Comparative Pathogenesis of Alkhumra Hemorrhagic Fever and Kyasanur Forest Disease Viruses in a Mouse Model

Kyasanur Forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (AHFV) are genetically closely-related, tick-borne flaviviruses that cause severe, often fatal disease in humans. Flaviviruses in the tick-borne encephalitis (TBE) complex typically cause neurological disease in humans whereas patients infected with KFDV and AHFV predominately present with hemorrhagic fever. A small animal model for KFDV and AHFV to study the pathogenesis and evaluate countermeasures has been lacking mostly due to the need of a high biocontainment laboratory to work with the viruses. To evaluate the utility of an existing mouse model for tick-borne flavivirus pathogenesis, we performed serial sacrifice studies in BALB/c mice infected with either KFDV strain P9605 or AHFV strain Zaki-1. Strikingly, infection with KFDV was completely lethal in mice, while AHFV caused no clinical signs of disease and no animals succumbed to infection. KFDV and high levels of pro-inflammatory cytokines were detected in the brain at later time points, but no virus was found in visceral organs; conversely, AHFV Zaki-1 and elevated levels of cytokines were found in the visceral organs at earlier time points, but were not detected in the brain. While infection with either virus caused a generalized leukopenia, only AHFV Zaki-1 induced hematologic abnormalities in infected animals. Our data suggest that KFDV P9605 may have lost its ability to cause hemorrhagic disease as the result of multiple passages in suckling mouse brains. However, likely by virtue of fewer mouse passages, AHFV Zaki-1 has retained the ability to replicate in visceral organs, cause hematologic abnormalities, and induce pro-inflammatory cytokines without causing overt disease. Given these striking differences, the use of inbred mice and the virus passage history need to be carefully considered in the interpretation of animal studies using these viruses.     

Newly recognized mosquito-associated viruses in mainland China, in the last two decades

There are four principal arboviruses in mainland China. Two kinds of them are mosquito-borne viruses, namely Japanese encephalitis virus and dengue virus, which lead to Japanese encephalitis, and dengue fever/dengue hemorrhagic fever respectively; the other two are tick-borne viruses, namely tick-borne encephalitis virus and Crimean-Congo hemorrhagic fever virus (also known as Xinjiang hemorrhagic fever virus), which contribute to tick-borne encephalitis and Xinjiang hemorrhagic fever respectively. With exception of these four main arboviruses, many other mosquito-associated viruses have been isolated and identified in recent years. These newly isolated and identified mosquito-associated viruses are probably responsible for human and animal infections and diseases. The purpose of this review is to describe the newly isolated mosquito-associated viruses in mainland China which belong to five viral families, including their virological properties, phylogenetic relationships, serological evidence, as well as to appeal the public health concentration worldwide.     

Ancient ancestry of KFDV and AHFV revealed by complete genome analyses of viruses isolated from ticks and mammalian hosts

Background

Alkhurma hemorrhagic fever virus (AHFV) and Kyasanur forest disease virus (KFDV) cause significant human disease and mortality in Saudi Arabia and India, respectively. Despite their distinct geographic ranges, AHFV and KFDV share a remarkably high sequence identity. Given its emergence decades after KFDV, AHFV has since been considered a variant of KFDV and thought to have arisen from an introduction of KFDV to Saudi Arabia from India. To gain a better understanding of the evolutionary history of AHFV and KFDV, we analyzed the full length genomes of 16 AHFV and 3 KFDV isolates.

Methodology/Principal Findings

Viral genomes were sequenced and compared to two AHFV sequences available in GenBank. Sequence analyses revealed higher genetic diversity within AHFVs isolated from ticks than human AHFV isolates. A Bayesian coalescent phylogenetic analysis demonstrated an ancient divergence of AHFV and KFDV of approximately 700 years ago.

