The Characterization and Pathogenicity of a Recombinant Porcine Epidemic Diarrhea Virus Variant ECQ1

Abstract

Porcine epidemic diarrhea virus (PEDV), a re-emerging enteropathogenic coronavirus, has become the predominant causative agent of lethal diarrhea in piglets, resulting in huge economic losses in many countries. Furthermore, the rapid variability of this virus has increased the emergence of novel variants with different pathogenicities. In this study, 633 fecal samples collected from diarrheic piglets in China during 2017-2019 were analyzed, and 50.08% (317/633) of these samples were PEDV-positive. The full-length spike (S) genes of 36 samples were sequenced, and a genetic evolution analysis was performed. The results showed that thirty S genes belonged to the GII-a genotype and six S genes belonged to the GII-b genotype. From the PEDV-positive samples, one strain, designated ECQ1, was successfully isolated, and its full-length genome sequence was determined. Interestingly, ECQ1 is a recombinant PEDV between the GII-a (major parent) and GII-b (minor parent) strains, with recombination occurring in the S2 domain of the S gene. The pathogenicity of ECQ1 was assessed in 5-day-old piglets and compared with that of the strain EHuB2, a representative of GII-a PEDV. Although both PEDV strains induced similar fecal viral shedding in the infected piglets, ECQ1 exhibited lower pathogenicity than did EHuB2, as evidenced by reduced mortality and less severe pathological changes in the intestines. These data suggest that PEDV strain ECQ1 is a potential live virus vaccine candidate against porcine epidemic diarrhea.

Keywords: epidemiology; pathogenicity; porcine epidemic diarrhea virus; recombination; variant.

Figure 1

Detection of PEDV infection in piglets in 19 provinces of China. (a) The overall prevalence of PEDV infection in piglets with diarrhea from 2017 to 2019. (b) The seasonal prevalence of PEDV infection in piglets with diarrhea from 2017 to 2019.

Figure 2

Phylogenetic tree analysis based on the S gene of PEDV. The maximum-likelihood phylogenetic tree (ML tree) was constructed using IQ-TREE software. The PEDV strains isolated in this study are indicated by red pentagons and blue circles.

Figure 3

Isolation and identification of PEDV strain ECQ1 and phylogenetic tree analysis based on its complete genome. (a) Cytopathic effects were observed in PEDV ECQ1-infected Vero cells. (b) IFA for identifying ECQ1 in Vero cells using monoclonal antibodies against the PEDV N protein. (c) RT-PCR for amplifying an expected product size of 563 bp for the PEDV N gene detection. M: DL 2000 marker; Lane 1: negative control; Lane 2: ECQ1. (d) Determination of the growth kinetics of PEDV strain ECQ1 in Vero cells. Vero cells were inoculated with ECQ1 strains (MOI = 0.1). Cells were harvested at different time points post-infection (6, 12, 18, 24, 30, 36, 42 and 48 hpi) and then subjected to a TCID₅₀ assay. (e) The maximum-likelihood phylogenetic tree was constructed using IQ-TREE software. The PEDV strain isolated in this study is indicated by red pentagons.

Figure 4

Recombination analysis of the complete genomic sequence of PEDV strain ECQ1. (a) Recombination detection program and SimPlot software were used to conduct the recombination analysis for the entire gene. Potential recombination events were identified based on strong p value (<10⁻¹⁰). (b) The PEDV ECQ1 genome exhibited recombination originating from two sources: 1. from positions 1 to 24,116 bp and 26,392 bp to the end of the PEDV-1C-like strain and 2. at positions 24,116–26,392 bp for the 17GXCZ-1ORF3c-like strain. (c) The maximum-likelihood phylogenetic tree was constructed using IQ-TREE software.

Figure 5

Pathogenicity of PEDV strain ECQ1 in piglets. (a) Clinical evaluation of piglets following PEDV challenge. Piglets, aged 5 days, were examined 2 days after inoculation with PEDV strains ECQ1 and EHuB2, with DMEM as a control. (b) Percentage of piglets that survived in each group. (c) Fecal excretion by 5-day-old piglets inoculated with different PEDV strains. ** p < 0.01, ECQ1 vs. EHuB2.

Figure 6

Intestinal lesions of PEDV-challenged piglets at 2 DPI. Macroscopic lesions found in piglets of ECQ1 (a), EHuB2 (b) and mock groups (c) in necropsy. H&E-stained jejunum (d,h,l) and ileum (e,i,m) tissue sections in the ECQ1 (d,e), EHuB2 (h,i) and mock groups (l,m). Immunohistochemically stained jejunum (f,j,n) and ileum (g,k,o) tissue sections in the ECQ1(f,g), EHuB2 (j,k) and mock groups (n,o).