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Decades after its discovery in East Africa, Zika virus (ZIKV) emerged in Brazil in 2013 and infected millions of people during intense urban transmission. Whether vertebrates other than humans are involved in ZIKV transmission cycles remained unclear. Here, we investigate the role of different animals as ZIKV reservoirs by testing 1723 sera of pets, peri-domestic animals and African non-human primates (NHP) sampled during 2013-2018 in Brazil and 2006-2016 in Côte d'Ivoire. Exhaustive neutralization testing substantiated co-circulation of multiple flaviviruses and failed to confirm ZIKV infection in pets or peri-domestic animals in Côte d'Ivoire ( n =259) and Brazil ( n =1416). In contrast, ZIKV seroprevalence was 22.2% (2/9, 95% CI, 2.8-60.1) in West African chimpanzees ( Pan troglodytes verus ) and 11.1% (1/9, 95% CI, 0.3-48.3) in king colobus ( Colobus polycomos ). Our results indicate that while NHP may represent ZIKV reservoirs in Africa, pets or peri-domestic animals likely do not play a role in ZIKV transmission cycles.

Sandfly-borne phleboviruses are distributed widely throughout the Mediterranean Basin, presenting a threat to public health in areas where they circulate. However, the true diversity and distribution of pathogenic and apathogenic sandfly-borne phleboviruses remains a key issue to be studied. In the Balkans, most published data rely on serology-based studies although virus isolation has occasionally been reported. Here, we report the discovery of two novel sandfly-borne phleboviruses, provisionally named Zaba virus (ZABAV) and Bregalaka virus (BREV), which were isolated in Croatia and North Macedonia, respectively. This constitutes the first isolation of phleboviruses in both countries. Genetic analysis based on complete coding sequences indicated that ZABAV and BREV are distinct from each other and belong to the genus Phlebovirus, family Phenuiviridae . Phylogenetic and amino acid modelling of viral polymerase shows that ZABAV and BREV are new members of the Salehabad phlebovirus species and the Adana phlebovirus species, respectively. Moreover, sequence-based vector identification suggests that ZABAV is mainly transmitted by Phlebotomus neglectus and BREV is mainly transmitted by Phlebotomus perfiliewi . BREV neutralizing antibodies were detected in 3.3% of human sera with rates up to 16.7% in certain districts, demonstrating that BREV frequently infects humans in North Macedonia. In vitro viral growth kinetics experiments demonstrated viral replication of both viruses in mammalian and mosquito cells. In vivo experimental studies in mice suggest that ZABAV and BREV exhibit characteristics making them possible human pathogens.

Japanese encephalitis remains the most important cause of viral encephalitis in humans in several southeast Asian countries, including Cambodia, causing at least 65 000 cases of encephalitis per year. This vector-borne viral zoonosis - caused by Japanese encephalitis virus (JEV) - is considered to be a rural disease and is transmitted by mosquitoes, with birds and pigs being the natural reservoirs, while humans are accidental hosts. In this study we report the first two JEV isolations in Cambodia from human encephalitis cases from two studies on the aetiology of central nervous system disease, conducted at the two major paediatric hospitals in the country. We also report JEV isolation from Culextritaeniorhynchus mosquitoes and from pig samples collected in two farms, located in peri-urban and rural areas. Out of 11 reverse-transcription polymerase chain reaction-positive original samples, we generated full-genome sequences from 5 JEV isolates. Five additional partial sequences of the JEV NS3 gene from viruses detected in five pigs and one complete coding sequence of the envelope gene of a strain identified in a pig were generated. Phylogenetic analyses revealed that JEV detected in Cambodia belonged to genotype I and clustered in two clades: genotype I-a, mainly comprising strains from Thailand, and genotype I-b, comprising strains from Vietnam that dispersed northwards to China. Finally, in this study, we provide proof that the sequenced JEV strains circulate between pigs, Culex tritaeniorhynchus and humans in the Phnom Penh vicinity.

A new phlebovirus, Medjerda Valley virus (MVV), was isolated from one pool of Phlebotomus sp. (Diptera; Psychodidae) sandflies trapped in the vicinity of the Utique site, northern Tunisia. Genetic analysis based on complete coding of genomic sequences of the three RNA segments indicated that MVV is most closely related to members of the Salehabad virus species, where it is the fourth virus for which the complete sequence is available. A seroprevalence study was performed to search for neutralizing antibodies in human sera in the same region. The results demonstrate that in this area, MVV can readily infect humans despite low seroprevalence rates. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our human seroprevalence results, together with high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece. Further studies are needed to investigate the capacity of each specific member of the Salehabad virus species to cause human or animal diseases.

