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.

To search for candidate IBD etiologic agents, we performed an unbiased high-throughput metagenomic analysis. RNA was extracted from frozen brain, lung, liver, kidney, heart, and gastrointestinal (GI) tissue from the animals for which multiple tissues were available (CAS02 to CAS07; Table 1 ), and libraries were prepared for deep sequencing (see Materials and Methods). Of these six animals, five had been diagnosed as IBD positive, and one (CAS04) was negative. Sequencing on the Illumina HiSeq platform generated approximately 1 Ï« 10 8 pairs of 100-nucleotide (nt) sequences, on average~6 million sequences for each of the 35 samples (no sample was available from the heart of snake CAS07). The complete data set for all samples is available from the NCBI Short Read Archive (accession no. SRA053624). To facilitate the search for viral sequences, we first removed low-quality, lowcomplexity, and host-derived sequences. To remove host sequences, including those deriving from possibly confounding endogenous retroviruses, we removed reads matching the recently sequenced boa constrictor genome (assembly no. 1 [16] ). These operations reduced the size of the data sets by an average of 93%. Identification of two distinct arenavirus-like genomes. With the remaining sequences, we performed BLASTX searches against a database of viral protein sequences (17) . This search revealed the presence of substantial numbers of sequences with similarity to arenavirus protein sequences in all of the IBD-diagnosed samples. We used these BLAST hits to nucleate complete genome assemblies using the PRICE de novo genome assembler (G. Ruby, freely available at http://derisilab.ucsf.edu/software/price/index.html). This analysis revealed that there were actually two distinct (59% pairwise nucleotide identity) but related viruses in the snakes from the aquarium: one from the IBD-positive annulated tree boas and one from the IBD-positive boa constrictors (Table 1 ; Fig. 2