Evaluation of a canarypox- vectored recombinant vaccine expressing canine distemper virus H gene using multiplex real time RT-PCR.

Canine distemper virus (CDV) is a highly contagious pathogen affecting domestic and wild carnivores, and vaccination remains the primary control strategy. While modified-live virus (MLV) CDV vaccines are traditionally assessed via infectivity-based assays, replication-defective canarypox-vectored recombinant vaccines cannot be evaluated using conventional tissue-culture titration. This study applied a multiplex real-time RT-PCR (multiplex RT-qPCR) assay targeting the CDV hemagglutinin (H) gene for in vitro molecular characterization of commercial CDV vaccine batches. Molecular quantification was contextualized using a TCID₅₀-characterized reference strain to maintain biological relevance without inferring infectivity. Fifteen commercial vaccine batches-five canarypox-vectored and ten MLV-were analyzed. The assay exhibited high linearity (R2 = 0.998), amplification efficiency (∼94%), and sensitive detection across a wide dynamic range. Canarypox-vectored vaccines showed a narrow Ct range (14.7-18.8), indicating low variability in RNA signal intensity, whereas MLV vaccines exhibited broader variation, including reduced RNA signal in selected batches. In MLV vaccines, Ct values showed strong inverse correlation with TCID₅₀ titers and were associated with virus neutralization titers (VNT), indicating that higher RNA signal intensity was observed alongside higher infectivity and stronger humoral responses. Correlation analyses were performed separately for MLV and canarypox-vectored vaccines to account for differences in biological mechanisms, and all associations were interpreted as correlative rather than causal. Post-vaccination shedding analysis revealed transient, low-level vaccinal RNA in MLV-vaccinated animals, while no shedding was detected for canarypox-vectored vaccines. Overall, multiplex RT-qPCR provides a sensitive and reproducible molecular approach for batch consistency assessment and supportive quality control of canarypox-vectored CDV vaccines. This method complements established biological assays and offers a practical in vitro alternative where conventional infectivity testing is not applicable.