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.

We then used PCR, rapid amplification of cDNA ends (RACE), and Sanger sequencing to validate the assemblies. Retrospective mapping of the sequences revealed that the individual IBDpositive tissue data sets contained between 8,422 and 227,134 viral sequences (between 0.13% and 3.8% of the total reads). Overall genome coverage ranged from 825-fold to 3,335-fold (the average number of sequences covering each base). The complete genomes for both viral species are available from GenBank (accession numbers JQ717261 to JQ717264). The data sets from IBD-negative CAS04 contained very low frequencies of sequences mapping to the two viruses (Ͻ5 ϫ 10 Ϫ5 ), a phenomenon that was also observed for the boa constrictor virus in the annulated tree boa samples and vice versa. These low-copy-number sequences likely resulted from intersample cross-contamination or bar code misregistration during sequencing (18) . This explanation was corroborated by quantitative reverse transcription-PCR (qRT-PCR), which demonstrated that all tissues of snake CAS04 were negative for viral RNA (there was no amplification) (Fig. 2 ). This analysis also confirmed that the two viruses segregated perfectly with the two snake species. By sequencing and by quantitative PCR (qPCR), viral RNA was detected in every tissue examined, a pattern reminiscent of the histopathological detection of inclusions in all of these tissues (Fig. 2) .