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., (© 2014 The Authors.)

Duck egg-drop syndrome virus (DEDSV) is a newly emerging pathogenic flavivirus causing avian diseases in China. The infection occurs in laying ducks characterized by a severe drop in egg production with a fatality rate of 5-15 %. The virus was found to be most closely related to Tembusu virus (TMUV), an isolate from mosquitoes in South-east Asia. Here, we have sequenced and characterized the full-length genomes of seven DEDSV strains, including the 5'- and 3'-non-coding regions (NCRs). We also report for the first time the ORF sequences of TMUV and Sitiawan virus (STWV), another closely related flavivirus isolated from diseased chickens. We analysed the phylogenetic and antigenic relationships of DEDSV in relation to the Asian viruses TMUV and STWV, and other representative flaviviruses. Our results confirm the close relationship between DEDSV and TMUV/STWV and we discuss their probable evolutionary origins. We have also characterized the cleavage sites, potential glycosylation sites and unique motifs/modules of these viruses. Additionally, conserved sequences in both 5'- and 3'-NCRs were identified and the predicted secondary structures of the terminal sequences were studied. Antigenic cross-reactivity comparisons of DEDSV with related pathogenic flaviviruses identified a surprisingly close relationship with dengue virus (DENV) and raised the question of whether or not DEDSV may have a potential infectious threat to man. Importantly, DEDSV can be efficiently recognized by a broadly cross-reactive flavivirus mAb, 2A10G6, derived against DENV. The significance of these studies is discussed in the context of the emergence, evolution, epidemiology, antigenicity and pathogenicity of the newly emergent DEDSV.

There has been an explosion in the discovery of 'insect-specific' flaviviruses and/or their related sequences in natural mosquito populations. Herein we review all 'insect-specific' flavivirus sequences currently available and conduct phylogenetic analyses of both the 'insect-specific' flaviviruses and available sequences of the entire genus Flavivirus. We show that there is no statistical support for virus-mosquito co-divergence, suggesting that the 'insect-specific' flaviviruses may have undergone multiple introductions with frequent host switching. We discuss potential implications for the evolution of vectoring within the family Flaviviridae. We also provide preliminary evidence for potential recombination events in the history of cell fusing agent virus. Finally, we consider priorities and guidelines for future research on 'insect-specific' flaviviruses, including the vast potential that exists for the study of biodiversity within a range of potential hosts and vectors, and its effect on the emergence and maintenance of the flaviviruses.

Sandflies are widely distributed around the Mediterranean Basin. Therefore, human populations in this area are potentially exposed to sandfly-transmitted diseases, including those caused by phleboviruses. Whilst there are substantial data in countries located in the northern part of the Mediterranean basin, few data are available for North Africa. In this study, a total of 1489 sandflies were collected in 2008 in Tunisia from two sites, bioclimatically distinct, located 235 km apart, and identified morphologically. Sandfly species comprised Phlebotomus perniciosus (52.2%), Phlebotomus longicuspis (30.1%), Phlebotomus papatasi (12.0%), Phlebotomus perfiliewi (4.6%), Phlebotomus langeroni (0.4%) and Sergentomyia minuta (0.5%). PCR screening, using generic primers for the genus Phlebovirus, resulted in the detection of ten positive pools. Sequence analysis revealed that two pools contained viral RNA corresponding to a novel virus closely related to sandfly fever Naples virus. Virus isolation in Vero cells was achieved from one pool. Genetic and phylogenetic characterization based on sequences in the three genomic segments showed that it was a novel virus distinct from other recognized members of the species. This novel virus was provisionally named Punique virus. Viral sequences in the polymerase gene corresponding to another phlebovirus closely related to but distinct from sandfly fever Sicilian virus were obtained from the eight remaining positive pools.

We analysed the complete coding sequences of all recognized species of Aedes-borne flavivirus, including previously uncharacterized viruses within the yellow fever virus (YFV), Spondweni virus (SPOV) and dengue virus (DENV) groups. Two major phylogenetic lineages were revealed: one included the YFV and Entebbe bat virus groups, and the other included the DENV, SPOV and Culex-borne flavivirus groups. This analysis supported previous evidence that Culex-borne flaviviruses have evolved from ancestral Aedes-borne viruses. However, the topology at the junction between these lineages remains complex and may be refined by the discovery of viruses related to the Kedougou virus. Additionally, viral evolution was found to be associated with the appearance of new biological characteristics; mutations that may modify the envelope protein structure were identified for seven viruses within the YFV group, and an expansion of host-vector range was identified in the two major evolutionary lineages, which in turn may facilitate the emergence of mosquito-borne flaviviruses.

