A boa constrictor with IBD, "mad snake disease".


Have you ever seen a sick boa constrictor? All of a sudden they start shedding, develop head tremors and secondary infections, twisting up into knots and wasting away. These poor animals may have acquired a fatal infectious disease called inclusion body disease (IBD). The disease can rapidly progress to the nervous system, with behavioral abnormalities such as disorientation, corkscrewing of the head and neck, holding the head in unnatural positions, or rolling onto the back. Affected snakes either die quickly or starve slowly over several years. The disease was first observed in captive snakes in zoos in the mid 1970s but the cause of the disease remained elusive. Unfortunately no treatment exists; snakes diagnosed with IBD are euthanized to stop transmission to other animals.


IBD is named after large eosinophilic inclusions (or “junk” in the form of huge protein aggregates) in the cytoplasms of nearly every cell in almost all tissues, possibly caused by replication of an unknown retrovirus. However it was unclear how the virus was transmitted.


Now the riddle has been solved and IBD treatments might be possible soon. And even more than that, by investigating the origin of IBD, the Joseph L. DeRisi lab at the University of California, San Francisco, identified a virus that shares characteristics with two known virus families that can cause fatal hemorrhagic fevers in humans!


It is well-established that some of the most medically important human diseases have origins in viruses from animal populations, or have animal reservoirs. Examples include HIV-1 and -2, influenza viruses, West Nile virus, severe acute respiratory virus (SARS), coronavirus, henipaviruses, rabies viruses, hantavirus, filoviruses, and arenaviruses. Therefore animal viruses and their hosts are excellent models for studying host-pathogen interactions and vaccine development.


To computationally identify the virus the researchers used high-throughput sequencing methods to search for candidate causes of IBD. Retroviruses are RNA viruses; each snake cell already contains 95% snake RNA needed for cell viability, plus the virus RNA. But how to separate the snake RNA from the virus RNA? The scientists simply compared sequences from the infected snake to sequences from a healthy snake to figure out what was foreign and therefore might belong to the virus. The problem was that the boa constrictor genome had not yet been sequenced. DeRisi organized the “Assemblathon 2” contest, in which teams competed to develop a computer program to assemble genetic sequences in a previously unknown animal genome, preferentially the boa constrictor genome.


The result of the RNA comparison shocked the scientists.


The foreign RNA sequences that were not present in the boa constrictor genome had several similarities to arenavirus genes. These similarities revealed the cause of the illness to be a completely new set of two arenaviruses. These viruses looked like distant relatives of other arenaviruses but had protein coats that were more similar to those of Ebola viruses. While nasty arenaviruses are common in rodents and cause infections in other mammals, we were unaware that they could infect reptiles. Like arenaviruses, Ebola viruses can cause fatal hemorrhagic fever or encephalitis when transmitted to humans. Neither of those viruses had ever been known to infect reptiles, and although it had been postulated that they shared a common ancestor, no link had ever been discovered.


The next step was genome assembly (building a complete genome out of raw data) using open access bioinformatics software, and then comparison with the RNA data. They found that the sequences from the snake virus belonged to four genes—one of which was most similar to genes found in filoviruses.


Turning back to the sick snakes, the scientists found the newly identified virus in six of eight snakes with IBD, and were able to isolate the virus.


Now the team had to find a way to grow the virus so that it could be studied further. They generated Boa constrictor cell lines to perform in vitro virus culture. When the virus was introduced into healthy boa constrictor cells, the virus replicated and the cells became clogged with giant protein aggregates like those in snakes with IBD. Antibodies aimed against the virus showed that these clumps were indeed derived from arenavirus protein, further strengthening the association of this new virus and the deadly disease. A final proof of this hypothesis will be a “challenge study,” where researchers intentionally infect boa constrictors and other captive snakes with the virus in order to induce and study IBD.


IBD is a very important disease of captive snakes. In solving this longstanding veterinary mystery and enabling the first steps towards treatment, vaccines, and perhaps even eradication of this disease, these scientists also discovered an unexpected new branch of virus biology: the viruses they found appear to be a combination of arenavirus and filovirus, neither of which had been known to infect reptiles. Their existence in reptiles raises an array of important questions about host range, evolution, basic biology and emergence of new diseases associated with this poorly understood branch of viral phylogeny.



Further information:


The article, “Identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: a candidate etiological agent for snake inclusion body disease (IBD)” by Mark D. Stenglein, Chris Sanders, Amy L. Kistler, J. Graham Ruby, Jessica Y. Franco, Drury R. Reavill, Freeland Dunker, and Joseph L. DeRisi is published in the open access journal mBio.


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