Genetic diversity of porcine reproductive and respiratory syndrome virus and evaluation of three one-step real-time RT-PCR assays in Korea

Abstract

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses in the global swine industry. Frequent genetic variations in this virus cause difficulties in controlling and accurately diagnosing PRRSV.

Methods: In this study, we investigated the genetic characteristics of PRRSV-1 and PRRSV-2 circulating in Korea from January 2018 to September 2021 and evaluated three one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assays.

Results: A total of 129 lung samples were collected, consisting of 47 samples for PRRSV-1, 62 samples for PRRSV-2, and 20 PRRSV-negative samples. Nucleotide sequence analysis of open reading frames (ORFs) 5, ORF6, and ORF7 genes from PRRSV samples showed that PRRSV-1 belonged to subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%), whereas PRRSV-2 was classified as lineage 1 (25/62, 40.32%), Korean lineage (Kor) C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%). Amino acid sequence analysis showed that the neutralizing epitope and T cell epitope of PRRSV-1, and the decoy epitope region and hypervariable regions of PRRSV-2 had evolved under positive selection pressure. In particular, the key amino acid substitutions were found at positions 102 and 104 of glycoprotein 5 (GP5) in some PRRSV-2, and at positions 10 and 70 of membrane protein (M) in most PRRSV-2. In addition, one-step real-time RT-PCR assays, comprising two commercial tests and one test recommended by the World Organization for Animal Health (OIE), were evaluated.

Conclusion: The results revealed that two of the real-time RT-PCR assays had high sensitivities and specificities, whereas the real-time RT-PCR assay of the OIE had low sensitivity due to mismatches between nucleotides of Korean PRRSVs and forward primers. In this study, we genetically characterized recent PRRSV occurrences and evaluated three one-step real-time RT-PCR assays used in Korea.

Keywords: Diagnostic method; Evaluation; Genetic diversity; Porcine reproductive and respiratory syndrome virus (PRRSV).

Fig. 1

Phylogenetic analysis of ORF5 of PRRSV-1 isolates. The tree was constructed by maximum likelihood method. Prototype viruses, vaccine viruses used in Korean swine farms were marked with  and , respectively. Subgroup A and C of field strains obtained from this study was marked with  and , respectively

Fig. 2

Phylogenetic analysis of ORF5 of PRRSV-2 isolates (A). The tree was constructed by maximum likelihood method. Prototype viruses, vaccine viruses used in Korean swine farms were marked with  and , respectively. Lineage 1, lineage 5, Kor A, Kor B, and Kor C of field strains obtained from this study was marked with , , , , and , respectively. Genotyping of PRRSV-2 lineage 1 circulating in Korea (B)

Fig. 3

The alignment and entropy plot (amino acid diversity) of GP5 and M of PRRSV-1 samples with Lelystad strain. Multiple alignments of GP5 (A) and M (B) protein of PRRSV-1 are numbered from start of the GP5 domain (aa 1–201) and M protein domain (aa 1–100), respectively. The sequence of Lelystad is shown at the top of (A) and (B). The size of each aa letter at each sequence logos is proportional to the frequency. Amino acids are color-coded: blank, nonpolar; green, polar uncharged; red, polar with a positive charge; blue, polar with negative charge. A red square box indicates a B-cell epitope region, and a red round box indicates a T-cell epitope region. NE: neutralizing epitope, T epitope: T-cell epitope

Fig. 4

The alignment and entropy plot (amino acid diversity) of GP5 and M of PRRSV-2 samples with VR2332 strain. Multiple alignments of GP5 (A) and M (B) protein of PRRSV-2 are numbered from start of the GP5 domain (aa 1–200) and M protein domain (aa 1–100), respectively. The sequence of VR2332 is shown at the top of (A) and (B). The meaning of the size and color of the sequence logos is explained in the legend to Fig. 3. A red square box indicates a B-cell epitope region, and a red round box indicates a T-cell epitope region. DE: decoy epitope, NE: neutralizing epitope, HVR: hypervariable region, B epitope: B-cell epitope, T epitope: T-cell epitope