The genus Flavivirus, which contains approximately 70 single-stranded, positive-sense RNA viruses, represents a unique model for studying the evolution of vector-borne disease, as it includes viruses that are mosquito-borne, tick-borne or have no known vector. Both theoretical work and field studies suggest the existence of a large number of undiscovered flaviviruses. Recently, the first isolation of cell fusing agent virus (CFAV) was reported from a natural mosquito population in Puerto Rico, and sequences related to CFAV have been discovered in mosquitoes from Thailand. CFAV had previously been isolated from a mosquito cell line in 1975 and represented the only known 'insect-only' flavivirus, appearing to replicate in insect cells alone. A second member of the 'insect-only' group, Kamiti River virus (KRV), was isolated from Kenyan mosquitoes in 2003. A third tentative member of the 'insect-only' group, Culex flavivirus (CxFV), was first isolated in 2007 from Japan and further strains have subsequently been reported from the Americas. We report the discovery, isolation and characterization of two novel 'insect-only' flaviviruses from Entebbe, Uganda: a novel lineage tentatively designated Nakiwogo virus (NAKV) and a new strain of CxFV. The individual mosquitoes from which these strains were isolated, identified retrospectively by using a reference molecular phylogeny generated using voucher specimens from the region, were Mansonia africana nigerrima and Culex quinquefasciatus, respectively. This represents the first isolation, to our knowledge, of a novel insect-only flavivirus from a Mansonia species and the first isolation of a strain of CxFV from Africa.

By using degenerate primers deduced from conserved patterns in the flavivirus polymerase gene, a novel RNA virus was discovered in Rhipicephalus ticks sampled from members of the family Bovidae in Senegal. It was named Ngoye virus (NGOV) after the location from which it was isolated. Viral particles could be observed by electron microscopy, but isolation in vertebrate or invertebrate cell lines or by intracerebral infection of newborn mice remained unsuccessful. This is atypical of recognized arboviruses. The characterization of 4176 nt of the non-structural genes revealed that NGOV is a novel flavivirus species. It forms a distinct phylogenetic lineage related distantly to previously identified members of the genus Flavivirus. Analysis of genetic data suggested that the processing of the NGOV polyprotein and the organization of its replication complex are similar to those of flaviviruses. Together with other recent data, these findings suggest that a large number of viruses related distantly to 'classical' arthropod-borne flaviviruses remain to be discovered.

Micromonas pusilla reovirus (MpRV) is an 11-segmented, double-stranded RNA virus isolated from the marine protist Micromonas pusilla. Sequence analysis (including conserved termini and presence of core motifs of reovirus polymerase), morphology and physicochemical properties confirmed the status of MpRV as a member of the family Reoviridae. Electron microscopy showed that intact virus particles are unusually larger (90-95 nm) than the known size of particles of viruses belonging to the family Reoviridae. Particles that were purified on caesium chloride gradients had a mean size of 75 nm (a size similar to the size of intact particles of members of the family Reoviridae), indicating that they lost outer-coat components. The subcore particles had a mean size of 50 nm and a smooth surface, indicating that MpRV belongs to the non-turreted Reoviridae. The maximum amino acid identity with other reovirus proteins was 21 %, which is compatible with values existing between distinct genera. Based on morphological and sequence findings, this virus should be classified as the representative of a novel genus within the family Reoviridae, designated Mimoreovirus (from Micromonas pusilla reovirus). The topology of the phylogenetic tree built with putative polymerase sequences of the family Reoviridae suggested that the branch of MpRV could be ancestral. Further analysis showed that segment 1 of MpRV was much longer (5792 bp) than any other reovirus segment and encoded a protein of 200 kDa (VP1). This protein exhibited significant similarities to O-glycosylated proteins, including viral envelope proteins, and is likely to represent the additional outer coat of MpRV.

