The first reptilian circovirus identified infects gut and liver tissues of black-headed pythons

Abstract

Viral metagenomic analysis of the liver of a black headed python (Aspidites melanocephalus) euthanized for a proliferative spinal lesion of unknown etiology yielded the first characterized genome of a reptile-infecting circovirus (black-headed python circovirus or BhPyCV). BhPyCV-specific in situ hybridization (ISH) showed that viral nucleic acids were strongly expressed in the intestinal lining and mucosa and multifocally in the liver. To investigate the presence of this virus in other snakes and its possible pathogenicity, 17 snakes in the python family with spinal disease were screened with ISH yielding a second BhP positive in intestinal tissue, and a Boelen's python (Morelia boeleni) positive in the liver. BhPyCV specific PCR was used to screen available frozen tissues from 13 of these pythons, four additional deceased pythons with and without spinal disease, and fecal samples from 37 live snakes of multiple species with unknown disease status. PCR detected multiple positive tissues in both of the ISH positive BhP and in the feces of another two live BhP and two live annulated tree boas (Corallus annulatus). Preliminary analysis indicates this circovirus can infect BhPs where it was found in 4/5 BhPs tested (2/2 with spinal disease, 2/3 live with unknown status), Boelen's python (1/2 with spinal disease), and annulated tree boa (2/6 live with unknown status) but was not detected in other python species with the same spinal lesions. This circovirus' causal or contributory role in spinal disease remains speculative and not well supported by these initial data.

Figure 1

Characteristics of BhPyCV genome. A Open reading frames and stem-loop origin of replication of BhPyCV genome. Conserved domains in Rep protein are highlighted. B Phylogenetic analysis of circovirus Rep proteins using Maximum Likelihood.

Figure 2

BhPyCV infection. A, B Longitudinal section of small intestine from index BhP at low (×4, A) and higher magnification (×10, B) showing a mucosal lymphoid aggregate (asterisk) and villi mildly expanded by edema and lymphocytes (arrows), H&E. D, E Serial sections from A–B showing abundant, deep red, punctate to coalescing ISH signal lining the mucosal epithelium, filling intestinal villi, and scattered in lymphoid follicle (asterisk). Hematoxylin counterstain, ×4 (D); ×10 (E). G, H Serial histologic sections from A–B, D–E. Negative control using bacterial DapB gene probe with no positive ISH signal, hematoxylin counterstain, ×4 (G); ×10 (H). C BhPyCV ISH probe on liver from index BhP showing discrete, punctate red staining within sinusoidal macrophages. Hematoxylin counterstain, ×40. F, I Medium–high magnification of intestinal villi from second BhP showing similar yet less dense and frequent staining within the epithelium (arrows) and scattered in the lamina propria (asterisk). Hematoxylin counterstain, ×20.