A reverse transcriptase-polymerase chain reaction assay for detecting highlands J virus

Highlands J (HJ) virus is an arbovirus frequently recovered at high rates i n 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 equin e 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. Ther efore, we developed a molecular-based assay by a reverse transcriptase (RT) -polymerase chain reaction (PCR) technique. Primers were constructed from c onserved sequences of the E1 coding region from 19 strains of HJ virus. PCR amplifications from serial dilutions of HJ virus-infected Vero cell cultur e supernatants indicated that this assay could detect viral RNA at concentr ations 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 pr oduct of the expected size. The RT-PCR technique developed was both sensiti ve and specific for detecting HJ virus from infected cell culture supernata nts, bird brain tissues, and mosquitoes. This new assay will permit rapid a nd accurate diagnosis of HJ virus, both enhancing surveillance activities f or EEE transmission risk and monitoring infections in domestic poultry and wild birds.