Conclusions/Significance

The high sequence diversity within tick populations and the presence of competent tick vectors in the surrounding regions, coupled with the recent identification of AHFV in Egypt, indicate possible viral range expansion or a larger geographic range than previously thought. The divergence of AHFV from KFDV nearly 700 years ago suggests other AHFV/KFDV-like viruses might exist in the regions between Saudi Arabia and India. Given the human morbidity and mortality associated with these viruses, these results emphasize the importance of more focused study of these significant public health threats.     

Crimean-Congo hemorrhagic fever virus genomics and global diversity

Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high case fatality that occurs in Africa, Europe, the Middle East, and Asia. The complete genomes of 13 geographically and temporally diverse virus strains were determined, and CCHF viruses were found to be highly variable with 20 and 8%, 31 and 27%, and 22 and 10% nucleotide and deduced amino acid differences detected among virus S (nucleocapsid), M (glycoprotein), and L (polymerase) genome segments, respectively. Distinct geographic lineages exist, but with multiple exceptions indicative of long-distance virus movement. Discrepancies among the virus S, M, and L phylogenetic tree topologies document multiple RNA segment reassortment events. An analysis of individual segment datasets suggests genetic recombination also occurs. For an arthropod-borne virus, the genomic plasticity of CCHF virus is surprisingly high.     

The Neglected Arboviral Infections in Mainland China

The major arboviral diseases in mainland China include Japanese encephalitis, dengue fever, Crimean-Congo hemorrhagic fever (also known as Xinjiang hemorrhagic fever), and tick-borne encephalitis. These and other newly found arbovirus infections due to Banna virus and Tahyna virus contribute to a large and relatively neglected disease burden in China. Here we briefly review the literature regarding these arboviral infections in mainland China with emphasis on their epidemiology, primary vectors, phylogenetic associations, and the prevention programs associated with these agents in China.     

In silico analysis for development of epitopes-based peptide vaccine against Alkhurma hemorrhagic fever virus

Abstract

Alkhurma hemorrhagic fever virus (ALKV) causes a fatal clinical disease in human beings of different tropical and sub-tropical regions. Recently, the ALKV epidemics have raised a great public health concern with the room for improvement in the essential therapeutic interventions. Despite increased realistic clinical cases of ALKV infection, the efficient vaccine or immunotherapy is not yet available to-date. Therefore, the current study aimed to analyze the envelope glycoprotein of ALKV for the development of B-cells and T-cells epitope-based peptide vaccine using the computational in silico method. Utilizing various immunoinformatics approaches, a total of 5 B-cells and 25 T-cells (MHC-I?=?17, MHC-II?=?8) epitope-based peptides were predicted in the current study. All predicted peptides had highest antigenicity and immunogenicity scores along with high binding affinity to human leukocyte antigen (HLA) class II alleles. Among 25T-cell epitopes, three peptides were found alike to have affinity to bind both MHC-I and MHC-II alleles. These outcomes suggested that these predicted epitopes could potentially be used in the development of an efficient vaccine against ALKV, which may enable to elicit both humoral and cell-mediated immunity. Although, these predicted peptides could be useful in designing a candidate vaccine for the prevention of ALKV; however, it’s in vitro and in vivo assessments are prerequisite.

Communicated by Ramaswamy H. Sarma     

RNA secondary structure in the coding region of dengue virus type 2 directs translation start codon selection and is required for viral replication

Dengue virus is a positive-strand RNA virus and a member of the genus Flavivirus, which includes West Nile, yellow fever, and tick-borne encephalitis viruses. Flavivirus genomes are translated as a single polyprotein that is subsequently cleaved into 10 proteins, the first of which is the viral capsid (C) protein. Dengue virus type 2 (DENV2) and other mosquito-borne flaviviruses initiate translation of C from a start codon in a suboptimal context and have multiple in-frame AUGs downstream. Here, we show that an RNA hairpin structure in the capsid coding region (cHP) directs translation start site selection in human and mosquito cells. The ability of the cHP to direct initiation from the first start codon is proportional to its thermodynamic stability, is position dependent, and is sequence independent, consistent with a mechanism in which the scanning initiation complex stalls momentarily over the first AUG as it begins to unwind the cHP. The cHP of tick-borne flaviviruses is not maintained in a position to influence start codon selection, which suggests that this coding region cis element may serve another function in the flavivirus life cycle. Here, we demonstrate that the DENV2 cHP and both the first and second AUGs of C are necessary for efficient viral replication in human and mosquito cells. While numerous regulatory elements have been identified in the untranslated regions of RNA viral genomes, we show that the cHP is a coding-region RNA element that directs start codon selection and is required for viral replication.     

