Development of universal and quadruplex real‐time RT‐PCR assays for simultaneous detection and differentiation of porcine reproductive and respiratory syndrome viruses

Abstract

Porcine reproductive and respiratory syndrome virus 1 (PRRSV1) and 2 (PRRSV2) (including 3 major subtypes: classical (CA‐PRRSV2), highly pathogenic (HP‐PRRSV2) and NADC30‐like (NL‐PRRSV2)) are currently coexisting in Chinese swine herds but with distinct virulence. Reliable detection and differentiation assays are crucial to monitor the prevalence of PRRSV and to adopt effective control strategies. However, current diagnostic methods cannot simultaneously differentiate the four major groups of PRRSV in China. In this study, universal and quadruplex real‐time RT‐PCR assays using TaqMan‐MGB probes were developed for simultaneous detection and differentiation of Chinese PRRSV isolates. The newly developed real‐time RT‐PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. In addition, the newly developed real‐time RT‐PCR assays were further validated by comparing with a universal PRRSV conventional RT‐PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China. Based on the clinical performance, the agreements between the universal and quadruplex real‐time RT‐PCR assays and the conventional RT‐PCR assay were 99.55% and 99.40%, respectively. Totally 90 samples were detected as PRRSV‐positive, including 2 samples that were determined to be co‐infected with NL‐PRRSV2 and HP‐PRRSV2 isolates by the quadruplex real‐time RT‐PCR assay. ORF5 sequencing confirmed the real‐time RT‐PCR results that 2, 6, 27 and 57 of the 92 sequences were PRRSV1, CA‐PRRSV2, NL‐PRRSV2 and HP‐PRRSV2, respectively. This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.

Chimeric HP-PRRSV2 containing an ORF2-6 consensus sequence induces antibodies with broadly neutralizing activity and confers cross protection against virulent NADC30-like isolate

“…To study the growth kinetics of the viruses in vitro, Marc-145 cells were infected with 200 median tissue culture infectious doses (TCID 50 ) of rJS-ORF2-6-CON, rJSTZ1712-12 or parental JSTZ1712-12 isolate, respectively. The multiple-step growth curves within 96 h post-infection (hpi) were determined by real-time RT-PCR assay [ 32 ]. The plaque morphology was examined in Marc-145 cells as previously described [ 33 ].…”

The infectious cDNA clone of commercial HP‐PRRS JXA1‐R‐attenuated vaccine can be a potential effective live vaccine vector

“…The in vitro growth kinetics of the parental JXA1‐R virus and cloned rJXA1‐R and rJXA1‐R‐EGFP viruses. The one‐step growth curves in Marc‐145 cells within 72hpi were determined by real‐time RT‐PCR assay (Chen, Ye, Xiao, et al., 2019). No significant difference was observed in the in vitro replication of the parental and cloned viruses…”

“…To compare the growth kinetics of cloned viruses with the parental JXA1‐R virus, Marc‐145 cells were infected with the parental JXA1‐R virus and the cloned rJXA1‐R (3th passage) and rJXA1‐R‐EGFP (3th passage) viruses at 200 median tissue culture infectious doses (TCID 50 ), respectively. Infected cells were collected at 0, 24, 48 and 72 hpi, and virus loads were determined by universal PRRSV real‐time RT‐PCR assay (Chen, Ye, Xiao, et al., 2019).…”

Identification of Two Porcine Reproductive and Respiratory Syndrome Virus Variants Sharing High Genomic Homology but with Distinct Virulence

“…In December 2017, a total of 18 sera were submitted from a clinically healthy pig farm with ~200 sows in Taizhou city, Jiangsu province, China for routine epidemiological investigation. The sera and lungs were used as templates for the routine detection of common swine viruses including PRRSV, classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine parvovirus (PPV) and porcine circovirus 2 (PCV2) by conventional and real-time RT-PCR assays [9,20,21,22].…”