title: Consensus nested PCR amplification and sequencing of diverse reptilian, avian, and mammalian orthoreoviruses.
abstract: 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.
authors.affiliation: Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA. wellehanj@vetmed.ufl.edu
authors.forename: James F X
authors.initials: JF
authors.lastname: Wellehan
authors.forename: April L
authors.initials: AL
authors.lastname: Childress
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Marschang
authors.forename: April J
authors.initials: AJ
authors.lastname: Johnson
authors.forename: Elaine W
authors.initials: EW
authors.lastname: Lamirande
authors.forename: John F
authors.initials: JF
authors.lastname: Roberts
authors.forename: Mary L
authors.initials: ML
authors.lastname: Vickers
authors.forename: Jack M
authors.initials: JM
authors.lastname: Gaskin
authors.forename: Elliott R
authors.initials: ER
authors.lastname: Jacobson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001499:Bayes Theorem; D001717:Birds; D016000:Cluster Analysis; D005784:Gene Amplification; D016679:Genome, Viral; D016013:Likelihood Functions; D008322:Mammals; D008969:Molecular Sequence Data; D018165:Orthoreovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D030101:Orthoreovirus, Avian / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D030102:Orthoreovirus, Mammalian / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*; D012088:Reoviridae Infections / Q000662:veterinary / Q000821:virology; D012104:Reptiles; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D012367:RNA, Viral