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.

Data sets did not contain consistent evidence of other possible bacterial or viral pathogens. Data sets contained retrovirus-like sequences most similar to that of Python molurus endogenous retrovirus (ERV) sequences (56) . These were present in nearly every case and control Python regius data set and most likely de- rived from ball python ERVs. Also, as with all metagenomic data sets from nonsterile sites, bacterial sequences were present. However, no particular bacterial taxon was associated with cases, in contrast to the nidovirus sequences, which were perfectly associated. Nevertheless, from these sequencing data sets alone, it is not possible to formally rule out a role in disease causality by other viral or bacterial pathogens. We next sought to determine the distribution of viral load within the tissues of individual infected snakes. Frozen samples from multiple tissues were available for 4 snakes. We extracted RNA from these tissues and used qRT-PCR to quantify viral RNA relative to expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (Fig. 6B) . Consistent with the pathology observed, viral load was consistently highest in respiratory tract tissue. Viral RNA levels were at approximately the same level in intestine-derived RNA from the single snake for which this tissue was available (no. 11), a finding consistent with respiratory/enteric tropism of many viruses in the Coronaviridae family (7, 8) . Viral RNA was also detectable in other tissues (liver, kidney, heart, spleen, and brain) at levels that were 3 to 5 orders of magnitude lower than in the lung. While these results support the notion that the respiratory tract is the primary location of viral replication, consistent with the disease pathology, additional systematic collection and analysis of samples from diseased animals will be necessary to definitively characterize tissue tropism for this virus.