Lyssavirus in Japanese Pipistrelle, Taiwan

A putative new lyssavirus was found in 2 Japanese pipistrelles (Pipistrellus abramus) in Taiwan in 2016 and 2017. The concatenated coding regions of the virus showed 62.9%–75.1% nucleotide identities to the other 16 species of lyssavirus, suggesting that it may be representative of a new species of this virus.

Bats are the natural hosts of most lyssaviruses, with the exceptions of MOKV and IKOV, which have not been identified in any bats (1)(2)(3)(4). Information about lyssaviruses in bats in Asia is limited. In Central Asia, ARAV was identified in the lesser mouse-eared bat (Myotis blythi) in Kyrgyzstan in 1991, and KHUV was identified in the whiskered bat (M. mystacinus) in Tajikistan in 2001 (5). In South Asia, GBLV was identified in the Indian flying fox (Pteropus medius) in Sri Lanka in 2015 (3). Although IRKV was identified in the greater tube-nosed bat (Murina leucogaster) in China in 2012 (6), knowledge of the exact species and locations of lyssaviruses in East Asia bat populations remains limited.
In this article, we report a putative new lyssavirus isolated during our surveillance program in Taiwan. Our discovery suggests that this lyssavirus may be representative of a new species, based on genetic distance.

The Study
Specimens for this study were collected under a permit issued by the Forestry Bureau, Council of Agriculture, Executive Yuan, Taiwan (document no. 1055104969). From 2014 through the end of May 2017, a total of 332 bat carcasses from 13 species were collected for lyssavirus surveillance. Of the collected individuals, 2 tested positive for the virus by direct fluorescent antibody testing and reverse transcription PCR (7-9). The first bat showing loss of appetite without specific clinical signs was found in Tainan City and died on July 2, 2016. The second bat was found dead in Yunlin County on April 12, 2017, and the carcass was shipped to the Animal Health Research Institute (AHRI) in New Taipei City. We obtained two 428-bp amplicons (N113F/N304R, containing the partial nucleoprotein [N] gene and phosphoprotein [P] gene) from these cases using lyssavirus screen primers (Table 1); we then subjected their sequences to BLAST (https://www.ncbi.nlm.nih.gov/ BLAST/), querying the GenBank database. Both sequences were similar to lyssaviruses, with nucleotide identities <79%.The 2 bats were identified as Japanese pipistrelle (Pipistrellus abramus), or Japanese house bat, by morphology (J.T. Wu, Taxonomic study of the genus Pipistrellus We isolated lyssavirus successfully from the 2 bats' brains; we confirmed the identity of lyssavirus morphologically by electron microscopy (11) and molecularly by nucleotide sequencing. In addition, after we isolated the viruses from the brain tissues, we performed reverse transcription PCR and virus isolation for visceral organs and salivary glands; both tests were positive for the salivary glands of both bats.

Conclusions
We report a lyssavirus, TWBLV, that is closely related to EBLV-1, IRKV, and DUVV. The full-length nucleotide sequence of the concatenated coding genes of TWBLV showed 62.9%-75.1% nucleotide identities to the other 16 lyssaviruses (Table 2), and TWBLV was the most closely related to EBLV-1. The demarcation criteria of lyssavirus species, established by the International Committee on the Taxonomy of Viruses, include genetic distance, topology, antigenic patterns, and additional characteristics (12). Based on genetic distance, no similarity to the other lyssavirus species of more than 75.1% nucleotide identities of the concatenated coding genes of TWBLVs suggested that the isolated TWBLV was a new lyssavirus species. The presence of TWBLV in the bats' salivary glands suggested that TWBLV may be shed through saliva. The study showed that the bat in East Asia could be infected with lyssavirus; however, because of the limited surveillance, the epidemiology of lyssavirus in Japanese pipistrelle and other bat species is still unclear. This uncertainty is likely to raise a public health concern in countries in Asia.
In conclusion, a lyssavirus, TWBLV, was identified in Japanese pipistrelle, and the infected bats may shed the virus through saliva. Japanese pipistrelle is a common insectivorous bat of low-altitude urban areas in East Asia (13,14). Persons in countries in Asia should be aware to seek proper prophylaxis immediately if bitten by a bat. Studies on the epidemiology and pathogenicity of TWBLV are necessary to further characterize the virus. Bats are sources of high viral diversity and high-profile zoonotic viruses worldwide. Although apparently not pathogenic in their reservoir hosts, some viruses from bats severely affect other mammals, including humans. Examples include severe acute respiratory syndrome coronaviruses, Ebola and Marburg viruses, and Nipah and Hendra viruses. Factors underlying high viral diversity in bats are the subject of speculation. The hypothesis is that flight, a factor common to all bats but to no other mammals, provides an intensive selective force for coexistence with viral parasites through a daily cycle that elevates metabolism and body temperature analogous to the febrile response in other mammals.