A pigtailed macaque model of Kyasanur Forest disease virus and Alkhurma hemorrhagic disease virus pathogenesis

Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.     

Characterization of the glycoproteins of Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus is the cause of an important tick-borne disease of humans throughout regions of Africa, Europe, and Asia. Like other members of the genus Nairovirus, family Bunyaviridae, the CCHF virus M genome RNA segment encodes the virus glycoproteins. Sequence analysis of the CCHF virus (Matin strain) M RNA segment revealed one major open reading frame that potentially encodes a precursor polyprotein 1,689 amino acids (aa) in length. Comparison of the deduced amino acid sequences of the M-encoded polyproteins of Nigerian, Pakistani, and Chinese CCHF virus strains revealed two distinct protein regions. The carboxyl-terminal 1,441 aa are relatively highly conserved (up to 8.4% identity difference), whereas the amino-terminal 243 to 248 aa are highly variable (up to 56.4% identity difference) and have mucin-like features, including a high serine, threonine, and proline content (up to 47.3%) and a potential for extensive O-glycosylation. Analysis of released virus revealed two major structural glycoproteins, G2 (37 kDa) and G1 (75 kDa). Virus protein analysis by various techniques, including pulse-chase analysis and/or reactivity with CCHF virus-specific polyclonal and antipeptide antibodies, demonstrated that the 140-kDa (which contains the mucin-like region) and 85-kDa nonstructural proteins are the precursors of the mature G2 and G1 proteins, respectively. The amino termini of the CCHF virus (Matin strain) G2 and G1 proteins were established by microsequencing to be equivalent to aa 525 and 1046, respectively, of the encoded polyprotein precursor. The tetrapeptides RRLL and RKPL are immediately upstream of the cleavage site for mature G2 and G1, respectively. These are completely conserved among the predicted polyprotein sequences of all the CCHF virus strains and closely resemble the tetrapeptides that represent the major cleavage recognition sites present in the glycoprotein precursors of arenaviruses, such as Lassa fever virus (RRLL) and Pichinde virus (RKLL). These results strongly suggest that CCHF viruses (and other members of the genus Nairovirus) likely utilize the subtilase SKI-1/S1P-like cellular proteases for the major glycoprotein precursor cleavage events, as has recently been demonstrated for the arenaviruses.     

Limited Effects of Type I Interferons on Kyasanur Forest Disease Virus in Cell Culture

BackgroundThe tick-borne flavivirus, Kyasanur Forest disease virus (KFDV) causes seasonal infections and periodic outbreaks in south-west India. The current vaccine offers poor protection with reported issues of coverage and immunogenicity. Since there are no approved prophylactic therapeutics for KFDV, type I IFN-α/β subtypes were assessed for antiviral potency against KFDV in cell culture.Conclusions/SignificanceTreatment of cell culture with IFN does not appear to be suitable for KFDV eradication and the assay used for such studies should be carefully considered. Further, it appears that the NS5 protein is sufficient to permit KFDV to bypass the antiviral properties of IFN. We suggest that other prophylactic therapeutics should be evaluated in place of IFN for treatment of individuals with KFDV disease.     

