title: Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction.
abstract: Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.
authors.affiliation: Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 300 West Drake Road, Fort Collins, CO 80523, USA.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Hussey
authors.forename: Rodney
authors.initials: R
authors.lastname: Clark
authors.forename: Katharine F
authors.initials: KF
authors.lastname: Lunn
authors.forename: Cormac
authors.initials: C
authors.lastname: Breathnach
authors.forename: Gisela
authors.initials: G
authors.lastname: Soboll
authors.forename: J Millar
authors.initials: JM
authors.lastname: Whalley
authors.forename: D Paul
authors.initials: DP
authors.lastname: Lunn
mesh_terms: D000818:Animals; D005260:Female; D004861:Herpesvirus 1, Equid / Q000235:genetics* / Q000302:isolation & purification*; D006734:Horse Diseases / Q000175:diagnosis / Q000821:virology*; D006736:Horses / Q000821:virology; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014766:Viremia / Q000662:veterinary* / Q000821:virology; D017735:Virus Latency; D017201:Virus Shedding