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.

The genus Orbivirus includes both insect and tick-borne viruses. The orbivirus genome, composed of 10 segments of dsRNA, encodes 7 structural proteins (VP1–VP7) and 3 non-structural proteins (NS1–NS3). An open reading frame (ORF) that spans almost the entire length of genome segment-9 (Seg-9) encodes VP6 (the viral helicase). However, bioinformatic analysis recently identified an overlapping ORF (ORFX) in Seg-9. We show that ORFX encodes a new non-structural protein, identified here as NS4. Western blotting and confocal fluorescence microscopy, using antibodies raised against recombinant NS4 from Bluetongue virus (BTV, which is insect-borne), or Great Island virus (GIV, which is tick-borne), demonstrate that these proteins are synthesised in BTV or GIV infected mammalian cells, respectively. BTV NS4 is also expressed in Culicoides insect cells. NS4 forms aggregates throughout the cytoplasm as well as in the nucleus, consistent with identification of nuclear localisation signals within the NS4 sequence. Bioinformatic analyses indicate that NS4 contains coiled-coils, is related to proteins that bind nucleic acids, or are associated with membranes and shows similarities to nucleolar protein UTP20 (a processome subunit). Recombinant NS4 of GIV protects dsRNA from degradation by endoribonucleases of the RNAse III family, indicating that it interacts with dsRNA. However, BTV NS4, which is only half the putative size of the GIV NS4, did not protect dsRNA from RNAse III cleavage. NS4 of both GIV and BTV protect DNA from degradation by DNAse. NS4 was found to associate with lipid droplets in cells infected with BTV or GIV or transfected with a plasmid expressing NS4.

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.

The complete nucleotide sequence of Great Island virus (GIV) genome was determined, along with genome segments (Seg) 1, 2 and 6 of Kemerovo (KEMV), Lipovnik (LIPV) and Tribec (TRBV) viruses. All four viruses, together with Broadhaven virus, are currently classified within the species Great Island virus and have been isolated from ticks, birds or humans. Sequence comparisons showed that Seg-4 of GIV encoded the outer-capsid protein responsible for cell attachment, although it was approximately half the length of its counterpart in the Culicoides or mosquitotransmitted orbiviruses. A second overlapping ORF (in the +2 reading frame) was identified in Seg-9 of GIV, encoding a putative dsRNA-binding protein. Phylogenetic analyses of the RNAdependent RNA polymerase (Pol) and T2 protein amino acid sequences indicated that the tickborne orbiviruses represent an ancestral group from which the mosquito-borne orbiviruses have evolved. This mirrors the evolutionary relationships between the arthropod vectors of these viruses, supporting a co-speciation hypothesis for these arboviruses and their arthropod-vectors. Phylogenetic analyses of the T2 proteins of KEMV, LIPV, TRBV and GIV (showing 82 % amino acid identity) correlated with the early classification of Great Island viruses as two distinct serocomplexes (Great Island and Kemerovo serocomplexes). Amino acid identity levels in the VP1(Pol) and T2 proteins between the two serocomplexes were 73 and 82 %, respectively, whilst those between previously characterized Orbivirus species are 53-73 % and 26-83 %, respectively. These data suggest that, despite limited genome segment reassortment between these two groups, their current classification within the same Orbivirus species could be re-evaluated.