Molecular Epidemiology of Crimean-Congo Hemorrhagic Fever Virus in Kosovo

Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic agent that causes severe, life-threatening disease, with a case fatality rate of 10–50%. It is the most widespread tick-borne virus in the world, with cases reported in Africa, Asia and Eastern Europe. CCHFV is a genetically diverse virus. Its genetic diversity is often correlated to its geographical origin. Genetic variability of CCHFV was determined within few endemic areas, however limited data is available for Kosovo. Furthermore, there is little information about the spatiotemporal genetic changes of CCHFV in endemic areas. Kosovo is an important endemic area for CCHFV. Cases were reported each year and the case-fatality rate is significantly higher compared to nearby regions. In this study, we wanted to examine the genetic variability of CCHFV obtained directly from CCHF-confirmed patients, hospitalized in Kosovo from 1991 to 2013. We sequenced partial S segment CCHFV nucleotide sequences from 89 patients. Our results show that several viral variants are present in Kosovo and that the genetic diversity is high in relation to the studied area. We also show that variants are mostly uniformly distributed throughout Kosovo and that limited evolutionary changes have occurred in 22 years. Our results also suggest the presence of a new distinct lineage within the European CCHF phylogenetic clade. Our study provide the largest number of CCHFV nucleotide sequences from patients in 22 year span in one endemic area.     

Domain III peptides from flavivirus envelope protein are useful antigens for serologic diagnosis and targets for immunization

The Flavivirus genus of the Flaviviridae family includes 70 enveloped single-stranded-RNA positive-sense viruses transmitted by arthropods. Among these viruses, there are a relevant number of human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV), Langat virus (LGTV) and Omsk hemorrhagic fever (OHFV). The flavivirus envelope (E) protein is a dominant antigen inducing immunologic responses in infected hosts and eliciting virus-neutralizing antibodies. The domain III (DIII) of E protein contains a panel of important epitopes that are recognized by virus-neutralizing monoclonal antibodies. Peptides of the DIII have been used with promising results as antigens for flavivirus serologic diagnosis and as targets for immunization against these viruses. We review here some important aspects of the molecular structure of the DIII as well as its use as antigens for serologic diagnosis and immunization in animal models.     

Geographical distribution of arboviruses in Yugoslavia.

Studies of arboviruses started in Yugoslavia in 1953 following the isolation of TBE virus which caused a severe epidemic that year. Until now the following viruses have been proven to circulate in the country: tick-borne encephalitis (TBE), Crimean-Congo hemorrhagic fever (CCHF), Bhanja (BHA), sandfly fever (SF), Tahyna (TAH), Calovo (CVO), West Nile (WN), dengue (DEN), Jug Bogdanovac (JB), and Hantaviruses. TBE virus is endemic in the north-west part of the country, causing also epidemics in cyclical intervals. Its typical clinical picture is aseptic meningitis, but severe cases with paralysis have also been described. The bite of ticks is confirmed in about 80% of cases. CCF caused a small epidemic with ten clinical cases in Macedonia in 1976. Bhanja virus was isolated on the Dalmatian island of Brac in 1977, the antibody rate there, determined by the HI method, being about 31%. The first human disease in the world was caused by the Yugoslav Bhanja virus strain. Sandfly fever is still active in the country. The Naples type is prevailing and has proved hazardous for newcomers. Hantaviruses have been studied since 1980. They caused severe epidemics (1967, 1980, 1989) and sporadic cases all over the country. Three different strains are in circulation. Further studies are needed for the rest of the above mentioned viruses to learn more about their significance in human pathology.     

