Consensus nested PCR amplification and sequencing of diverse reptilian, avian, and mammalian orthoreoviruses

The orthoreoviruses are segmented RNA viruses that infect diverse vertebrate host species. While the most common human orthoreovirus, Mammalian Reovirus, is not typically associated with significant disease, the majority of Orthoreovirus species have been shown to cause significant and often fatal disease in reptiles, birds, and primates. There is significant potential for jumping species. A consensus nested-PCR method was designed for investigation of the RNA-dependent RNA polymerase gene of Orthoreovirus and Aquareovirus. This protocol was used to obtain sequencing template from reoviruses of three different vertebrate classes. Bayesian and maximum likelihood phylogenetic analysis found that all viruses analyzed clustered in the genus Orthoreovirus, that reptile reoviruses formed three distinct clusters, and that an African grey parrot reovirus clustered with Nelson Bay virus from bats. This PCR method may be useful for obtaining templates for initial sequencing of novel orthoreoviruses from diverse vertebrate hosts.