Ball Python Nidovirus: a Candidate Etiologic Agent for Severe Respiratory Disease in Python regius

Stenglein, Mark D.; Jacobson, Elliott R.; Wozniak, Edward J.; Wellehan, James F. X.; Kincaid, Anne; Gordon, Marcus; Porter, Brian F.; Baumgartner, Wes; Stahl, Scott; Kelley, Karen; Towner, Jonathan S.; DeRisi, Joseph L.

Phylogenetic analysis. We performed phylogenetic analyses to understand the evolutionary relationship between this and other nidoviruses. We obtained protein sequences from 33 representa-tive species, and created multiple sequence alignments of the relatively conserved protease, RdRp, and helicase domains of pp1ab (see Table S3 and Fig. S5 in the supplemental material) (42) . Bayesian and maximum-likelihood (ML) phylogenies based on individual domain and concatenated alignments had nearly identical topologies ( Fig. 7 ; see also Fig. S6 ). In these phylogenies, the 5 major groups of nidoviruses (Torovirinae, Coronavirinae, Roniviridae, Mesoniviridae, and Arteriviridae) formed well-separated branches. Ball python nidovirus (BPNV) formed a monophyletic clade with the viruses in the Torovirinae subfamily of the Coronaviridae family. The snake virus is approximately equally distant from the two genera in the subfamily, the toroviruses, which infect mammals, and the bafiniviruses, which infect ray-finned fish ( Fig. 7 ; see also Fig. S6 ). We noted in the protease alignment that two putative active site motifs previously identified by Ulferts et al. (57) , one containing a histidine and a GX[S/C] G motif, aligned in all sequences. A third putative Asp/Glu-containing motif shifted drastically in alignments, even within Coronavirinae. Nevertheless, the topology of the tree generated using the protease alignment was in close agreement with other phylogenies. This analysis did not find a monophyletic grouping of the Torovirinae and Coronavirinae subfamilies of the family Coronaviridae. This paraphyly was found to be significant (P Ͻ 0.01) by log likelihood (Shimodaira-Hasegawa) testing (58) .