Phylogeny of the Genus Flavivirus

We undertook a comprehensive phylogenetic study to establish the genetic relationship among the viruses of the genus Flavivirus and to compare the classification based on molecular phylogeny with the existing serologic method. By using a combination of quantitative definitions (bootstrap support level and the pairwise nucleotide sequence identity), the viruses could be classified into clusters, clades, and species. Our phylogenetic study revealed for the first time that from the putative ancestor two branches, non-vector and vector-borne virus clusters, evolved and from the latter cluster emerged tick-borne and mosquito-borne virus clusters. Provided that the theory of arthropod association being an acquired trait was correct, pairwise nucleotide sequence identity among these three clusters provided supporting data for a possibility that the non-vector cluster evolved first, followed by the separation of tick-borne and mosquito-borne virus clusters in that order. Clades established in our study correlated significantly with existing antigenic complexes. We also resolved many of the past taxonomic problems by establishing phylogenetic relationships of the antigenically unclassified viruses with the well-established viruses and by identifying synonymous viruses.The genus Flavivirus of the family Flaviviridae comprises over 70 viruses, many of which, such as the dengue (DEN) viruses, Japanese encephalitis (JE) virus, St. Louis encephalitis (SLE) virus, and yellow fever (YF) virus are important human pathogens (22, 31). Dengue and its severe and sometimes fatal forms, dengue hemorrhagic fever and dengue shock syndrome, alone affect nearly 80 million people a year (30). As demonstrated in recent outbreaks of meningitis by West Nile (WN) virus in Algeria and Romania, viruses of this group sometimes cause serious public health concern in unexpected locations (27).Most of these viruses were serologically classified into eight antigenic complexes, but many viruses, including the prototype of this group, YF virus, could not be affiliated with any complexes (6). Furthermore, many new viruses have been documented since the establishment of the serological classification, but their overall relationship with the other viruses has not been determined. The difficulty encountered with flavivirus classification partly derives from the extensive geographic distribution and the diversity of the arthropod vectors or vertebrates hosts associated with biological transmission of these viruses. Also, it derives from a confusion in virus nomenclature. For example, tick-borne encephalitis virus strains isolated primarily in western parts of Eurasia have been called TBE viruses, but, as clearly pointed out by Calisher (5), no such virus as tick-borne encephalitis virus (or TBE) has ever been registered to an international body dedicated to virus taxonomy. To compound the problem further, an increasing number of viruses have been added as new members of so-called TBE complex without a virus definition provided (16, 18, 29, 42, 52). This practice clearly demonstrates a need for establishing objective criteria for a better classification of those viruses.Molecular genetic classification of these viruses has been attempted before. In all previous studies, fewer than one-third of the members, primarily mosquito-borne and tick-borne viruses, were used to create phylogenetic trees, which showed evolution of mosquito-borne and tick-borne viruses from the presumed ancestor (3, 11). Since few sequence data were available from other viruses, in particular the viruses without known vectors (hereafter called the non-vector group), those phylogenetic trees provided only partial information.To establish a comprehensive phylogeny of the genus Flavivirus, we attempted to obtain the genomic sequence of a 1.0-kb segment at the 3′ terminus of the NS5 gene from all viruses whose sequences were not available. We analyzed, together with the other sequence data already published, the genetic relationships among the members of this group. Quantitative criteria based on a combination of the bootstrap support level and the pairwise nucleotide sequence identity were established to define subgeneric taxa. These included cluster, clade, and species. With our new taxonomic definitions, we then compared our genetic classification with the traditional system based on serological data.     

Evaluation of the cell culture based and the mouse brain derived inactivated vaccines against Crimean-Congo hemorrhagic fever virus in transiently immune-suppressed (IS) mouse model

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the Nairoviridae family within the Bunyavirales order of viruses. Crimean-Congo hemorrhagic fever (CCHF) is the most widespread among tick-borne human viral diseases. It is endemic in many areas of Africa, Asia, the Middle East, in the Balkans, Russia and countries of the former Soviet Union. The confirmed CCHF cases were seen in Spain in 2016 to signify expansion of the virus into new geographical areas. CCHFV causes a viral human disease characterized by sudden onset of fever, headache, abdominal pain, nausea, hypotension, hemorrhage, and hepatic dysfunction with fatality rates up to 30%. Currently, there are no spesific treatments or licensed vaccines available for CCHFV. The absence of a susceptible animal model for CCHFV infection was severely hindered work on the development of vaccines. However, several animal models of CCHFV infection have been recently developed and used to assess vaccine efficacy. In this study, we have used the transiently immune-suppressed (IS) mouse model that MAb-5A3 was used to block IFN-I signaling in immune intact, wild-type mice at the time of CCHFV infection to evaluate the immune response and efficacy of the cell culture based and the mouse brain derived inactivated vaccines against CCHFV. Both vaccine preparations have provided complete protection but the cell culture based vaccine more effectively induced to CCFHV spesific antibodies and T cell responses. This is the first comparison of the cell culture based and the mouse brain derived vaccines for assessing the protective efficacy and the immunogenicity in the IS mouse CCHFV model.     

