the genetic diversity of CHIKV within the country. Estimates of average evolutionary divergence within states showed varying divergence among the sequences both within the states and between the states. We identified distinct molecular signatures of the different genotypes of CHIKV revealing emergence of a new signature in the New Delhi clade. Statistical analyses and construction of evolutionary path of the virus within the country revealed gradual spread of one specific strain all over the country. CONCLUSION: This study has identified unique mutations in the E1 and E2 genes and has revealed the presence of ancestral CHIKV population with maximum diversity circulating in Maharashtra. The study has further revealed the trend of CHIK spread in India since its first report in 1963 and its subsequent reappearance in 2005.     

Molecular characterization of viral G gene in emerging and Re-emerging areas of rabies in China, 2007 to 2011

In recent years (2007 to 2011),although the overall number of rabies cases in China has decreased,there is evidence of emerging or re-emerging cases in regions without previous rabies cases or with low...     

Deleterious effects of hepatitis delta virus replication on host cell proliferation

Hepatitis delta virus (HDV) infection and spread in vivo are dependent upon coinfection by hepatitis B virus (HBV), and dual HDV/HBV infection is frequently more severe than HBV infection alone, raising the possibility that HDV infection may be deleterious to cells. Here we have examined the effects of HDV replication on the long-term growth of cultured cells. Our results show that most cells transfected with HDV cDNA do not give rise to stable cell lines expressing viral antigens or replicative intermediates; in addition, cotransfection of HDV replicons with a plasmid vector expressing a hygromycin resistance marker results in a dose-dependent impairment of hygromycin-resistant colony formation. When cells transfected with replication-competent HDV cDNA are followed prospectively, a progressive decline in viral RNA replication and a steady decrease in the proportion of cells expressing delta antigen are observed. However, in transient transfection assays, no evidence was found to link HDV replication to apoptosis or to cell cycle arrest, nor did HDV replication confer on host cells enhanced sensitivity to inducers of apoptosis. Thus, HDV replication does not appear to be acutely cytotoxic. However, in dividing cells HDV replication is associated with a subtler growth disadvantage, leading to selection in culture for cells displaying diminished HDV expression. This effect would not be expected to cause hepatitis in vivo but might contribute to impaired liver regeneration in the setting of ongoing hepatocellular injury.     

Genetic diversity of Usutu virus

Usutu virus is a mosquito-borne virus first isolated from Culex naevei in South Africa in 1959. The first emergence of Usutu virus outside Africa was recorded in Austria. Here, a phylogenetic analysis targeting the E5 and NS5 genes was carried out on the viral strains circulating in Europe. The NS5 gene tree showed two main clades, one of which included the Italian sequences. In the E gene tree all sequences grouped into the same main clade, with sequences from Austria divided into two separate clusters. Only sites under negative selective pressure were found in E and NS5 proteins. The results suggest that Usutu virus circulating in Europe has a degree of genetic diversity higher than expected and that infection may arise from different sources.     

Hepatitis D Virus Infection of Mice Expressing Human Sodium Taurocholate Co-transporting Polypeptide

Hepatitis D virus (HDV) is the smallest virus known to infect human. About 15 million people worldwide are infected by HDV among those 240 million infected by its helper hepatitis B virus (HBV). Viral hepatitis D is considered as one of the most severe forms of human viral hepatitis. No specific antivirals are currently available to treat HDV infection and antivirals against HBV do not ameliorate hepatitis D. Liver sodium taurocholate co-transporting polypeptide (NTCP) was recently identified as a common entry receptor for HDV and HBV in cell cultures. Here we show HDV can infect mice expressing human NTCP (hNTCP-Tg). Antibodies against critical regions of HBV envelope proteins blocked HDV infection in the hNTCP-Tg mice. The infection was acute yet HDV genome replication occurred efficiently, evident by the presence of antigenome RNA and edited RNA species specifying large delta antigen in the livers of infected mice. The resolution of HDV infection appears not dependent on adaptive immune response, but might be facilitated by innate immunity. Liver RNA-seq analyses of HDV infected hNTCP-Tg and type I interferon receptor 1 (IFNα/βR1) null hNTCP-Tg mice indicated that in addition to induction of type I IFN response, HDV infection was also associated with up-regulation of novel cellular genes that may modulate HDV infection. Our work has thus proved the concept that NTCP is a functional receptor for HDV infection in vivo and established a convenient small animal model for investigation of HDV pathogenesis and evaluation of antiviral therapeutics against the early steps of infection for this important human pathogen.     

Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein.

