Influence of age and body condition on astrovirus infection of bats in Singapore: An evolutionary and epidemiological analysis

Mendenhall, Ian H.; Skiles, Maggie M.; Neves, Erica Sena; Borthwick, Sophie A.; Low, Dolyce H.W.; Liang, Benjamin; Lee, Benjamin P.Y.-H.; Su, Yvonne C.F.; Smith, Gavin J.D.

As Astroviruses replicate in the gastro-intestinal tract and are Table 2 Pooled prevalence estimates using frequentist and Bayesian methods with a Gibbs sampler compared to individual prevalence rates. fecally-orally transmitted, the relative viral load may be higher in feces compared to swabs. This may explain our higher detection rate from E. spelaea feces, but as bat viruses may be shed differentially over time, we cannot exclude changes in viral load affecting our results. We found variable rates of shedding across our sampling period. In Singapore, temperatures are not variable month to month, but there are two monsoon seasons, which may be associated with bat borne virus shedding [43] . A study on Myotis myotis in Germany demonstrated distinct astrovirus amplification peaks that correlated with the formation of a suitable roost size and also a post-parturition period [45] . In the tropics, where there is lower seasonal variation and an absence of torpor, the reproductive behavior of bats may drive virus persistence [46] . This may not always be the case as astrovirus and coronavirus shedding in Borneo was not associated with reproductive status [43] . We analyzed the astrovirus RdRp sequences from a broad range of hosts including bats and non-human primates [44, 47, 48] . Consistent with previous studies, our results indicate that bats harbor a genetically diverse group of astroviruses that are typically polyphyletic and predominantly situated in a basal position of the phylogenetic tree. Interestingly, we detected diverse lineages of bat astroviruses in our [27] [28] [29] [30] [31] [32] [33] samples that were collected from a small geographic area. We demonstrated that the astroviruses from Rhinolophus lepidus and Eonycteris spelaea in Singapore were phylogenetically distinct and clearly segregated into two distantly-related lineages. This is reflected by the low percentage (55.3%-56.9%) of nucleotide sequence identity between Rhinolophus and Eonycteris RdRp sequences. We also observed that the E. spelaea astroviruses from Singapore and Laos exhibited high levels of sequence variation and at least two clades were identified. Clade 1 is genetically more diverse than Clade 2, and further split into smaller sub-groups. This includes all E. spelaea astroviruses collected from Singapore, although these are not monophyletic. The majority of E. spelaea astroviruses detected in Singapore are more closely related with Rousettus astroviruses from Laos. As Rousettus and Eonycteris are commonly encountered in the same roosting environment, and there is evidence that Rousettus bat coronaviruses can infect other genera of bats [49] , it is unsurprising that closely related astroviruses were detected in these species. In addition, we observed that some astroviruses of E. spelaea in Singapore are closely related with Pteropus giganteus astroviruses detected in Bangladesh. The close genetic relationship of these viruses from Bangladesh, Laos and Singapore indicates an important role for bats in the dispersal of astroviruses over a wide geographical range, consistent with previous observations for lineage D betacoronaviruses [30] . Clade 2 is composed of astroviruses from several bat species from Cambodia and Laos that are most closely related to astroviruses from other mammalian species. Notably, E. spelaea astroviruses from Singapore (Clade 1) are only distantly related to E. spelaea astroviruses from Laos (Clade 2), indicating these astroviruse