The genus Flavivirus contains approximately 70 single-stranded, positive-sense RNA viruses that are mosquito-borne, tick-borne or have no known vector. Two discoveries support previous suggestions of the existence of a large number of unsampled flaviviruses: (i) a new flavivirus, Kamiti River virus, was recently isolated from Kenyan mosquitoes, and (ii) sequences with high similarity to those of flaviviruses have been found integrated into the genome of Aedes mosquitoes, suggesting a past infection with a virus (or viruses) that has yet to be discovered. These sequences were related most closely to a flavivirus that infects insects alone, cell fusing agent virus (CFAV). CFAV was originally isolated in the laboratory from an Aedes aegypti cell line. To date, this virus had not been found in the wild. In the present study, over 40 isolates of a novel strain of CFAV were discovered from mature mosquitoes sampled from the wild in Puerto Rico. The viral strain was present in a range of mosquito species, including Aedes aegypti, Aedes albopictus and Culex sp., from numerous locations across the island and, importantly, in mosquitoes of both sexes, suggesting vertical transmission. Here, results from viral screening, and cell culture and molecular identification of the infected mosquitoes are presented. Experimental-infection tests were also conducted by using the original CFAV strain and a highly efficient reverse-transcription mechanism has been documented, in which initiation of copying occurs at the 3' terminus of either the genomic RNA or the intermediate of replication, potentially elucidating the mechanism by which flaviviral sequences may have integrated into mosquito genomes.

Seadornaviruses are emerging arboviral pathogens from the south-east of Asia. The genus Seadornavirus contains two distinct species, Banna virus (BAV) isolated from humans with encephalitis and Kadipiro virus. BAV replicates within insect cells and mice but not in cultured mammalian cells. Here, the discovery of Liao ning virus (LNV), a new seadornavirus from the Aedes dorsalis mosquito, which was completely sequenced and was found to be related to BAV and Kadipiro virus, is reported. Two serotypes of LNV could be distinguished by a serum neutralization assay. According to amino acid identity with other seadornaviruses, and to criteria set by the ICTV for species delineation, LNV was identified as a member of a new species of virus. Its morphology was characterized by electron microscopy and found to be similar to that of BAV. LNV is the first reported seadornavirus that replicates in mammalian cells, leading to massive cytopathic effect in all transformed or embryonic cell lines tested. LNV- and BAV-infected mice producing a viraemia lasting for 5 days was followed by viral clearance. Mice infection generated virus quasi-species for LNV (the first reported observation for quasi-species in the family Reoviridae) but not for BAV. Challenge with BAV in mice immunized against BAV did not lead to productive infection. However, challenge with LNV in mice immunized against LNV was lethal with a new phase of viraemia and massive haemorrhage.

An orbivirus designated Yunnan orbivirus (YUOV) was isolated from Culex tritaeniorhynchus mosquitoes collected in the Yunnan province of China. Electron microscopy showed particles with typical orbivirus morphology. The YUOV genome was sequenced completely and compared with previously characterized orbivirus genomes. Significant identity scores were detected between proteins encoded by the segments (Seg-1 to Seg-10) of YUOV and those encoded by their homologues in insect-borne and tick-borne orbiviruses. Analysis of VP1 (Pol) and VP2 (T2, which correlates with the virus serogroup) indicated that YUOV is a new species of the genus Orbivirus that is unrelated to the other insect-borne orbiviruses. The replication of YUOV in mosquito cell lines was restricted to Aedes albopictus cells and the virus failed to replicate in mammalian cell lines. However, intraperitoneal injection of virus into naïve mice resulted in productive, non-lethal virus replication and viraemia. Infected mice developed serum neutralizing antibodies and were protected against a new infection challenge. Sequence analysis of clones from the segments encoding outer coat proteins (Seg-3 and Seg-6) of YUOV recovered from mouse blood did not show significant changes in the sequences. The availability of the complete genome sequence will facilitate the development of sequence-specific PCR assays for the study of YUOV epidemiology in the field.

Banna virus (BAV) particles contain seven structural proteins: VP4 and VP9 form an outer-capsid layer, whilst the virus core contains three major proteins (VP2, VP8 and VP10) and two minor proteins (VP1 and VP3). Sequence analysis showed that VP3 contains motifs [Kx(I/V/L)S] and (Hx(n)H) that have previously been identified in the guanylyltransferases of other reoviruses. Incubation of purified BAV-Ch core particles with [alpha-32P]GTP resulted in exclusive covalent labelling of VP3, demonstrating autoguanylation activity (which is considered indicative of guanylyltransferase activity). Recombinant VP3 prepared in a cell-free expression system was also guanylated under similar reaction conditions, and products were synthesized (in the presence of non-radiolabelled GDP) that co-migrated with GMP, GDP and GpppG during TLC. This reaction, which required magnesium ions for optimum activity, demonstrates that VP3 possesses nucleoside triphosphatase (GTPase) activity and is the BAV guanylyltransferase (RNA 'capping' enzyme).

