title: A reverse transcriptase-polymerase chain reaction assay for detecting Highlands J virus.
abstract: Highlands J (HJ) virus is an arbovirus frequently recovered at high rates in mosquitoes collected in the eastern United States. HJ virus is primarily a veterinary pathogen causing disease in domestic birds including turkeys, chickens, and partridges. It has an enzootic cycle similar to eastern equine encephalitis (EEE) virus and is often used as an indicator species in EEE surveillance programs. Current immunologic techniques to identify HJ virus are often inefficient and can involve cross-reactivity of antibodies. Therefore, we developed a molecular-based assay by a reverse transcriptase (RT)-polymerase chain reaction (PCR) technique. Primers were constructed from conserved sequences of the E1 coding region from 19 strains of HJ virus. PCR amplifications from serial dilutions of HJ virus-infected Vero cell culture supernatants indicated that this assay could detect viral RNA at concentrations of 10 plaque-forming units per reaction. Extracted RNAs from western equine encephalitis, EEE, LaCrosse, and Jamestown Canyon viruses were not detected with this assay. RNA extracted directly from the brain tissue of a dead house sparrow and from a pool of Culiseta mosquitoes yielded a PCR product of the expected size. The RT-PCR technique developed was both sensitive and specific for detecting HJ virus from infected cell culture supernatants, bird brain tissues, and mosquitoes. This new assay will permit rapid and accurate diagnosis of HJ virus, both enhancing surveillance activities for EEE transmission risk and monitoring infections in domestic poultry and wild birds.
authors.affiliation: Center for Vector-Borne Disease, University of Rhode Island, Kingston 02881, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Whitehouse
authors.forename: A
authors.initials: A
authors.lastname: Guibeau
authors.forename: D
authors.initials: D
authors.lastname: McGuire
authors.forename: T
authors.initials: T
authors.lastname: Takeda
authors.forename: T N
authors.initials: TN
authors.lastname: Mather
mesh_terms: D000524:Alphavirus / Q000235:genetics / Q000302:isolation & purification*; D018354:Alphavirus Infections / Q000175:diagnosis / Q000662:veterinary*; D000818:Animals; D000835:Animals, Wild; D001483:Base Sequence; D001715:Bird Diseases / Q000175:diagnosis; D001717:Birds; D002645:Chickens; D002522:Chlorocebus aethiops; D003429:Cross Reactions; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary; D011201:Poultry Diseases / Q000175:diagnosis*; D012367:RNA, Viral / Q000032:analysis* / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014709:Vero Cells
chemical_list: D012367:RNA, Viral