Identification, Characterization, and In Vitro Culture of Highly Divergent Arenaviruses from Boa Constrictors and Annulated Tree Boas: Candidate Etiological Agents for Snake Inclusion Body Disease

Stenglein, Mark D.; Sanders, Chris; Kistler, Amy L.; Ruby, J. Graham; Franco, Jessica Y.; Reavill, Drury R.; Dunker, Freeland; DeRisi, Joseph L.

Sanger sequencing and RACE. Virus genome sequences assembled from deep sequencing reads were validated using Sanger sequencing and RACE. Primer sequences are listed in Table S1 in the supplemental material. PCR mixtures contained 1ϫ reaction buffer, 0.25 M primer, 0.2 mM dNTPs, 2 U Taq DNA polymerase, and 0.25 l cDNA. Thermocycling consisted of 95°C for 2 min and then 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 2 min. Amplicons were purified from agarose gels using the PureLink gel extraction kit (Invitrogen) and cloned into the pCR2.1 TOPO vector (Invitrogen) according to the manufacturer's protocols. Cloned amplicons were Sanger sequenced (Quintara Biosciences). Because the viral genome segments are predicted to exist in genomic and antigenomic forms, 5= RACE was used to obtain both end sequences, essentially as described elsewhere (63), with primers listed in Table S1 . Multiple RACE amplicons were cloned and sequenced as described above. In cases where RACE amplicons did not extend to the end of the deep sequencing assemblies, the deep sequence coverage was sufficient to determine the terminal sequences. The complete genome sequences of CASV and GGV have been deposited with the NCBI under accession numbers JQ717261 to JQ717264. Antibody production. A peptide corresponding to the C-terminal 14 amino acids (SGGKKTKDPTPATI) of the nucleoprotein of GGV was synthesized and injected into rabbits for polyclonal antibody production (Pacific Immunology).