Banna virus (BAV) is the type species of the genus Seadornavirus within the family Reoviridae. The Chinese BAV isolate (BAV-Ch), which causes encephalitis in humans, was shown to have a structural organization and particle morphology reminiscent of that of rotaviruses, with fibre proteins projecting from the surface of the particle. Intact BAV-Ch virus particles contain seven structural proteins, two of which (VP4 and VP9) form the outer coat. The inner (core) particles contain five additional proteins (VP1, VP2, VP3, VP8 and VP10) and are 'non-turreted', with a relatively smooth surface appearance. VP2 is the 'T = 2' protein that forms the innermost 'subcore' layer, whilst VP8 is the 'T = 13' protein forming the core-surface layer. Sequence comparisons indicate that BAV VP9 and VP10 are equivalent to the VP8* and VP5* domains, respectively, of rotavirus outer-coat protein VP4 (GenBank accession no. P12976). VP9 has also been shown to be responsible for virus attachment to the host-cell surface and may be involved in internalization. These similarities reveal a previously unreported genetic link between the genera Rotavirus and Seadornavirus, although the expression of BAV VP9 and VP10 from two separate genome segments, rather than by the proteolytic cleavage of a single gene product (as seen in rotavirus VP4), suggests a significant evolutionary jump between the members of these two genera.

Genome segment 9 (Seg-9) of Colorado tick fever virus (CTFV) is 1884 bp long and contains a large open reading frame (ORF; 1845 nt in length overall), although a single in-frame stop codon (at nt 1052-1054) reduces the ORF coding capacity by approximately 40 %. However, analyses of highly conserved RNA sequences in the vicinity of the stop codon indicate that it belongs to a class of 'leaky terminators'. The third nucleotide positions in codons situated both before and after the stop codon, shows the highest variability, suggesting that both regions are translated during virus replication. This also suggests that the stop signal is functionally leaky, allowing read-through translation to occur. Indeed, both the truncated 'termination' protein and the full-length 'read-through' protein (VP9 and VP9', respectively) were detected in CTFV-infected cells, in cells transfected with a plasmid expressing only Seg-9 protein products, and in the in vitro translation products from undenatured Seg-9 ssRNA. The ratios of full-length and truncated proteins generated suggest that read-through may be down-regulated by other viral proteins. Western blot analysis of infected cells and purified CTFV showed that VP9 is a structural component of the virion, while VP9' is a non-structural protein.

Flavivirus-related sequences have been discovered in the dsDNA genome of Aedes albopictus and Aedes aegypti mosquitoes, demonstrating for the first time an integration into a eukaryotic genome of a multigenic sequence from an RNA virus that replicates without a recognized DNA intermediate. In the Aedes albopictus C6/36 cell line, an open reading frame (ORF) of 1557 aa with protease/helicase and polyprotein processing domains characteristic of flaviviruses was identified. It is closely related to NS1-NS4A genes of the Cell Fusing Agent and Kamiti River virus and the corresponding mRNAs were detected. Integrated sequences homologous to the envelope, NS4B and polymerase genes of flaviviruses were identified. Overall, approximately two-thirds of a flavivirus-like genome were characterized. In the Aedes aegypti A20 cell line, a 492 aa ORF related to the polymerase of the Cell Fusing Agent and Kamiti River virus was identified. These flavivirus-related integrated DNA sequences were detected in laboratory-bred and wild Aedes albopictus and Aedes aegypti mosquitoes, demonstrating that their discovery is not an artefact resulting from the manipulation of mosquito cell lines, since they exist under natural conditions. This finding has major implications regarding evolution, as it represents an entirely different mechanism by which genetic diversity may be generated in eukaryotic cells distinct from accepted processes.

This paper describes the study of hepatitis C virus (HCV) evolution in the largest cohort of HCV-infected blood donors (BDs)/blood recipients (BRs) reported to date (25 pairs). A molecular analysis of partial sequences in the E1 (envelope) and NS5-B (polymerase) genes was performed. Phylogenetic reconstruction showed that the evolution of dominant strains was qualitatively and quantitatively different in BDs and BRs. The evolutionary rate was significantly higher in BRs, in which, in addition, most substitutions observed were antonymous. These findings corroborate the hypothesis that a large part of virus evolution - which was evaluated to be equivalent to approximately 20 years of chronic evolution - is acquired during the early phase of infection. These findings should be taken into account for the modelling of the long-term evolution of HCV and their possible contribution to improve our understanding of HCV natural history is discussed.

