Rapid Identification of Known and New RNA Viruses from Animal Tissues

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Abstract
Viral surveillance programs or diagnostic labs occasionally obtain infectious samples that fail to be typed by available cell culture, serological, or nucleic acid tests. Five such samples, originating from insect pools, skunk brain, human feces and sewer effluent, collected between 1955 and 1980, resulted in pathology when inoculated into suckling mice. In this study, sequence-independent amplification of partially purified viral nucleic acids and small scale shotgun sequencing was used on mouse brain and muscle tissues. A single viral agent was identified in each sample. For each virus, between 16% to 57% of the viral genome was acquired by sequencing only 42–108 plasmid inserts. Viruses derived from human feces or sewer effluent belonged to the Picornaviridae family and showed between 80% to 91% amino acid identities to known picornaviruses. The complete polyprotein sequence of one virus showed strong similarity to a simian picornavirus sequence in the provisional Sapelovirus genus. Insects and skunk derived viral sequences exhibited amino acid identities ranging from 25% to 98% to the segmented genomes of viruses within the Reoviridae family. Two isolates were highly divergent: one is potentially a new species within the orthoreovirus genus, and the other is a new species within the orbivirus genus. We demonstrate that a simple, inexpensive, and rapid metagenomics approach is effective for identifying known and highly divergent new viruses in homogenized tissues of acutely infected mice. Viral surveillance programs aim to identify circulating viruses to safeguard the public and livestock from viral outbreaks. Occasionally, samples suspected of harboring a virus cause severe disease in laboratory animals, but the identity of the virus eludes researchers. Here, we applied a simple viral discovery technique to identify viruses directly from the tissues of inoculated symptomatic mice and found a single virus in each sample using a rapid viral particle purification and random nucleic acid amplification method. Two viruses appear to be closely related to the members of the Picornaviridae family. In three other samples, originally collected from pools of crushed mosquitoes and the brain of a sick skunk, a known and novel viruses related to members of the Reoviridiae family were identified. Reoviruses are considered major pathogens of livestock. Our studies provide the groundwork for further analysis of the prevalence and pathogenesis of these divergent viruses and illustrate the ease with which new viral species can be identified in tissues of acutely infected animals.