Molecular-based identification and phylogeny of genomic and proteomic sequences of mosquito-borne flavivirus

Mosquito-borne flaviviruses (MBFVs) are important cause of emerging and re-emerging human diseases nearly worldwide, transmitted by arthropod vectors (mostly aedes and culex mosquitoes), with particular reference to yellow fever virus, Japanese encephalitis virus, dengue fever virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, etc. In over 100 countries, more than 2.5 billion people are at risk of infection, and approximately 20 million infections are reported annually. Through the analysis of gene sequence data of these virus populations it is possible to infer phylogenetic relationships, which in turn can yield important epidemiological information, including their demographic history. Early attempts to define the evolutionary relationships and origins of viruses in the genus flavivirus are hampered by the lack of genetic information particularly amongst the MBFVs. In this study, complete genome, translated polyprotein, structural and non-structural proteins of MBFVs have been targeted and revealed an extensive series of clades defined by their epidemiology and disease associations. The branching patterns of at the deeper nodes of the resultant trees were different from those reported in the previous study. The significance of these observations is discussed.     

Organization of the Karshi virus strain Leiv-2247 Uz genome and its phylogenetic relationships with other representatives of the Flavivirus genus

For the first time, the Karshi virus complete genome sequence has been determined and analyzed in this work. The Karshi virus was attributed to the Tick-borne encephalitis group on the basis of phylogenetic analysis of polyprotein sequencing, as well as partial NS5 of the Flavivirus genus. Comprehensive analysis of the longed to its own cluster, which includes also the Royal Farm virus.     

Re-emergence of Crimean-Congo hemorrhagic fever virus in Central Africa

Background

Crimean-Congo hemorrhagic fever (CCHF) is a severe tick-borne disease well recognized through Europe and Asia where diagnostic tests and medical surveillance are available. However, it is largely neglected in Africa, especially in the tropical rainforest of Central Africa where only sporadic human cases have been reported and date back to more than 30 years. We describe here an isolated human case that occurred in the Democratic Republic of the Congo in 2008 and performed phylogenetic analysis to investigate whether it resulted from a regional re-emergence or from the introduction of a novel virus in the area.

Methods and Findings

Near complete segment S and partial segment M sequences were characterized. Bayesian phylogenetic analysis and datation were performed to investigate the relationship between this new strain and viral strains from Africa, Europe and Asia. The new strain is phylogenetically close to the previously described regional genotype (II) that appears to be specific to Central Africa. Phylogenetic discrepancy between segment S and M suggested genetic exchange among local sublineages, which was dated back to 130–590 years before present.

Conclusions

The phylogenetic analyses presented here suggest ongoing CCHF virus circulation in Central Africa for a long time despite the absence of reported human cases. Many infections have most probably been overlooked, due to the weakness of healthcare structures and the absence of available diagnostic procedure. However, despite the lack of accurate ecological data, the sporadic reporting of human cases could also be partly associated with a specific sylvatic cycle in Central Africa where deforestation may raise the risks of re-emergence. For these reasons, together with the high risk of nosocomial transmission, public health authorities'' attention should be drawn to this etiological agent.     

The complete coding sequence of a European isolate of GB-C/hepatitis G virus

We determined the full-length coding sequence of a GB-C/hepatitis G (GBV-C/HGV) virus isolate (AUQ) obtained from a French blood donor. The genome of AUQ strain contains a long open reading frame encoding 2842 amino acid residues. Comparison with the 33 complete genome sequences so far available from the databases indicates that the full-length sequence of GBV-C/HGV AUQ showed 9.0-14.1% nucleotide sequence divergence but only 1.6-5.3% at the amino acid level. Analysis of the potential cleavage sites of the polyprotein found that they were remarkably conserved among all sequences. Although phylogenetic studies based on partial genomic sequences suggested a clusterization according to the geographical origin, analysis based on full-length polyprotein did not provide similar conclusions.