Tamana bat virus (TABV, isolated from the bat Pteronotus parnellii) is currently classified as a tentative species in the genus FLAVIVIRUS: We report here the determination and analysis of its complete coding sequence. Low but significant similarity scores between TABV and member-viruses of the genus Flavivirus were identified in the amino acid sequences of the structural, NS3 and NS5 genes. A series of cysteines located in the envelope protein and the most important enzymatic domains of the virus helicase/NTPase, methyltransferase and RNA-dependent RNA polymerase were found to be highly conserved. In the serine-protease domain, the catalytic sites were conserved, but variations in sequence were found in the putative substrate-binding sites, implying possible differences in the protease specificity. In accordance with this finding, the putative cleavage sites of the TABV polyprotein by the virus protease are substantially different from those of flaviviruses. The phylogenetic position of TABV could not be determined precisely, probably due to the extremely significant genetic divergence from other member-viruses of the family FLAVIVIRIDAE: However, analysis based on both genetic distances and maximum-likelihood confirmed that TABV is more closely related to the flaviviruses than to the other genera. These findings have implications for the evolutionary history and taxonomic classification of the family as a whole: (i) the possibility that flaviviruses were derived from viruses infecting mammals rather than from mosquito viruses cannot be excluded; (ii) using the current criteria for the definition of genera in the family Flaviviridae, TABV should be assigned to a new genus.

Montana Myotis leukoencephalitis virus (MMLV), a virus isolated from bats, causes an encephalitis in small rodents reminiscent of flavivirus encephalitis in humans. The complete MMLV genome is 10690 nucleotides long and encodes a putative polyprotein of 3374 amino acids. The virus contains the same conserved motifs in genes that are believed to be interesting antiviral targets (NTPase/helicase, serine protease and RNA-dependent RNA polymerase) as flaviviruses of clinical importance. Phylogenetic analysis of the entire coding region has confirmed the classification of MMLV in the clade of the flaviviruses with no known vector (NKV) and within this clade to the Rio Bravo branch (both viruses have the bat as their vertebrate host). We have provided for the first time a comparative analysis of the RNA folding of the 3' UTR of the NKV flaviviruses (Modoc, Rio Bravo and Apoi viruses, in addition to MMLV). Structural elements in the 3' UTR that are preserved among other flaviviruses have been revealed, as well as elements that distinguish the NKV from the mosquito- and tick-borne flaviviruses. In particular, the pentanucleotide sequence 5' CACAG 3', which is conserved in all mosquito- and tick-borne flaviviruses, is replaced by the sequence 5' C(C/U)(C/U)AG 3' in the loop of the 3' long stable hairpin structure of all four NKV flaviviruses. The availability of this latter sequence motif allows us to designate a virus as either an NKV or a vector-borne flavivirus.

Full-length and partial genome sequences of four members of the genus Aquareovirus, family Reoviridae (Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus) were characterized. Based on sequence comparison, the unclassified Grass carp reovirus was shown to be a member of the species Aquareovirus C. The status of golden ide reovirus, another unclassified aquareovirus, was also examined. Sequence analysis showed that it did not belong to the species Aquareovirus A or C, but assessment of its relationship to the species Aquareovirus B, D, E and F was hampered by the absence of genetic data from these species. In agreement with previous reports of ultrastructural resemblance between aquareoviruses and orthoreoviruses, genetic analysis revealed homology in the genes of the two groups. This homology concerned eight of the 11 segments of the aquareovirus genome (amino acid identity 17-42%), and similar genetic organization was observed in two other segments. The conserved terminal sequences in the genomes of members of the two groups were also similar. These data are undoubtedly an indication of the common evolutionary origin of these viruses. This clear genetic relatedness between members of distinct genera is unique within the family Reoviridae. Such a genetic relationship is usually observed between members of a single genus. However, the current taxonomic classification of aquareoviruses and orthoreoviruses in two different genera is supported by a number of characteristics, including their distinct G+C contents, unequal numbers of genome segments, absence of an antigenic relationship, different cytopathic effects and specific econiches.

Recombination events are known to occur in non-segmented RNA viruses like polioviruses or alphaviruses. Analysis of the subgenomic sequences of dengue virus type 1 (DENV-1) structural genes has recently allowed the identification of possible recombination breakpoints. Because DENV is a major human pathogen, this discovery might have important implications for virus pathogenicity, vaccine safety and efficiency, or diagnosis and, therefore, requires clear confirmation. We report the complete sequence determination of one Asian and two African strains of DENV-1 isolated from human patients. Rigorous sequence analysis provided strong evidence for the occurrence of intragenomic recombination events between DENV-1 strains belonging to different lineages. Singapore S275/90 strain appears to be the evolutionary product of a recombination event between viruses belonging to two distinct lineages: one lineage includes an African strain isolated in Abidjan (Ivory Coast) and the other includes isolates from Djibouti and Cambodia. The 'Recombination Detection Program', bootscanning and analysis of diversity plots provided congruent results concerning the existence of a two-switch recombination event and the localization of recombination breakpoints. Thus, the 5' and 3' genomic ends of the Singapore S275/90 strain were inherited from a Djibouti/Cambodia lineage ancestor and an internal fragment located in the envelope/NS1 region originated from an Abidjan lineage ancestor.

