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.

Fecal samples were vortexed for 15 s and then centrifuged at 4000 rpm for 1 min. Oral and rectal swabs were vortexed and then pooled for each individual. Samples collected between 12 April 2011 to 2 March 2012 were pooled by date (5 individual samples per pool) to test for pooled prevalence. RNA extraction cDNA synthesis, PCR amplification, cloning and sequencing were performed as previously described [31, 32] . 5′ and 3′ RACE was attempted to generate cDNA from the ends (Life Technologies, Carlsbad, CA), but neither approach was successful. RACE generally requires high amounts and high quality of target nucleic acids and these fecal samples may have relatively few astrovirus reads compared to the host and bacterial background. Over 700 global astrovirus RdRp sequences were downloaded from the NCBI GenBank and analyzed with 38 bat astrovirus sequences generated from this study (GenBank accession numbers MF983815-MF983852) using Geneious 7.1.7 software [33] . The final dataset was reduced to a total of 449 astrovirus sequences after the removal of short sequences and phylogenies were reconstructed using RAxML v8.0.14 [34] as previously described [30] .