Development and Validation of a Viability RT-qPCR Assay for Detecting Infectious Spring Viraemia of Carp Virus (SVCV)

Area of Science:

• Aquatic animal health
• Virology
• Molecular diagnostics

Background:

• Spring viremia of carp virus (SVCV) causes significant losses in freshwater aquaculture.
• Standard RT-qPCR cannot differentiate between infectious and non-infectious SVCV particles, potentially overestimating infection risk.

Purpose of the Study:

• To develop and validate a viability RT-qPCR (vqPCR) assay for detecting infectious SVCV.
• To assess the assay's ability to distinguish live SVCV from inactivated virus and free RNA.

Main Methods:

• Designed primers and probes targeting the SVCV glycoprotein (G) gene for broad genetic coverage.
• Optimized the assay using PMAxx to inhibit amplification from non-infectious viral material.
• Evaluated assay performance using analytical sensitivity, specificity, and simulated environmental samples.

Main Results:

• The vqPCR assay demonstrated high analytical sensitivity (LoD95% of 6.82 copies/reaction) and specificity.
• Optimized vqPCR effectively distinguished infectious SVCV from inactivated virus and free RNA.
• vqPCR detected earlier loss of SVCV infectivity in simulated freshwater compared to standard qPCR.

Conclusions:

• The developed vqPCR assay accurately quantifies infectious SVCV in freshwater environments.
• This method offers improved risk assessment for SVCV in aquaculture by detecting only viable virus.
• vqPCR shows potential as a complementary tool for monitoring infectious SVCV, complementing cell culture and PCR methods.