Characterization of Nipah Virus from Naturally Infected Pteropus vampyrus Bats, Malaysia

We isolated and characterized Nipah virus (NiV) from Pteropus vampyrus bats, the putative reservoir for the 1998 outbreak in Malaysia, and provide evidence of viral recrudescence. This isolate is monophyletic with previous NiVs in combined analysis, and the nucleocapsid gene phylogeny suggests that similar strains of NiV are co-circulating in sympatric reservoir species.

at 98 nt positions; these nucleotide changes translated into amino acid changes at 44 positions. Subsequent analysis of the deduced amino acid sequences of the open reading frames of the nucleocapsid, phosphoprotein, matrix, fu-sion, attachment, and polymerase genes showed high sequence similarities (98%-99%) between nucleocapsid, matrix, fusion, attachment, and polymerase proteins of NiV P. vampyrus and other previously sequenced NiV isolates from Malyasia. However, phosphoprotein shares the lowest homology (96%). Table 2 shows a summary of the specifi c deduced amino acid changes compared with other NiV sequences.
Phylogenetic analyses were generated by using maximum-likelihood methods (8). Sequences were analyzed with henipavirus sequences available in GenBank. The analysis of the combined nucleotide dataset shows that NiV P. vampyrus forms a monophyletic clade with other NiV isolates from Malaysia, yet it differs from human, pig, and P. hypomelanus bat isolates. NiV from humans in Bangladesh is more distantly related and basal to all NiV sequences from Malaysia ( Figure 1). This relationship is further supported by the polymerase gene analysis (data not shown). When the nucleocapsid gene alone was analyzed, including 56 NiV sequences from P. lylei bats in Thailand, NiV P. vampyrus phylogenetically grouped most closely with NiV P. lylei (AY858110), and the monophyly of NiV sequences from Malysia was lost ( Figure 2). This sister relationship between NiV P. lylei and NiV P. vampryus is also evident in analysis of the atttachment gene (data not shown).

Conclusions
Our evidence suggests that NiV can recrudesce in previously infected adult bats, thus providing a new potential mechanism for maintenance in natural hosts. We isolated NiV from a seropositive adult bat at the time of capture, and therefore it was unlikely to have had remnant maternal antibodies. Also, it is unlikely that infection could have been introduced by other wild bats because other bats could not access the enclosure where the study bats were kept. The antibodies waned during the bat's captivity, and subsequent seroconversion correlated with our fi nding of NiV in the individual bat's urine. The bats were isolated from contact with wild bats, and all other bats placed in the colony were negative for henipavirus by culture; only bats 24, 38, and 48 subsequently seroconverted. Recrudescence of NiV infection in bats is not completely unexpected because NiV infection has resulted in (fatal) relapsing illness in humans, several months to 4 years after initial exposure (9). Other paramyxoviruses, including canine distemper (10) and measles virus (11), can persist in tissues for some years.
The seroconversion of the 3 bats in this colony is consistent with recent viral challenge (6) and a scenario in which bat 24 underwent recrudescence of a latent infection. The seroconversion supports the conclusion that bats 38 and 48 were infected through exposure to urine, feces, or saliva from bat 24.
NiV was not isolated from the 2 male bats, which may have been because of a low amount of virus excreted, a very narrow time frame for excretion, or both; however, these fi ndings suggest that they did not undergo recrudescence. Evidence for NiV recrudescence adds to our understanding of henipavirus ecology and transmission dynamics. Repeated shedding of NiV through recrudescence may enhance viral maintenance in isolated colonies without the boom and bust dynamics, typical of acute viral infections with long-term immunity and reduce the necessity of intercolony migration for maintenance.
Our phylogenetic analyses help address some longstanding questions regarding the natural history of henipaviruses (12). Close homology between NiV P. vampyrus and a NiV P. lylei isolates and evidence from nucleocapsid and polymerase gene analysis suggest that NiV is naturally transmitted between these 2 species, which roost together in Thailand and parts of Cambodia. Furthermore, NiV diversity in isolates obtained from P. lylei bats demonstrates that multiple strains co-circulate within populations and that the ecology and sympatry of Pteropus spp., not coevolutionary patterns, determine NiV strain diversity in reservoir hosts.
During the 1998 outbreak, NiV isolates from P. hypomelanus bats was found to be nearly identical to those from pigs and humans (5 6 nt changes) (13,14). However, this species is only found on offshore islands, has limited dispersal, and does not overlap with the index farms. Thus, P. vampyrus bats are likely the putative spillover hosts in Malaysia, not P. hypomelanus bats; nonetheless, our isolate differed markedly from others in the outbreak. Because laboratory contamination of the NiV P. hypomelanus isolate seems unlikely (14), the co-circulation of multiple strains in P. vampyrus bats is probable. Alternatively, the differences we observed in NiV P. vampyrus may be the result of rapid RNA virus evolution during the 7-year period between sampling of NiV P. vampyrus (2005) and sampling of the other isolates from Malaysia (1998)(1999). Our data support this hypothesis. Assuming a known henipavirus genome length of ≈18,000 nt (15), the average substitution rate for Paramyxoviridae of 0.50 × 10 -3 substitutions/ site/year, and a constant molecular clock, the 98-nt changes observed correspond broadly to the time frame (≈7 years) between sampling of these isolates.