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.

CASV and GGV encode a predicted transmembrane glycoprotein at the same genomic position as arenavirus GPCs. However, like Z, these proteins did not contain detectable homology to arenavirus glycoproteins as measured by BLAST (versus nr) or HHPred. Instead, by BLASTP analysis the snake virus proteins were related to the glycoproteins of filoviruses (e.g., Ebola and Marburg viruses) and avian retroviruses (e.g., avian leukosis virus) and the cellular syncytin, a repurposed endogenous retroviral gene (Fig. 3D) (35, 36) . The predicted GPs of CASV and GGV are 393 and 427 amino acids long and, like other class 1 viral fusion proteins, may be proteolytically cleaved into two functional domains: the GP1 receptor binding and the GP2 transmembrane/ fusion domains (37, 38) . Amino acids 222 to 393 of CASV GP and 256 to 427 of GGV GP are 82% identical. This relatively conserved domain likely corresponds to the GP2 domain and was the region with detectable similarity by BLAST. Using HHPred, the top hit for this domain of both CASV and GGV was to the Sudan Ebola virus GP2 (PDB accession no. 3S88; probability of homologous relationships, 99.98%). Like filovirus GP2s, the CASV and GGV domains are predicted to form C-terminal transmembrane do-mains that anchor the protein in the viral membrane. In contrast to the relatively conserved GP2 domain, the predicted N-terminal GP1 domains of CASV and GGV glycoproteins are only 31% identical, and neither domain contains detectable homology to known proteins (by BLASTP versus nr with an E value of Ͻ1). Analysis of the predicted GP1/GP2 boundaries did not reveal obvious candidate protease cleavage sites present in both the GGV and CASV sequences. In vitro virus culture. To enable further characterization of these viruses, we developed in vitro cell culture growth conditions that permitted virus propagation. We first attempted to infect reptile cell lines available from the ATCC (viper heart VH-2 and iguana heart IGH-2). Inoculum was prepared from the kidney and liver of the boa constrictors CAS06 and CAS07, the samples from which sufficient frozen tissue remained. Viral RNA levels in culture supernatant were monitored by qRT-PCR. Viral RNA was initially detectable in the supernatant but rapidly disappeared as culture medium was replaced and was undetectable by 7 days postinoculation and over the course of 18 days. We observed similar negative results with an African green monkey-derived Vero cell line, known to be permissive for the replication of many arenaviruses. This suggested that these lines or the culture conditions were not permissive for replication or that the inoculum that we used lacked replication-competent virus.