The genetic and antigenic variability of the G glycoproteins from 76 human respiratory syncytial (RS) viruses (subgroup A) isolated during six consecutive epidemics in either Montevideo, Uruguay, or Madrid, Spain, have been analyzed. Genetic diversity was evaluated for all viruses by the RNase A mismatch cleavage method and for selected strains by dideoxy sequencing. The sequences reported here were added to those published for six isolates from Birmingham, United Kingdom, and for two reference strains (A2 and Long), to derive a phylogenetic tree of subgroup A viruses that contained two main branches and several subbranches. During the same epidemic, viruses from different branches were isolated. In addition, closely related viruses were isolated in distant places and in different years. These results illustrate the capacity of the virus to spread worldwide, influencing its mode of evolution. The antigenic analysis of all isolates was carried out with a panel of anti-G monoclonal antibodies that recognized strain-specific (or variable) epitopes. A close correlation between genetic relatedness and antigenic relatedness in the G protein was observed. These results, together with an accumulation of amino acid changes in a major antigenic area of the G glycoprotein, suggest that immune selection may be a factor influencing the generation of RS virus diversity. The pattern of RS virus evolution is thus similar to that described for influenza type B viruses, expect that the level of genetic divergence among the G glycoproteins of RS virus isolates is the highest reported for an RNA virus gene product.     

Analysis of host range phenotypes of primate hepadnaviruses by in vitro infections of hepatitis D virus pseudotypes

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.     

Efficient Hepatitis Delta Virus RNA Replication in Avian Cells Requires a Permissive Factor(s) from Mammalian Cells

Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian cells, avian cells do not support efficient HDV RNA replication and that this defect cannot be rescued by provision of HDV gene products in trans. Contrary to earlier assertions, this defect is not due to enhanced apoptosis triggered in avian cells by HDV. Fusion of avian cells to mammalian cells rescues HDV replication in avian nuclei, indicating that the nonpermissive phenotype of avian cells is not due to the presence of dominantly acting inhibitors of replication. Rather, avian cells lack one or more essential permissive factors present in mammalian cells. These results set the stage for the identification of such factors and also explain the failure of earlier efforts to transmit HDV infection to avian hosts harboring indigenous hepadnaviruses.     

Non-ribozyme sequences enhance self-cleavage of ribozymes derived from Hepatitis delta virus.

Analysis of the self-cleavage of ribozymes derived from the genomic RNA of Hepatitis delta virus (HDV) has revealed that certain co-transcribed vector sequences significantly affect the activity of the ribozyme. Specifically, the t1/2 of self-cleavage for a 135 nucleotide HDV RNA varied, at 42 degrees C, from 5 min to 88 min, depending on the vector-derived sequences flanking the 5' end of the ribozyme. Further analysis suggested that this phenomenon was most likely due to the interaction of vector-derived sequences with a 16 nucleotide region found at the 3' end of the ribozyme. These findings have implications for studies of ribozymes transcribed from cDNA templates, and may provide information regarding the catalytic structure of the HDV ribozyme.     

Characterization and phylogenetic analysis of Varicella-zoster virus strains isolated from Korean patients

Varicella-zoster virus (VZV) is a causative agent of chickenpox in primary infection and shingles after its reactivation from latency. Complete or almost-complete genomic DNA sequences for various VZV strains have been reported. Recently, clinical VZV strains were isolated from Korean patients whose genome was sequenced using high-throughput sequencing technology. In this study, we analyzed single nucleotide polymorphism (SNP) of VZV strains to genetically characterize Korean clinical isolates. Phylogenetic analyses revealed that three Korean strains, YC01, YC02, and YC03, were linked to clade 2. Comprehensive SNP analysis identified 86 sites specific for the 5 VZV clades. VZV strains isolated from Korea did not form a phylogenetic cluster. Rather, YC02 and YC03 clustered strongly with Chinese strain 84-7 within clade 2, more specifically cluster 2a. Signature sequences for the cluster 2a were identified and found to play an important role in the separation of cluster 2a strains from other clade 2 strains, as shown in substitution studies. Further genetic analysis with additional strains isolated from Japan, China, and other Asian countries would provide a novel insight into the significance of two distinct subclades within clade 2.     

A novel chromosome region maintenance 1-independent nuclear export signal of the large form of hepatitis delta antigen that is required for the viral assembly

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus, as it requires hepatitis B virus for virion production and transmission. We have previously demonstrated that sequences within the C-terminal 19-amino acid domain flanking the isoprenylation motif of the large hepatitis delta antigen (HDAg-L) are important for virion assembly. In this study, site-directed mutagenesis and immunofluorescence staining demonstrated that in the absence of hepatitis B virus surface antigen (HBsAg), the wild-type HDAg-L was localized in the nuclei of transfected COS7 cells. Nevertheless, in the presence of HBsAg, the HDAg-L became both nuclei- and cytoplasm-distributed in about half of the cells. An HDAg-L mutant with a substitution of Pro-205 to alanine could neither form HDV-like particles nor shift the subcellular localization in the presence of HBsAg. In addition, nuclear trafficking of HDAg-L in heterokaryons indicated that HDAg-L is a nucleocytoplasmic shuttling protein. A proline-rich HDAg peptide spanning amino acid residues 198 to 210, designated NES(HDAg-L), can function as a nuclear export signal (NES) in Xenopus oocytes. Pro-205 is critical for the NES function. Furthermore, assembly of HDV is insensitive to leptomycin B, indicating that the NES(HDAg-L) directs nuclear export of HDAg-L to the cytoplasm via a chromosome region maintenance 1-independent pathway.     