An orbivirus identified as St Croix River virus (SCRV) was isolated from cells of Ixodes scapularis ticks. Electron microscopy showed particles with typical orbivirus morphology. The SCRV genome was sequenced completely and compared to previously characterized orbivirus genomes. Significant identity scores (21-38%) were detected between proteins encoded by segments S1, S2, S4, S5, S6, S8, S9 and S10 of SCRV and those encoded by segments S1, S3, S4, S5, S6, S7, S9 and S10, respectively, of Bluetongue virus (BTV), the prototype orbivirus species. The protein encoded by SCRV genome segment 3 (VP3) is thought to be the equivalent of VP2 of BTV. Segment 7 encodes a protein homologous to non-structural protein NS2(ViP) of BTV. Analysis of VP1(Pol) (segment 1) shows that SCRV is an orbivirus, distantly related to the other sequenced species. Blot hybridizations and sequence comparisons of the conserved protein encoded by genome segment 2 (the T2 subcore shell protein) with previously identified orbiviruses confirm that SCRV is a distinct orbivirus species, unrelated to another tick-borne species, Great Island virus. The presence of SCRV in cells prepared from tick eggs suggests that transovarial transmission of SCRV may occur in ticks.

The phylogenetic relationship between the complete genomic sequences of ten Japanese and one French isolate of TT virus-like mini virus (TLMV) was investigated. Analysis of the variability of the nucleotide sequences and the detection of signature patterns for overlapping genes suggested that ORFs 1 and 2 are probably functional. However, this was not the case for a putative third ORF, ORF3. Throughout the viral genome, several nucleotide or amino acid motifs that are conserved in circoviruses such as TT virus (TTV) and chicken anaemia virus were identified. Phylogenetic analysis distinguished three main groups of TLMV and allowed the identification of putative recombination breakpoints in the untranslated region. TLMV genomes were detected by PCR in the plasma of 38/50 French blood donors tested and were also identified in peripheral blood mononuclear cells, faeces and saliva. A phylogenetic study of 37 TLMV strains originating from France, Japan and Brazil showed that groupings were not related to geographical origin.

Arboviruses with genomes composed of 12 segments of double-stranded (ds) RNA have previously been classified as members or probable members of the genus Coltivirus within the family REOVIRIDAE: A number of these viruses have been isolated in North America and Europe and are serologically and genetically related to Colorado tick fever virus, the Coltivirus type species. These isolates constitute subgroup A of the coltiviruses. The complete genome sequences are now presented of two Asian arboviruses, Kadipiro virus (KDV) and Banna virus (BAV), which are currently classified as subgroup B coltiviruses. Analysis of the viral protein sequences shows that all of the BAV genome segments have cognate genes in KDV. The functions of several of these proteins were also indicated by this analysis. Proteins with dsRNA-binding domains or with significant similarities to polymerases, methyltransferases, NTPases or protein kinases were identified. Comparisons of amino acid sequences of the conserved polymerase protein have shown that BAV and KDV are only very distantly related to the subgroup A coltiviruses. These data demonstrate a requirement for the subgroup B viruses to be reassigned to a separate new genus, for which the name Seadornavirus is proposed.

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 non-vectored flaviviruses. Using a novel protocol for sequence determination, the first complete coding sequence of St Louis encephalitis virus and those of two representative non-vectored flaviviruses, Rio Bravo (isolated from bat) and Apoi (isolated from rodent), are reported. The encoded polyproteins of Rio Bravo and Apoi virus are the smallest described to date within the genus FLAVIVIRUS: The highest similarities with other flaviviruses were found in the NS3 and NS5 genes. The proteolytic cleavage sites for the viral serine protease were highly conserved among the flaviviruses completely sequenced to date. Comparative genetic amino acid alignments revealed that p-distance cut-off values of 0.330-0.470 distinguished the arthropod-borne viruses according to their recognized serogroups and Rio Bravo and Apoi virus were assigned to two distinct non-vectored virus groups. Within these serogroups, cladogenesis based on the complete ORF sequence was similar to trees based on envelope and NS5 sequences. In contrast, branching patterns at the deeper nodes of the tree were different from those reported in the previous study of NS5 sequences. The significance of these observations is discussed.

