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

Abstract

Inclusion body disease (IBD) is an infectious fatal disease of snakes typified by behavioral abnormalities, wasting, and secondary infections. At a histopathological level, the disease is identified by the presence of large eosinophilic cytoplasmic inclusions in multiple tissues. To date, no virus or other pathogen has been definitively characterized or associated with the disease. Using a metagenomic approach to search for candidate etiologic agents in snakes with confirmed IBD, we identified and de novo assembled the complete genomic sequences of two viruses related to arenaviruses, and a third arenavirus-like sequence was discovered by screening an additional set of samples. A continuous boa constrictor cell line was established and used to propagate and isolate one of the viruses in culture. Viral nucleoprotein was localized and concentrated within large cytoplasmic inclusions in infected cells in culture and tissues from diseased snakes. In total, viral RNA was detected in 6/8 confirmed IBD cases and 0/18 controls. These viruses have a typical arenavirus genome organization but are highly divergent, belonging to a lineage separate from that of the Old and New World arenaviruses. Furthermore, these viruses encode envelope glycoproteins that are more similar to those of filoviruses than to those of other arenaviruses. These findings implicate these viruses as candidate etiologic agents of IBD. The presence of arenaviruses outside mammals reveals that these viruses infect an unexpectedly broad range of species and represent a new reservoir of potential human pathogens.

Importance: Inclusion body disease (IBD) is a common infectious disease of captive snakes. IBD is fatal and can cause the loss of entire animal collections. The cause of the disease has remained elusive, and no treatment exists. In addition to being important to pet owners, veterinarians, breeders, zoological parks, and aquariums, the study of animal disease is significant since animals are the source of virtually every emerging infectious human disease. We searched for candidate causative agents in snakes diagnosed with IBD and found a group of novel viruses distantly related mainly to arenaviruses but also to filoviruses, both of which can cause fatal hemorrhagic fevers when transmitted from animals to humans. In addition to providing evidence that strongly suggests that these viruses cause snake IBD, this discovery reveals a new and unanticipated domain of virus biology and evolution.

FIG 1

Histology showing eosinophilic inclusions in IBD-positive tissue samples. Liver and kidney frozen tissue sections from boa constrictors subsequently diagnosed with or without IBD were H&E stained and imaged. Nuclei appear blue/purple, and eosinophilic structures, including cytoplasmic inclusions (some of which are marked with arrowheads), appear orange/brown. Bars, 10 µm.

FIG 2

Viral RNA is detected in all tissues of IBD-diagnosed snakes. The 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.

FIG 3

New viruses mix arenavirus characteristics and filovirus-like glycoproteins. (A) Genome organization of the viruses recovered from CAS (California Academy of Sciences) snakes. Features are to scale, and segments are in the predicted genomic sense. Predicted coding regions and intergenic hairpins are depicted. (B) An alignment of the terminal 19 5′ nucleotides of the indicated genome (vRNA) or antigenome (vcRNA) segments. Variable residues are highlighted. For comparison, terminal sequences from three reference rodent arenaviruses are shown: TCRV (NC_004292.1), LASV (NC_004296.1), and LCMV (NC_004294.1). (C) Matrices of pairwise percent amino acid identity between snake viruses and reference rodent arenaviruses. Values are based on multiple sequence alignments generated using ClustalW software (see Materials and Methods). Sequence accession numbers are the same as those for panel B. (D) Phylogenies of predicted snake virus NP, L, and GP2 protein sequences and related sequences. Old World and New World designate major clades of previously described arenaviruses. Bars, 0.2 substitutions per site. The bootstrap percentages for select nodes are indicated.

FIG 4

Viral replication in a boa constrictor continuous cell line. Relative levels of viral L and S segment RNA measured by qRT-PCR in the supernatant of JK cells inoculated with homogenate from snake CAS06 kidney and liver. Inset, image of JK cells.

FIG 5

An antibody to the viral nucleoprotein stains cytoplasmic aggregates in infected cells and tissues. (A) Western blot with a polyclonal anti-NP peptide antibody against infected and mock-infected JK cells and against liver and kidney tissue homogenates from one uninfected and two infected boa constrictors. Numbers represent molecular masses in kDa. (B) Immunofluorescence using the same antibody on infected and mock-infected JK cells. Images show merged Hoechst stain and anti-NP signals. Bar, 10 µm. (C) Immunofluorescence of liver and kidney tissue sections from an infected and an uninfected boa constrictor. Rows 2 and 4 show the boxed areas of the images in rows 1 and 3. Bars, 50 µm (rows 1 and 3) and 10 µm (rows 2 and 4).

FIG 6

In infected tissues, viral nucleoprotein localizes to eosinophilic inclusion bodies. The same tissue sections were first imaged by fluorescent microscopy and then H&E stained and reimaged. Rows 2 and 4 show the boxed areas of the images in rows 1 and 3. Closed arrowheads, eosinophilic inclusions fully staining; open arrowheads, inclusions ringed by NP fluorescence (see text). Bars, 50 µm (rows 1 and 3) and 10 µm (rows 2 and 4).

FIG 7

Models for evolution of arenavirus glycoprotein genes. S segments of extant and presumed ancestral arenaviruses are shown. See text for additional explanation. O/NWA, Old/New World arenavirus.