Development and Validation of a Viability <scp>RT</scp> ‐ <scp>qPCR</scp> Assay for Detecting Infectious Spring Viraemia of Carp Virus ( <scp>SVCV</scp> )

Spring viremia of carp virus (SVCV) is a contagious pathogen associated with significant mortality and economic losses in freshwater aquaculture. Although reverse transcription quantitative PCR (RT-qPCR) enables rapid detection, it does not distinguish between infectious and non-infectious viral particles, which may lead to overestimation of infection risk. In this study, a viability RT-qPCR (vqPCR) assay targeting the glycoprotein (G) gene of SVCV was developed and analytically evaluated. Primers and probes were designed based on 45 full-length G-gene sequences representing genogroups Ia-Id to ensure broad genetic coverage. The assay achieved a 95% limit of detection (LoD_95%) of 6.82 copies per reaction and showed no cross-reactivity with other tested aquatic pathogens. Optimisation using 25 μM PMAxx effectively reduced amplification from inactivated virus and free viral RNA while preserving detection of infectious SVCV. Under controlled laboratory conditions, the vqPCR assay successfully differentiated varying proportions of infectious virus in mixed samples. In laboratory-simulated environmental freshwater, both qPCR and vqPCR signals declined with increasing temperature; notably, vqPCR reflected the loss of infectivity earlier than qPCR. The assay demonstrated good repeatability and reproducibility across three independent laboratories and showed overall agreement with cell culture and WOAH-recommended nested PCR results. Although further validation under diverse environmental conditions is warranted, these findings indicate that the proposed vqPCR approach may serve as a complementary method for monitoring infectious SVCV in freshwater aquaculture.