Diagnostic application of H3N8-specific equine influenza real-time reverse transcription polymerase chain reaction assays for the detection of Canine influenza virus in clinical specimens

The objective of the current study was to determine the capability of 3 recently described onestep TaqMan real time reverse transcription polymerase chain reaction (real-time RT-PCR) assays targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of H3N8 Equine influenza virus (EIV NP, EIV M, and EIV HA3 assays, respectively) to detect Canine influenza virus (CIV). The assays were initially evaluated with nucleic acid extracted from tissue culture fluid (TCF) containing the A/canine/FL/43/04 strain of Influenza A virus associated with the 2004 canine influenza outbreak in Florida. The EIV NP, EIV M, and EIV HA3 assays could detect CIV nucleic acid at threshold cycle (Ct) values of 16.31, 23.71, and 15.28, respectively. Three assays using TCF or allantoic fluid (AF) samples containing CIV (n = 13) and archived canine nasal swab samples (n = 20) originally submitted for laboratory diagnosis of CIV were further evaluated. All TCF and AF samples, together with 10 nasal swab samples that previously tested positive for virus by attempted isolation in embryonated hens' eggs or Madin-Darby canine kidney cells, were positive in all 3 real-time RT-PCR assays. None of the 3 assays detected the H1N1 Swine influenza virus strain in current circulation. These findings demonstrate that previously described real-time RT-PCR assays targeting NP, M, and H3 HA gene segments of H3N8 EIV are also valuable for the diagnosis of CIV infection in dogs. The assays could expedite the detection and identification of CIV.