Two species of porcine parainfluenza viruses (PPIV), PPIV1 and PPIV5, are globally distributed in pig herds and associated with porcine respiratory diseases, and a diagnostic tool for the simultaneous detection of the two viruses is required. In this study, a TaqMan probe-based duplex real-time reverse transcription polymerase chain reaction (dqRT-PCR) assay was first developed for the differential detection of PPIV1 and PPIV5 nucleocapsid protein (NP) genes in porcine clinical samples. The dqRT-PCR assay was highly sensitive, its limit of detection was approximately 10 RNA copies/reaction, it specifically amplified the targeted NP genes of PPIV1 and PPIV5 without cross-reacting with other porcine pathogens, and their clinical detection rates were 15.2% and 0.7%, respectively. The results from 441 clinical samples taken from 278 Korean domestic pig farms showed that the prevalence of PPIV1 and PPIV5 was 11.2% and 1.1%, respectively, and co-infection of both viruses was confirmed in a farm, suggesting that PPIV1 and PPIV5 are co-circulating in current Korean pig herds. Phylogenetic analysis based on the partial NP genes suggested that genetically diverse PPIV1 strains are circulating in Korean pig herds. The developed dqRT-PCR assay was found to be an accurate, reliable, and quantitative detection tool for PPIV1 and PPIV5 RNA in clinical pig samples and will be useful for etiological and epidemiological studies and the control of viral infections in the field.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been frequently reported in companion dogs and cats worldwide during the ongoing coronavirus disease. However, RT-qPCR methods developed for humans have been used for the diagnosis of SARS-CoV-2 infections in suspected companion dogs and cats owing to the lack of the companion animal-tailored methods. Therefore, we developed a multiplex RT-qPCR (mRT-qPCR) using newly designed primers and probes targeting RdRp and N genes of all currently circulating SARS-CoV-2 variants as well as the canine or feline 16S rRNA gene as an endogenous internal positive control (EIPC) for reliable diagnosis of SARS-CoV-2 infection from suspected dogs and cats. The developed mRT-qPCR assay specifically detected the target genes of SARS-CoV-2 but no other canine or feline pathogens. Furthermore, canine and feline EIPCs were stably amplified by mRT-qPCR in samples containing canine- or feline-origin cellular materials. This assay has high repeatability and reproducibility, with an optimal limit of detection (<10 RNA copies per reaction) and coefficients of variation (<1.0%). The detection rate of SARS-CoV-2 of the developed mRT-qPCR was 6.6% for canine and feline nasopharyngeal samples, which was consistent with that of a commercial mRT-qPCR kit for humans. Collectively, the newly developed mRT-qPCR with canine and feline EIPC can efficiently diagnose and evaluate the viral load in field specimens and will be a valuable tool for etiological diagnosis, epidemiological study, and controlling SARS-CoV-2 infections in canine and feline populations.