Molecular Detection of Porcine Parainfluenza Viruses 1 and 5 Using a Newly Developed Duplex Real-Time RT-PCR in South Korea

Abstract

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.

Keywords: dqRT-PCR; porcine parainfluenza virus 1; porcine parainfluenza virus 5.

Figure 1

Limit of detection (LOD) and standard curves for the monoplex and duplex real-time reverse transcription polymerase chain reactions (dqRT-PCR) for porcine parainfluenza virus 1 (PPIV1) and PPIV5. LODs and standard curves of monoplex qRT-PCR for PPIV1 (A,B) and PPIV5 (C,D), and dqRT-PCR for PPIV1 and PPIV5 (E,F). Lines 6–0 are 10-fold serial dilutions of the PPIV1 and PPIV5 standard RNAs (10⁶–10⁰ copies, respectively). Standard curves of the assays were generated using 10-fold serial dilutions for PPIV1 and PPIV5 standard RNA (10⁶–10⁰ copies). The coefficient of determination (R²) and regression curve equations (y) were evaluated using the CFX Manager Software (Bio-Rad, Hercules, CA, USA).

Figure 2

Alignments of the partial nucleocapsid protein gene sequences for porcine parainfluenza virus 1 (PPIV1) obtained from eleven discordant and six concordant clinical samples. Primers and probe binding sites of Schuele’s real-time reverse transcription polymerase chain reaction (qRT-PCR) (A) and the newly developed duplex qRT-PCR (B) are indicated by black arrows and lines. A dot indicates the same base, and a letter with a red background indicates a mismatched base.

Figure 3

Phylogenetic tree for 30 partial nucleocapsid protein gene (184 bp) sequences from porcine parainfluenza virus 1 (PPIV1), including 17 sequences obtained in this study and 13 previously reported PPIV1 strains from GenBank. The Maximum Likelihood trees were inferred with 1000 rapid bootstrap replicates in MEGA X software, version 10.2.4. Red and blue circles indicate discordant and concordant clinical samples, respectively.