Boid Inclusion Body Disease Is Also a Disease of Wild Boa Constrictors

Abstract

Reptarenaviruses cause boid inclusion body disease (BIBD), a potentially fatal disease, occurring in captive constrictor snakes boas and pythons worldwide. Classical BIBD, characterized by the formation of pathognomonic cytoplasmic inclusion bodies (IBs), occurs mainly in boas, whereas in pythons, for example, reptarenavirus infection most often manifests as central nervous system signs with limited IB formation. The natural hosts of reptarenaviruses are unknown, although free-ranging/wild constrictor snakes are among the suspects. Here, we report BIBD with reptarenavirus infection in indigenous captive and wild boid snakes in Costa Rica using histology, immunohistology, transmission electron microscopy, and next-generation sequencing (NGS). The snakes studied represented diagnostic postmortem cases of captive and wild-caught snakes since 1989. The results from NGS on archival paraffin blocks confirm that reptarenaviruses were already present in wild boa constrictors in Costa Rica in the 1980s. Continuous sequences that were de novo assembled from the low-quality RNA obtained from paraffin-embedded tissue allowed the identification of a distinct pair of reptarenavirus S and L segments in all studied animals; in most cases, reference assembly could recover almost complete segments. Sampling of three prospective cases in 2018 allowed an examination of fresh blood or tissues and resulted in the identification of additional reptarenavirus segments and hartmanivirus coinfection. Our results show that BIBD is not only a disease of captive snakes but also occurs in indigenous wild constrictor snakes in Costa Rica, suggesting boa constrictors to play a role in natural reptarenavirus circulation. IMPORTANCE The literature describes cases of boid inclusion body disease (BIBD) in captive snakes since the 1970s, and in the 2010s, others and ourselves identified reptarenaviruses as the causative agent. BIBD affects captive snakes globally, but the origin and the natural host of reptarenaviruses remain unknown. In this report, we show BIBD and reptarenavirus infections in two native Costa Rican constrictor snake species, and by studying archival samples, we show that both the viruses and the disease have been present in free-ranging/wild snakes in Costa Rica at least since the 1980s. The diagnosis of BIBD in wild boa constrictors suggests that this species plays a role in the circulation of reptarenaviruses. Additional sample collection and analysis would help to clarify this role further and the possibility of, e.g., vector transmission from an arthropod host.

Keywords: arenavirus; boa constrictor; boid inclusion body disease; inclusion body disease; veterinary microbiology; viral pathogenesis; wild snake.

FIG 1

Animal C5, captive annulated tree boa (C. annulatus) with BIBD. (A, B) Brain stem. Cytoplasmic inclusion bodies (IBs) (arrows) in neurons that are comprised of reptarenavirus nucleoprotein (B). (C) Pancreas with IBs in epithelial cells (arrows). There are also individual cells with diffuse cytoplasmic viral antigen expression (right image, arrowhead). (D) Liver with IBs in hepatocytes (arrowheads). (E) Blood smear. Red blood cells with IBs (arrowheads) and hemogregarine infection (arrow). May-Grünwald-Giemsa stain. (A, C [left], and D) HE stain; (B, C [right]) immunohistology, hematoxylin counterstain. Bars = 20 μm.

FIG 2

Gross findings in two wild-caught boa constrictors that were euthanized due to incurable disease. (A, B) Animal W3. (A) Head with severe traumatic injuries. (B) Liver with focal granulomatous inflammation in association with urate crystals (gout). (C) Animal W4. Head with multifocal necrotic dermatitis (arrows).

FIG 3

Wild-caught boa constrictor (animal W5) with BIBD. Inclusion body (IB) formation and reptarenavirus nucleoprotein (NP) expression in tissues. (A, B) Cerebral cortex with abundant cytoplasmic IBs in neurons. Some neurons exhibit more than one IB (arrowheads), and others show large square IBs (A, arrows) in which reptarenavirus NP expression is restricted to a focal punctate reaction (B, left image, arrows). (C, D) Liver with variably sized IBs in hepatocytes (arrowheads). (E, F) Kidney with variably sized IBs in tubular epithelial cells (arrows). Nota bene (NB): The coarse brown pigment in the epithelial cells represents lipofuscin-like degradation products frequently observed in the kidney of boas. (A, C, E) hematoxylin and eosin (H&E) stain; (B, D, F) immunohistology, hematoxylin counterstain. Bars = 20 μm.

FIG 4

Wild-caught boa constrictor (animal W5) with BIBD. Light and transmission electron microscopy findings in a cell pellet prepared from the boa constrictor (B. constrictor) kidney-derived cell line I/1Ki at 6 days postinoculation with brain homogenate. (A, B) Inclusion body (IB) formation (A, arrowheads) and reptarenavirus nucleoprotein (NP) expression (B) in the cells. Like the neurons in the cerebral cortex (Fig. 3A), individual cells exhibit large square IBs (arrows). (C to F) Ultrastructural features. The cells exhibit smaller and irregular early (arrow) and more complex older (arrowhead) IBs (C) and occasional large square IBs (D) with a grid-like, directed structure (E, F).

FIG 5

The distribution of reads in Lazypipe (33) analysis after the removal of reads matching to Python bivitattus genome. The y axis represents percentage of reads. The reads matching viruses, bacteria, bacteriophages, eukaryota, and unknown (i.e., those not without match) are shown in colors ranging from black to white as indicated in bottom left corner. The number of reads matching each category are presented in written format (% of total reads of the sample and the number of reads in brackets) under each respective sample name.

FIG 6

Phylogenetic analysis of hartmaniviruses identified in the study. The maximum clade credibility trees were inferred using the Bayesian MCMC method with Cprev Blosum and WAG amino acid substitution models for RdRp, GPC, and NP, respectively. (A) A phylogenetic tree based on the RdRp amino acid sequences of the viruses identified in this study and those available in GenBank. (B) Phylogenetic tree based on the NP amino acid sequences of the viruses identified in this study and those available in GenBank. (C) A phylogenetic tree based on the GPC amino acid sequences of the viruses identified in this study and those available in GenBank.