Journal of General Virology (2002), 83, 2443–2454. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Genome sequence analysis of Tamana bat virus and its relationship with the genus Flavivirus X. de Lamballerie, 1 S. Crochu, 1 F. Billoir, 1 J. Neyts, 2 P. de Micco, 1 E. C. Holmes 3 and E. A. Gould 4 1 Unite ! des Virus Emergents, EA3292-IFR48, Universite ! de la Me ! diterrane ! e, Faculte ! de Me ! decine, 27 Bd J. Moulin, F13005 Marseille, France 2 Rega Institute for Medical Research, Minderbroedersstraat 10, K. U. Leuven, B-3000 Leuven, Belgium 3 Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK 4 Centre for Ecology and Hydrology, Mansfield Road, Oxford OX1 3SR, UK 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. Introduction Tamana bat virus (TABV) was isolated in 1973 by J. L. Price in Trinidad from the salivary glands, saliva and spleen of the insectivorous bat Pteronotus parnellii (Price, 1978). Some characteristics of the virus (sensitivity to ether, pathogenicity for suckling mice and ability to haemagglutinate goose erythrocytes) were those of (enveloped) ‘ arboviruses ’, i.e. viruses currently classified as flaviviruses or alphaviruses, but no evidence for the existence of an arthropod vector could be found. Despite extensive investigations, no serological re- Author for correspondence : X. de Lamballerie. Fax ›33 4 91 32 44 95. e-mail xndl-virophdm!gulliver.fr The GenBank accession number for the complete sequence of the TABV ORF is AF285080. lationship with known arboviruses or other viruses could be detected and TABV remained unclassified. More recently, Kuno et al. (1998) published electron micrographs of TABV propagated in Vero cells that showed virus particles with the typical size and morphology of flaviviruses. However, a combination of PCR primers designed by these authors with the ability to amplify a portion of the NS5 gene of a large variety of flaviviruses yielded no amplicon when applied to TABV. Therefore, after more than 25 years and both serological and molecular investigations, the taxonomic pos- ition of TABV remains undetermined and quite intriguing. Here, we present the complete coding sequence of this hitherto unclassified virus and report our attempts to elucidate the genetic structure and evolutionary relationships of TABV. This analysis allowed us to identify TABV as a new and divergent member of the family Flaviviridae and had implications for the 0001-8388 # 2002 SGM CEED