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Metagenomic Sequencing IDs Viral Candidates in Snake Wasting Disease

NEW YORK (GenomeWeb News) – Using a sequencing-based approach, researchers have uncovered a previously unknown group of viruses in samples from snakes with a fatal infectious condition known as inclusion body disease.

The University of California at San Francisco-led team did metagenomic sequencing on tissues from infected annulated tree boas and boa constrictors. From the RNA sequences present in these samples, the investigators tracked down viruses that are most closely related to mammal-infecting arenaviruses. The viruses were present in the majority of infected animals tested for the study and appear to produce inclusion body disease-like features in boa constrictor cell lines. The study appears online today in mBio.

"The findings presented here raise the possibility of improved [inclusion body disease] diagnostics, prevention, and treatment," UCSF researchers Mark Stenglein and Joe DeRisi, the study's co-corresponding authors, and their colleagues wrote.

"Ultimately," they added, "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."

Inclusion body disease, a condition most often noted in snakes from the boa and python families living in captivity, is characterized by unusual behavior, wasting, and elevated infection risk. These symptoms are accompanied by the presence of intra-cellular protein clumps known as large eosinophilic cytoplasmic inclusions in various tissues from infected animals, the study authors explained, but the cause of the disease remained mysterious.

As part of their own efforts to better understand inclusion body disease, researchers used the Illumina HiSeq 2000 to sequence RNA from brain, liver, kidney, heart, and gastrointestinal tissue that had been collected from snakes that died from the disease at a California Academy of Sciences aquarium.

After tossing out sequences stemming from the red-tailed boa constrictor genome, the team was left with sequences representing two related but independent viruses. One of the viruses turned up in samples from infected annulated tree boas, while the other was found in boa constrictors with the disease.

After generating additional data using Sanger sequencing, PCR, and rapid amplification of cDNA ends approaches, the researchers were able to put together complete genome sequences for the two viruses.

Their comparisons with known viral sequences indicated that the potential pathogens are most closely related to arenaviruses, a type of virus that typically infects mammals. But the new viruses also appear to have coat protein features that overlap with those found in filoviruses such as Ebola and Marburg or in avian retroviruses, they noted.

Similar but slightly different sequences turned up in an infected boa constrictor from Tennessee that was tested by RT-PCR as part of a screen on 21 more snakes with or without inclusion body disease.

"Although the complete genome of this third virus has not yet been fully determined," researchers explained, "alignments of the recovered sequences reveal that this third virus is more closely related to the virus recovered from the [California Academy of Sciences] boa constrictors."

Overall, researchers tracked down arenavirus-related viral sequences from six of the eight inclusion body disease-infected snakes tested for the discovery and follow-up stages of the study. The viral RNA did not turn up in any of the 18 uninfected control snakes.

Meanwhile, the team's boa constrictor cell line studies supported the notion that these newly detected viruses contribute to inclusion body disease, with infected cells producing aggregates reminiscent of those observed in infected snakes.

The study authors cautioned that more research is needed to verify such apparent disease ties. Still, they say, the identification of these potential viral culprits is expected to improve researchers' understanding of inclusion body disease as well as the ability to limit its spread.

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