Development of an mRNA-specific real-time PCR for the detection of replicating cyprinid herpesvirus-3 (CyHV-3) in carp and non-target species

Cyprinid herpesvirus 3 (CyHV-3) is a highly contagious virus that causes high mortalities in common and koi carp worldwide. The molecular detection of this double-stranded DNA virus has been extensively researched. Nonetheless, there are currently no real-time PCR assays available for detecting CyHV-3 mRNA, which could serve as an indicator of active virus replication, aiding in the evaluation of the susceptibility of non-target species. This study describes a probe-based reverse transcription, real-time PCR (RT-qPCR) assay that was designed to detect CyHV-3 mRNA for efficient, high-throughput detection. The assay is highly specific for CyHV-3 mRNA, with no detection in samples from non-infected fish, closely related viruses or CyHV-3 DNA. The analytical sensitivity of the assay was examined using dilutions of a plasmid control and nucleic acid from a CyHV-3-infected cell line, where the CyHV-3 mRNA limit of detection was approximately 1 copy per reaction. Testing of diluted CyHV-3 mRNA demonstrated comparable sensitivity of the RT-qPCR with an existing reverse transcription PCR assay. Progressive monitoring of positive control samples revealed that the assay had a high level of repeatability. The assay was used to provide further evidence that non-target species silver perch and Murray cod were not susceptible to developing disease when experimentally exposed to CyHV-3. The novel RT-qPCR assay is an invaluable tool for detection of the replication phase of CyHV-3.