A Strategy To Estimate Unknown Viral Diversity in Mammals

Anthony, Simon J.; Epstein, Jonathan H.; Murray, Kris A.; Navarrete-Macias, Isamara; Zambrana-Torrelio, Carlos M.; Solovyov, Alexander; Ojeda-Flores, Rafael; Arrigo, Nicole C.; Islam, Ariful; Ali Khan, Shahneaz; Hosseini, Parviez; Bogich, Tiffany L.; Olival, Kevin J.; Sanchez-Leon, Maria D.; Karesh, William B.; Goldstein, Tracey; Luby, Stephen P.; Morse, Stephen S.; Mazet, Jonna A. K.; Daszak, Peter; Lipkin, W. Ian

Samples and PCR screening. Samples (n ϭ 1,897) were collected from apparently healthy P. giganteus bats throughout Bangladesh between 2006 and 2010, as described previously (48) . This included urine (n ϭ 926), throat swabs (n ϭ 806), feces (n ϭ 78), and roost urine (n ϭ 97). All samples were collected by trained veterinarians, and all animals were released unharmed. Samples were collected directly into lysis buffer (bio-Mérieux, Inc.) and stored at Ϫ80°C until transfer to the Center for Infection and Immunity at Columbia University. Roost urine samples were also obtained by suspending 3-by 2-m polyethylene sheets underneath roosting colonies, which collected urine (with possible fecal contamination) from the bats roosting above. Total nucleic acid was extracted from all samples using the EasyMag (bioMérieux, Inc.) platform, and cDNA synthesis performed using SuperScript III first-strand synthesis supermix (Invitrogen), all according to the manufacturer's instructions. Viral discovery was performed using broadly reactive consensus PCR assays targeting coronaviruses (49) , paramyxoviruses (50) , astroviruses (51), influenza A viruses (38) , adenoviruses (52) , polyomaviruses (53), bocaviruses (54) , and herpesviruses (55) . Consensus primers for hantaviruses were modified from an existing protocol (56) in order to increase the degeneracy of the assay, and the assay validated for its ability to detect diverse hantaviruses, including Andes, Puumala, Sin Nombre, Prospect Hill, Seoul, and Thottapalayam hantaviruses. The modified primer sequences were UHantaF1 (GWGGVCARACWGCHGAYT) and UHantaR1 (CCW GGTGTDADYTCHTCWGC) (expected amplicon, 250 bp), and the annealing temperature was 52°C. All PCR products of the expected size were cloned into Strataclone PCR cloning vector, and 12 white colonies sequenced using standard M13R primers.