Phylogenetic Analysis of Porcine Epidemic Diarrhea Virus (PEDV) during 2020-2022 and Isolation of a Variant Recombinant PEDV Strain

Abstract

Porcine epidemic diarrhea (PED) is an acute, highly contagious, and infectious disease caused by porcine epidemic diarrhea virus (PEDV). PEDV can affect pigs of all ages, with 50~100% mortality in neonatal piglets and substantial economic losses in the swine industry. In the present study, 347 fecal and intestinal samples were collected from seven regions in China during 2020-2022. A comprehensive molecular investigation of the spike (S) gene of PEDV strains was carried out, which included phylogenetic analysis of the obtained PEDV sequences. Epidemiological surveillance data indicate that the GIIc subgroup strains are widely distributed among pigs. A PEDV strain was successfully isolated from positive small intestine samples and identified through RT-PCR detection using specific N gene primers of PEDV, indirect immunofluorescence assay (IFA), TEM analysis, genome sequencing, and full-length S gene analysis, named PEDV/SC/2022. RDP and SimPlot analysis showed that the isolate originated from the recombination of PEDV/AH2012 and PEDV/AJ1102. In conclusion, our findings contribute to the current understanding of PEDV epidemiology and provide valuable information for the control of PED outbreaks in China.

Keywords: PEDV; phylogeny; recombination; variant; virus isolation.

Figure 1

ML trees of the PEDV S gene. Scale bar: 0.01 (model: GTR + G + I). The sequences collected in this study were marker in red.

Figure 2

Amino acid sequence alignment of the PEDV S gene (aa 1–250). The first row represents the site of the amino acid. The black letters represents the name of the sequences. The conserved amino acid sites are marked with the red background and blue boxes.

Figure 3

The results of PEDV-infected Vero cells. (A) PEDV-infected Vero cells (100×). (B) Normal Vero cells (100×). (C) Amplification results of RT-PCR. M: DNA Marker 2000. 1: PEDV N gene. 2: Negative control.

Figure 4

PEDV isolate replicated in Vero cells. (A) Growth curve of PEDV isolate at in Vero. Data are presented as mean ± SD of triplicates. (B) Detection of PEDV infection in Vero cells by IFA. Cells were immunostained for PEDV (green) and dsRNA (red). Nuclei were stained with DAPI (blue). (C) Electron micrograph of purified isolate negatively stained with 2% phosphotungstic acid (×50 K).

Figure 5

ML trees based on the nucleotide sequences of the whole genome (A) and full-length S gene (B) of the PEDV isolate in this study, and other representative strains. The black circle represents the isolate in this study.

Figure 6

Nucleotide sequence homology analysis of the whole genome (A) and full-length S gene (B) of the PEDV isolate in this study, and other representative strains.

Figure 7

Recombination analysis of PEDV strains. The results of RDP4 are supported by ≥6 programs (A). The Y-axis shows the pairwise identity, and the X-axis indicates the positions in alignment. The dotted green line in (A) indicates the regions where recombination events may occur. The breakpoint was identified with SimPlot (B). The green and yellow lines in (B) represents KC21014_AH2012 and JX188454_AJ1102, respectively.