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.

The recovery of high numbers of arenavirus-related sequences from the IBD-diagnosed snakes but not the single IBD-negative relative amount of L segment viral RNA detected by qRT-PCR for each tissue in each snake is shown. Results are normalized to the detected levels of two snake mRNAs (glyceraldehyde-3-phosphate dehydrogenase and RPS2). Virtually identical results were obtained for S segment RNA. Each bar represents one tissue: from left to right, brain, GI tissue, heart, kidney, liver, lung, serum, and blood cells. The snakes' histopathology-based IBD diagnoses are indicated. Snake designations are given in Table 1 . A sample was unavailable for CAS07 heart tissue, and data were unavailable for the CAS06 kidney. snake suggested that the viruses might play a role in disease pathogenesis (Fig. 2) . To investigate this possibility, we obtained and screened 21 additional samples from IBD-positive and -negative snakes of various species for viral RNA (Table 1) . We performed RT-PCR with multiple sets of primers, including consensus primers designed to amplify sequence from both novel viruses, and by additional deep sequencing in some cases. A third related virus was detected by RT-PCR in a boa constrictor from Collierville, TN, diagnosed with IBD in 2008. Although the complete genome of this third virus has not yet been fully determined, alignments of the recovered sequences reveal that this third virus is more closely related (~80% nucleotide identity) to the virus recovered from the CAS boa constrictors (see Fig. S1 in the supplemental material). Viral RNA was not detected in the other three confirmed IBD cases. Thus, 6/8 IBD-positive samples were positive for one of these divergent but related viruses. We did not detect viral RNA in any of 18 control samples (Table 1 ). These results show that multiple related arenavirus-like viruses are associated with geographically and temporally widespread IBD cases and also reveal a high degree of diversity in this clade. Until all available samples can be analyzed by deep sequencing, we cannot rule out the presence of additional divergent species in the samples that have tested negative by RT-PCR.