Here we described the discovery and characterization of two complete viruses and partial sequence from a related, yet potentially distinct third virus, isolated from cases of snake inclusion body disease. These viruses share a typical arenavirus-like genome organization, but the protein sequences of Z and GPC imply a more complicated evolutionary relationship to previously characterized arenaviruses. These viruses were detected in 6/8 snakes with confirmed IBD diagnoses and 0/18 IBD-negative animals. In infected animals, viral RNA and protein were detected in tissues with cytoplasmic inclusions, and indeed, viral nucleoprotein was found to localize to the same eosinophilic inclusions that give the disease its name. Although formal confirmation that these novel arenavirus-like agents cause disease in snakes will require experimental challenge studies, their detection in reptiles raises an array of intriguing questions about the host range, evolution, basic biology, and mechanisms of pathogenesis associated with this unusual branch of the virus phylogeny.
The arenaviruses are a family of viruses that had been previously believed to infect only mammals (21
). Rodents are thought to be the natural hosts of arenaviruses, and individual virus species are associated with specific hosts. Infection is typically chronic and asymptomatic in rodents. However, when arenaviruses zoonotically infect humans or other mammals, severe disease can result. Scientific study has focused on these viruses for two principal reasons: (i) some of these viruses (e.g., LASV) can cause fatal hemorrhagic fever in humans, and (ii) the arenavirus LCMV provides an excellent tool to study the immune response in its natural host, Mus musculus
). Infection of members of the Reptilia
class of animals demonstrates that arenavirus infection is not limited to mammals.
The discovery of viruses that are in most ways arenavirus-like with filovirus-like GP2 domains relates to the previously stated hypothesis that envelope glycoproteins from filoviruses and arenaviruses share an ancient common ancestor (44
). There are several possible models to explain the configurations of the extant arenavirus species. One possibility is that the GPC gene of the arenavirus common ancestor was more similar to the GPC gene of CASV and GGV and therefore to those of filoviruses and avian retroviruses (). In this scenario, recombination or significant divergence occurred on the lineage leading to the Old World (OW) and New World (NW) arenaviruses. An alternate model is that the GP gene of the ancestral arenavirus was similar to that of the present-day rodent arenaviruses and may itself have derived from an ancestor common with the filoviruses (). Then, along the lineage leading to snake arenaviruses, recombination with a filovirus or retrovirus introduced a new GP gene. Intrasegmental recombination between arenaviruses leading to speciation has been documented, so a precedent for these models exists (46
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.
Whether infection by these viruses causes disease in snakes is perhaps the principal open question following from this study. It is possible that—as is often the case in rodents—arenavirus infection of snakes is chronic and subclinical. Indeed, the CAS snakes diagnosed as IBD and virus positive () were not showing any of the typical signs of regurgitation, anorexia, or central nervous system abnormalities at the time of sample collection, though it could have been relatively early in the course of infection. In contrast, the snake from Tennessee that was diagnosed as IBD and arenavirus positive () was diagnosed in a postmortem exam. This snake had produced a clutch of infertile eggs (slugs), developed dysecdysis (incomplete shedding), continued to decline, and ultimately died. It is clear that arenavirus infection does result in large viral protein-containing inclusions, a diagnostic finding believed to be prognostic of a fatal outcome, although disease progression has proven variable, with animals surviving for weeks to months after first clinical manifestation, and with the caveat that “IBD” in different snake species may ultimately prove attributable to different causes.
We do not know if snakes are the natural host of these viruses or if snakes are infected adventitiously, in a manner akin to the zoonotic transmission of rodent arenaviruses to humans. Likewise, it is unclear how the virus is transmitted. One possibility is that virus is transmitted from snake to snake by blood-feeding mites, infestations of which have anecdotally been associated with IBD outbreaks (1
). Snakes eat rodents; thus, another possibility is that these viruses are transmitted when snakes eat infected mice or rats. This possibility is not unprecedented: “callitrichid hepatitis virus” was originally identified as the agent responsible for outbreaks of fatal hepatitis in captive marmosets and tamarins in zoos (47
). This virus was subsequently shown to be identical to LCMV, which was being transmitted to the zoo animals via infected mice that they were fed (48
). In contrast, the viruses identified here are highly divergent from Old and New World arenaviruses. Additional experiments to test the viruses’ host range in cell lines and animals will help answer these questions.
Two of the eight snakes diagnosed as IBD positive in this study tested negative for arenavirus infection (). There are a number of other possible causes of the pathology observed in these cases. One obvious alternative is infection by other viruses not detected by our methods, including additional divergent arenaviruses. A precedent for this possibility is the case of avian proventricular dilatation disease, where follow-up studies identified numerous additional genogroups of avian bornavirus, the causative agent (15
). Deep sequencing of additional IBD-positive samples will help resolve this question. Alternatively, infection by nonviral pathogens may be responsible in some cases, though we did not detect any obvious such organisms in our metagenomic analyses. Alternatively, the cytoplasmic inclusions may be a by-product of some other disease state or cellular stress, although the localization of viral nucleoprotein to these inclusions that we observed would appear to contradict this alternate hypothesis. Expanded association studies will more firmly determine the proportion of IBD cases attributable to arenavirus infection and may identify etiologic agents responsible for nonarenavirus IBD diagnoses.
The findings presented here raise the possibility of improved IBD diagnostics, prevention, and treatment. IBD is currently diagnosed by relatively insensitive blood analysis or invasive biopsy and histopathology. Viral RNA was as abundant in blood cells as in infected tissues (), so RT-PCR using RNA purified from whole blood is the approach offering the best combination of specificity, sensitivity, and ease of sample collection, though proper controls and procedures will have to be used to minimize the possibility of PCR false positives due to contamination. Primers targeting the region of the genomes conserved among the three viruses described here are listed in Table S1
in the supplemental material. Antibodies against viral proteins offer an additional diagnostic approach, but antibodies against conserved viral epitopes would have to be generated and validated.
IBD is an important disease of captive snakes. While the specific mode of transmission is unknown, it is likely that the elimination of diseased snakes will greatly reduce the likelihood of transmission, even if there exists an intermediate vector. The availability of a diagnostic test, whether by RT-PCR or by virus-specific antibodies, will prevent the introduction of infected, but possibly asymptomatic, snakes into healthy collections. Ultimately, diagnostically driven surveillance by veterinarians will likely identify outbreaks or hot spots of the disease and perhaps one day lead to adequate control of this previously vexing condition. Furthermore, vaccines exist or are in development for some arenaviruses (21
), and ribavirin and other drugs have been shown to be effective in decreasing the severity of disease caused by hemorrhagic fever-causing arenaviruses (21
). This offers hope that arenavirus-targeting vaccines or treatments could be effective against snake IBD.