Molecular evolution of hepatitis A virus: a new classification based on the complete VP1 protein

Hepatitis A virus (HAV) is a positive-stranded RNA virus in the genus Hepatovirus in the family Picornaviridae So far, analysis of the genetic variability of HAV has been based on two discrete regions, the VP1/2A junction and the VP1 N terminus. In this report, we determined the nucleotide and deduced amino acid sequences of the complete VP1 gene of 81 strains from France, Kosovo, Mexico, Argentina, Chile, and Uruguay and compared them with the sequences of seven strains of HAV isolated elsewhere. Overall strain variation in the complete VP1 gene was found to be as high as 23.7% at the nucleotide level and 10.5% at the amino acid level. Different phylogenetic methods revealed that HAV sequences form five distinct and well-supported genetic lineages. Within these lineages, HAV sequences clustered by geographical origin only for European strains. The analysis of the complete VP1 gene allowed insight into the mode of evolution of HAV and revealed the emergence of a novel variant with a 15-amino-acid deletion located on the VP1 region where neutralization escape mutations were found. This could be the first antigenic variant of HAV so far identified.     

Recent Emergence and Spread of an Arctic-Related Phylogenetic Lineage of Rabies Virus in Nepal

Rabies is a zoonotic disease that is endemic in many parts of the developing world, especially in Africa and Asia. However its epidemiology remains largely unappreciated in much of these regions, such as in Nepal, where limited information is available about the spatiotemporal dynamics of the main etiological agent, the rabies virus (RABV). In this study, we describe for the first time the phylogenetic diversity and evolution of RABV circulating in Nepal, as well as their geographical relationships within the broader region. A total of 24 new isolates obtained from Nepal and collected from 2003 to 2011 were full-length sequenced for both the nucleoprotein and the glycoprotein genes, and analysed using neighbour-joining and maximum-likelihood phylogenetic methods with representative viruses from all over the world, including new related RABV strains from neighbouring or more distant countries (Afghanistan, Greenland, Iran, Russia and USA). Despite Nepal''s limited land surface and its particular geographical position within the Indian subcontinent, our study revealed the presence of a surprising wide genetic diversity of RABV, with the co-existence of three different phylogenetic groups: an Indian subcontinent clade and two different Arctic-like sub-clades within the Arctic-related clade. This observation suggests at least two independent episodes of rabies introduction from neighbouring countries. In addition, specific phylogenetic and temporal evolution analysis of viruses within the Arctic-related clade has identified a new recently emerged RABV lineage we named as the Arctic-like 3 (AL-3) sub-clade that is already widely spread in Nepal.     

Sequence diversity within the reovirus S2 gene: reovirus genes reassort in nature, and their termini are predicted to form a panhandle motif.

To better understand genetic diversity within mammalian reoviruses, we determined S2 nucleotide and deduced sigma 2 amino acid sequences of nine reovirus strains and compared these sequences with those of prototype strains of the three reovirus serotypes. The S2 gene and sigma 2 protein are highly conserved among the four type 1, one type 2, and seven type 3 strains studied. Phylogenetic analyses based on S2 nucleotide sequences of the 12 reovirus strains indicate that diversity within the S2 gene is independent of viral serotype. Additionally, we found marked topological differences between phylogenetic trees generated from S1 and S2 gene nucleotide sequences of the seven type 3 strains. These results demonstrate that reovirus S1 and S2 genes have distinct evolutionary histories, thus providing phylogenetic evidence for lateral transfer of reovirus genes in nature. When variability among the 12 sigma 2-encoding S2 nucleotide sequences was analyzed at synonymous positions, we found that approximately 60 nucleotides at the 5' terminus and 30 nucleotides at the 3' terminus were markedly conserved in comparison with other sigma 2-encoding regions of S2. Predictions of RNA secondary structures indicate that the more conserved S2 sequences participate in the formation of an extended region of duplex RNA interrupted by a pair of stem-loops. Among the 12 deduced sigma 2 amino acid sequences examined, substitutions were observed at only 11% of amino acid positions. This finding suggests that constraints on the structure or function of sigma 2, perhaps in part because of its location in the virion core, have limited sequence diversity within this protein.