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A search for the natural host of Nipah virushas led to the isolation of a previously unknown member of the family Paramyxoviridae. Tioman virus(TiV) was isolated from the urine of fruit bats (Pteropus hypomelanus) found on the island of the same name off the eastern coast of peninsular Malaysia. An electron microscopic study of TiV-infected cells revealed spherical and pleomorphic-enveloped viral particles (100–500 nm in size) with a single fringe of embedded peplomers. Virus morphogenesis occurred at the plasma membrane of infected cells and morphological features of negative-stained ribonucleoprotein complexes were compatible with that of viruses in the family Paramyxoviridae.Serological studies revealed no cross-reactivity with antibodies against a number of known Paramyxoviridaemembers except for the newly described Menangle virus(MenV), isolated in Australia in 1997. Failure of PCR amplification using MenV-specific primers suggested that this new virus is related to but different from MenV. For molecular characterization of the virus, a cDNA subtraction strategy was employed to isolate virus-specific cDNA from virus-infected cells. Complete gene sequences for the nucleocapsid protein (N) and phosphoprotein (P/V) have been determined and recombinant N and V proteins produced in baculovirus. The recombinant N and V proteins reacted with porcine anti-MenV sera in Western blot, confirming the serological cross-reactivity observed during initial virus characterization. The lack of a C protein-coding region in the P/V gene, the creation of P mRNA by insertion of 2-G residues, and the results of phylogenetic analyses all indicated that TiV is a novel member of the genus Rubulavirus.