GB viruses A and C (GBV-A and GBV-C) have been isolated from humans and non-human primates. Phylogenetic analysis based on full-length polyproteins suggests that these two viruses have a common ancestor. It has now been determined that analysis of subgenomic amino acid sequences in the E2 and NS5 regions of GBV-A and a 345 nucleotide segment in the 5' non-coding (5'NC) region was able to reproduce the phylogenetic relationships obtained by complete polyprotein sequences analysis. Using 5'NC sequences from databases, GBV-A isolates were discriminated into eight genetic groups, each one closely associated with specific primate hosts. Phylogenetic analyses performed on sequences from the epsilon-globin genes of primate hosts on one hand and complete polyprotein sequences from GBV-A and GBV-C isolates on the other suggest that a mechanism of cospeciation could be involved in virus evolution over a period of 35 million years.

Sera from French in-patients were tested for the presence of the TT virus (TTV) genome using PCR and degenerate primers located in ORF1. Thirty-six sequences were determined and compared with those deposited in databases, revealing a high degree of genetic variability between TTV isolates (up to 47% for amino acid sequences). Phylogenetic analysis demonstrated the existence of three main groups corresponding to the previously described genotypes 1 and 2 and to a new genotype 3. Isolates could be assigned to distinct genotypes if their genetic distance was > 27%. No comparable genetic criteria were found for the definition of sub-types in the region studied. A 15-31 month follow-up of three haemodialysis patients proved the existence of chronic infection by TTV. In one patient, two strains belonging to different genotypes were detected at the same time. Sequences of both ORF1 and ORF2 remained unchanged for a given strain during the follow-up.

In this study, the basis for the classification of virus isolates grouped within the genus Coltivirus, family Reoviridae, is discussed. Sequences of dsRNA segments from American (segments 9-12), European (segment 12) and Asian (segments 7-12) isolates were characterized and polythetic criteria were defined for their taxonomic classification. These criteria (including sequence analysis) permitted the different species to be distinguished and classified into two groups. In both groups, subgroups were defined according to the degree of homology between the genomic sequences. American and European isolates are classified within group A, which includes subgroups A1 (Colorado tick fever virus species) and A2 (Eyach virus species). Asian isolates are classified in group B, which includes subgroups B1 (JKT-7075 virus species) and B2 (JKT-6423 virus species). The proteins encoded by the sequenced genomic segments were analysed. This allowed the identification of dsRNA binding domains in the proteins encoded by segment 8 of subgroup B1 isolates and segment 12 of subgroup B2 isolates. A conserved pattern of amino acids in segment 7 of group B isolates matched sequences found in the catalytic domains of protein kinases.

The nucleotide sequences of the tenth (M6), eleventh (S1) and twelfth (S2) dsRNA genomic segments of the Florio strain (N-7180) of Colorado tick fever virus were determined and found to be 675, 998 and 1884 bp, respectively, in length. A nonanucleotide motif and a hexanucleotide motif were found to be highly conserved in the 5' and 3' non-coding regions (NCRs), respectively, of the three segments. The first and last three nucleotides of each segment were of inverted complementarity, and segment-specific inverted terminal repeats were detected in the NCRs of the three segments. These findings suggest the occurrence of intracellular panhandle structures for the RNA transcripts. A readthrough phenomenon is suspected in segment M6. The environment surrounding the opal codon (position 1052-1054) of segment M6 conforms to that of leaky opal codons described in the literature.

The phylogenetic status of recently described isolates of hepatitis C virus (HCV) from Vietnam, Thailand and Indonesia (previously classified as types 7, 8, 9, 10 and 11) was re-analysed by the neighbour-joining method instead of the unweighted pair-group method with arithmetic mean (UPGMA) that was first used by the discoverers of these strains. The analysis of complete amino acid sequences and of nucleotide sequences of the envelope 1 (672 nt) and nonstructural 5B (1092 nt) genomic regions permitted the re-assignment of the type 7, 8, 9 and 1 1 isolates to type 6, and that of type 10 strains to type 3. Finally, this study made possible the classification of the previously described HCV strains (including these South-East Asian isolates) in six major types and at least 30 subtypes. It confirms that analysis of the E 1 and NS5B genomic regions using the neighbour-joining method is a reliable tool for the assignment of most new isolates.