Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay

Area of Science:

• Aquatic Animal Health
• Molecular Diagnostics
• Virology

Background:

• Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) are OIE-listed rhabdoviruses causing significant losses in aquaculture and wild fish populations.
• Accurate and rapid detection methods are crucial for managing these economically important fish diseases.

Purpose of the Study:

• To develop and validate a novel multiplex real-time quantitative reverse transcription PCR (mqRT-PCR) assay.
• To enable simultaneous detection, identification, and quantification of SVCV, IHNV, and VHSV.

Main Methods:

• Design of primers and probes targeting conserved regions of the glycoprotein (G) gene for SVCV and VHSV, and the nucleoprotein (N) gene for IHNV.
• Optimization and validation of the mqRT-PCR assay for sensitivity, specificity, and interference.
• Testing of 80 clinical fish samples, with results confirmed by antigen-capture ELISA and cell culture.

Main Results:

• The mqRT-PCR assay demonstrated high sensitivity, detecting as few as 100 copies of SVCV, 220 copies of IHNV, and 140 copies of VHSV.
• No cross-reactivity was observed with other fish viruses, including pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV), and grass carp reovirus (GCRV).
• The assay successfully identified target viruses in 80 clinical fish samples, correlating with confirmatory diagnostic methods.

Conclusions:

• The developed mqRT-PCR assay is a sensitive, specific, and efficient tool for the simultaneous detection and quantification of SVCV, IHNV, and VHSV.
• This assay holds significant potential for routine diagnostics and research applications in fish health management.
• The method provides a valuable advancement for controlling OIE-listed rhabdoviral diseases in farmed and wild fish.