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

• Published in: Veterinary microbiology 2009, Vol.133 (1), p.34-42
• Main Authors: Wellehan, James F.X., Childress, April L., Marschang, Rachel E., Johnson, April J., Lamirande, Elaine W., Roberts, John F., Vickers, Mary L., Gaskin, Jack M., Jacobson, Elliott R.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; animal diseases; Animals; Aquareovirus; Avian orthoreovirus; Bayes Theorem; Biological and medical sciences; bird diseases; Birds; Cluster Analysis; Consensus PCR; consensus sequence; Fundamental and applied biological sciences. Psychology; Gene Amplification; genetic variation; Genome, Viral; Likelihood Functions; Mammalian orthoreovirus; Mammals; microbial genetics; Microbiology; Miscellaneous; molecular genetics; Molecular Sequence Data; Nelson Bay orthoreovirus; Nelson Bay virus; Orthoreovirus; Orthoreovirus - classification; Orthoreovirus - genetics; Orthoreovirus - isolation & purification; Orthoreovirus, Avian - classification; Orthoreovirus, Avian - genetics; Orthoreovirus, Avian - isolation & purification; Orthoreovirus, Mammalian - classification; Orthoreovirus, Mammalian - genetics; Orthoreovirus, Mammalian - isolation & purification; Phylogeny; Polymerase; polymerase chain reaction; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - veterinary; Primates; rapid methods; Reoviridae; Reoviridae Infections - veterinary; Reoviridae Infections - virology; Reovirus; Reptiles; Reptilian orthoreovirus; RNA; RNA, Viral - chemistry; RNA, Viral - genetics; Sequence Alignment; sequence analysis; Sequence Homology, Amino Acid; species differences; Species Specificity; viral diseases of animals and humans; Virology
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2008.06.011

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.