Development and Validation of Quantitative PCR Assays for Strigid Alphaherpesvirus 1 and 2 in Great Horned Owls (Bubo virginianus).

Abstract

Great Horned Owls (Bubo virginianus; GHOW) are a true owl species (family Strigidae) widely distributed across the Americas. In GHOWs, herpesviral disease is best known from fatal infections with columbid alphaherpesvirus 1, which is endemic in rock pigeons (Columba livia). Recently, two novel alphaherpesviruses, strigid alphaherpesvirus 1 (StrAHV1) and strigid alphaherpesvirus 2 (StrAHV2), have been identified in GHOWs displaying ocular lesions and upper respiratory signs, respectively. To date, these are the only potentially endemic herpesviruses described in this species. This study aimed to develop and validate quantitative PCR (qPCR) assays for rapid detection of StrAHV1 and StrAHV2. Two singleplex probe-hybridization qPCR assays were developed for each virus, StrAHV1 and StrAHV2. Each primer-probe combination was cross-validated against the other. Whole blood and individual conjunctival, choanal, and cloacal swabs were obtained from 39 GHOWs hospitalized at three institutions in California, Colorado, and Florida in the USA and were run in duplicate for each assay. The highest number of copies of StrAHV1 detected and the greatest percentage of positive samples were from the choana (assay 1A: 15 of 35 [43%] positive, mean 194,030 copies, median 0 copies, range 0-3,411,469 copies; assay 1B: 16 of 35 [46%] positive, mean 197,622 copies, median 0 copies, range 0-3,554,915 copies). Conjunctiva had the next highest number of copies detected and positivity rate, followed by cloaca, with the number of copies detected and positivity rate both lowest in blood samples. A Kruskal-Wallis test using the choana results from the StrAHV1 assay 1A comparing the results from the three states found no significant differences (P=0.3443). No animals were positive for StrAHV2, suggesting that it may not be an endemic pathogen in GHOW. The qPCR assays developed can be used to further evaluate the epidemiology of StrAHV1 and StrAHV2 along with their clinical significance in GHOW.