Candidate Aetiological Agent for Snake Inclusion Body Disease

Inclusion body disease has been recognised in a number of countries in captive snakes from the 1980s

Posted by Stephen on August 26, 2012

Inclusion body disease has been recognised in a number of countries in captive snakes from the 1980s. It is common progressive, often fatal, infectious disease of captive snakes, with those of the family Boidae (boas and pythons) being the most susceptible. No aetiological agent has been definitively characterized and it has previously been regarded as a retrovirus-like agent.

Mark Stenglein and Joseph DeRisi et al, in the open-access journal mBio, documented their identification and characterisation of a virus from the family Arenaviridae in captive snakes; the first of this family to be isolated from reptiles. This family contains viruses which cause fatal haemorrhagic fevers such as Lassa fever in humans, which kills thousands of people every year in Africa. The natural host of arenaviruses is rodents with occasional spread to humans. In rodents the disease is typically chronic and asymptomatic.

On histological examination of infected tissue the classical feature is eosinophilic intracytoplasmic inclusion bodies in epithelial cells of all visceral organs, which gives the disease its name. On post mortem gross changes are usually as a result of secondary infection such as stomatitis, pneumonia and granulomas within the kidneys and liver. Evaluation of the brain often reveals non-suppurative meningoencephalitis with intra-cytoplasmic inclusions present in degenerating neurons. A more severe inflammatory response is noted in pythons, in which the disease is often more fatal.

Chronic weight loss or an acute onset of neurological signs is a common presentation of this disease with affected animals commonly described as stargazing. Regurgitation, flaccid paralysis, head tremors and dysecdysis can all occur with this disease. No clinical presentation is pathognomic for IBD. Infected snakes may be unable to right themselves or exhibit in-coordination resulting in owners describing their animals as ‘drunk’ or ‘tying themselves in knots’.

Antemortem diagnosis can be based on neurological signs, however, other tests can be used for confirmation such as white blood cell analysis, organ function (liver and kidney), inclusion bodies in WBCs or through the biopsy specimens. The various clinical signs indicate the use of multiple tests.

No treatment is available and infected snakes are often euthanised at onset of neurological signs.

The method of transmission is not known. A number of ways have been postulated such as through consumption of infected rodents or via mites, such as the snake mite Ophionyssus natricis, however only anecdotal evidence is available.

Deep sequencing and de novo sequence assembly following viral RNA extraction from organs resulted in the discovery of two complete viruses, GGV (Golden Gate virus) and CASV (California Academy of Sciences virus) with their subsequent characterisation, and the partial sequence of a distinct, possibly related, third virus from cases of IBD. This revealed a highly distinct genome from typical arenaviruses due to the complicated evolutionary relationship. GGV was detected in boa constrictors and CASV was found in diseased annulated tree boas. CASV and GGV glycoproteins were ascertained to be closely related to filovirus or retrovirus.

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.

To investigate the relationship between previously described arenavirus and these novel arenaviruses a phylogenetic analyses was carried out. The four major open reading frames (L, NP, Z, and GPC) present on CASV and GGV were used for the comparison. The results suggested that a long time ago a recombination event occurred between the genomes of arenaviruses and filoviruses or retroviruses resulting in a separate biological taxa from Old World (Lassa/LCMV serocomplex) and New World (Tacaribe serocomplex) arenaviruses, as illustrated in the figure above.

Infected tissue was introduced to cultured boa constrictor kidney cells and five days post-inolculation cell supernatant was found to contain viral RNA.

Viral RNA and protein were isolated from infected cells with viral nucleoprotein found to result in similar intracytoplasmic eosinophilic inclusions. No virus was present in two of the eight snakes diagnosed with IBD or the 18 disease free controls. Clearly more research is needed to prove causality – that this candidate aetiological agent actually results in inclusion body disease in captive snakes.

Is this virus an ancestor of both arenaviruses and filoviruses which consequently infected rodents, or is it a result of the aptly named ‘rodent revenge hypothesis’ in which a rodent passed the virus to its reptilian adversaries?

To hear Mark Stenglein and Joseph DeRisi discuss their discovery with the hosts of TWiV (This Week in Virology) visit TWiV episode 196 here.

References:

Stenglein, M. et al. 2012, ‘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’, mBio, Vol. 3, No. 4. (2012), doi:10.1128/mBio.00180-12