Differential Disease Susceptibilities in Experimentally Reptarenavirus-Infected Boa Constrictors and Ball Pythons

Abstract

Inclusion body disease (IBD) is an infectious disease originally described in captive snakes. It has traditionally been diagnosed by the presence of large eosinophilic cytoplasmic inclusions and is associated with neurological, gastrointestinal, and lymphoproliferative disorders. Previously, we identified and established a culture system for a novel lineage of arenaviruses isolated from boa constrictors diagnosed with IBD. Although ample circumstantial evidence suggested that these viruses, now known as reptarenaviruses, cause IBD, there has been no formal demonstration of disease causality since their discovery. We therefore conducted a long-term challenge experiment to test the hypothesis that reptarenaviruses cause IBD. We infected boa constrictors and ball pythons by cardiac injection of purified virus. We monitored the progression of viral growth in tissues, blood, and environmental samples. Infection produced dramatically different disease outcomes in snakes of the two species. Ball pythons infected with Golden Gate virus (GoGV) and with another reptarenavirus displayed severe neurological signs within 2 months, and viral replication was detected only in central nervous system tissues. In contrast, GoGV-infected boa constrictors remained free of clinical signs for 2 years, despite high viral loads and the accumulation of large intracellular inclusions in multiple tissues, including the brain. Inflammation was associated with infection in ball pythons but not in boa constrictors. Thus, reptarenavirus infection produces inclusions and inclusion body disease, although inclusions per se are neither necessarily associated with nor required for disease. Although the natural distribution of reptarenaviruses has yet to be described, the different outcomes of infection may reflect differences in geographical origin.IMPORTANCE New DNA sequencing technologies have made it easier than ever to identify the sequences of microorganisms in diseased tissues, i.e., to identify organisms that appear to cause disease, but to be certain that a candidate pathogen actually causes disease, it is necessary to provide additional evidence of causality. We have done this to demonstrate that reptarenaviruses cause inclusion body disease (IBD), a serious transmissible disease of snakes. We infected boa constrictors and ball pythons with purified reptarenavirus. Ball pythons fell ill within 2 months of infection and displayed signs of neurological disease typical of IBD. In contrast, boa constrictors remained healthy over 2 years, despite high levels of virus throughout their bodies. This difference matches previous reports that pythons are more susceptible to IBD than boas and could reflect the possibility that boas are natural hosts of these viruses in the wild.

Keywords: arenavirus; inclusion body disease; pathogenesis; reptarenavirus; veterinary pathogens.

FIG 1

Timeline of experimental reptarenavirus infection of boa constrictors and ball pythons. The times of collection of the pre- and postinfection biopsy samples (bx) and blood samples tested are indicated. weeks post inf., weeks postinfection. (Insets) Images of a representative infected boa constrictor and representative infected ball python at the end of their respective study periods.

FIG 2

Boa constrictors had persistently high viral loads in all tissues. Viral RNA (vRNA) levels were quantified by qRT-PCR. (A) Blood viral RNA levels. (B and C) Tissue viral RNA levels for snake G (B) and snake H (C). Samples from the uninfected boa constrictor were negative. w.p.i., weeks postinfection; S2 and L2, viral genome segment genotypes.

FIG 3

Viral RNA is detectable in feces, urates, and shed skin from infected boa constrictors. Viral RNA was detected by qRT-PCR. Viral RNA was not detected in any fecal, urate, or skin sample collected from ball pythons. *, the positive result for this fecal sample from the control snake may have resulted from sample mislabeling; no other sample from this control animal ever tested positive.

FIG 4

Viral RNA was detectable in infected ball python brains. Viral and cellular RNA levels were quantified by qRT-PCR. (A) Reptarenavirus RNA was detected in brain but not other tissues. (B and C) Viral RNA levels were normalized to the levels of GAPDH mRNA in ball python L (B) and M (C) brains. Samples from uninfected snakes were negative. Controls were the virus 37 inoculum (virus 37), the GoGV inoculum (GoGV), and uninfected python J brain (J brain). nd, not detected.

FIG 5

Reptarenavirus nucleoprotein-positive inclusions were detected in tissues of infected boa constrictors throughout infection. (A) Biopsy and necropsy liver sections from infected and uninfected boa constrictors were stained with anti-NP antibody (green) and DAPI (blue). (B) Necropsy heart, kidney, and intestine sections were stained as described in the legend to panel A. Bars = 10 μm.

FIG 6

Reptarenavirus nucleoprotein detected in the brains of infected snakes. Brain sections were stained with anti-NP antibody (green) and with DAPI (blue). Sections were obtained from uninfected ball pythons J (A), infected ball python L (B), and infected boa constrictor H (C). The top, lower left, and lower right panels display increasingly zoomed images of the same sections, in which the bars are 2,000, 200, and 20 μm, respectively. Contrast speckled cytoplasmic staining in infected ball python cells (B) with inclusions in infected boa constrictor cells (C).

FIG 7

Reptarenavirus nucleoprotein was not detected in non-CNS tissues of infected ball pythons. Necropsy tissues from infected ball python L were stained with anti-NP antibody (green) and DAPI (blue). These images are representative of negative staining of all non-CNS tissues from all ball pythons. Bars = 50 µm.

FIG 8

Inclusions were evident in infected boa constrictor brains. Images of hematoxylin and eosin (H&E)-stained brain sections from the indicated boa constrictors are shown. Infected boa constrictors (boas G and H) had numerous, brightly eosinophilic, cytoplasmic viral inclusion bodies (arrowheads) within neuronal cell bodies and glial cells of the brain. Similar inclusions were found within cells of nearly every organ examined. No inflammation was associated with the inclusions. The uninfected boa constrictor (boa I) did not have inclusions. (Insets) Magnified views of the boxed regions. Bars = 50 μm.

FIG 9

Inflammation in infected ball python central nervous system tissues. Arenavirus-infected pythons (pythons L and M) had moderate lymphocytic, histiocytic, and granulocytic inflammation (asterisks) within the brain, spinal cord, and ganglia. Necrotic neurons were occasionally seen (arrows). No inflammation was detected in the uninfected pythons. C, central canal. H&E-stained tissue section. Bars = 50 μm.

FIG 10

Bile duct inclusions in ball python M. Small eosinophilic cytoplasmic inclusions (arrows) were seen in rare bile duct epithelial cells of the infected ball python M. H&E-stained tissue section. Bar = 50 μm.