A novel array of real-time RT-PCR assays for the rapid pathotyping of type I avian paramyxovirus (APMV-1)

Newcastle disease (ND) caused by virulent avian paramyxovirus type I (APMV-1) is a WOAH and EU listed disease affecting poultry worldwide. ND exhibits different clinical manifestations that may either be neurological, respiratory and/or gastrointestinal, accompanied by high mortality. In contrast, mild or subclinical forms are generally caused by lentogenic APMV-1 and are not subject to notification. The rapid discrimination of virulent and avirulent viruses is paramount to limit the spread of virulent APMV-1. The appropriateness of molecular methods for APMV-1 pathotyping is often hampered by the high genetic variability of these viruses that affects sensitivity and inclusivity. This work presents a new array of real-time RT-PCR (RT-qPCR) assays that enable the identification of virulent and avirulent viruses in dual mode, i.e., through pathotype-specific probes and subsequent Sanger sequencing of the amplification product. Validation was performed according to the WOAH recommendations. Performance indicators on sensitivity, specificity, repeatability and reproducibility yielded favourable results. Reproducibility highlighted the need for assays optimization whenever major changes are made to the procedure. Overall, the new RT-qPCRs showed its ability to detect and pathotype all tested APMV-1 genotypes and its suitability for routine use in clinical samples.