Comparative Genomic Analysis of Classical and Variant Virulent Parental/Attenuated Strains of Porcine Epidemic Diarrhea Virus

Abstract

Since 2010, the variant porcine epidemic diarrhea virus (PEDV) has been the etiological agent responsible for the outbreak of porcine epidemic diarrhea (PED) worldwide. In this study, a variant PEDV strain YN1 was isolated, serially propagated on the Vero cells and was characterized for 200 passages. To better elucidate the molecular basis of Vero cell adaptation of variant PEDV strains, we sequenced, compared, and analyzed the full-genome sequences of parental YN1 and passages 15, 30, 60, 90, 144, and 200. The results showed that the variations increased with the viral passage. The nucleotides sequences of non-structural protein (NSP)2, NSP4-7, NSP10, NSP12 and NSP13 genes did not change during the Vero cell adaptation process. After comparison of the variation characteristic of classical, variant virulent/attenuated strains, it was found that attenuation of PEDV virus was associated with 9-26 amino acid (aa) changes in open reading frames (ORF) 1a/b and S protein, early termination in ORF3, 1-3 aa changes in E, M and N protein and some nucleotide sequences' synonymous mutations. The aa deletion at about 144 aa of S protein could be the attenuation marker for the PEDV. The pig study showed that the early termination in ORF3 was more important for virus cell adaptation than virus attenuation.

Keywords: genomic analysis; variant PEDV; virus attenuation; virus cell adaptation.

Figure 1

Virus identification, virus titration and propagation kinetic. (A) Control Vero cells; (B) The cytopathic effects (CPE) of Vero cells infected with YN15 at 24 h post-inoculation; (C) Blank control cells in immunofluorescence assay (IFA); (D) Fluorescent signal in porcine epidemic diarrhea virus (PEDV)-infected cells; (E) Virus titer of YN1, YN15, YN60 and YN144; (F) One-step growth curve of YN1, YN15, YN60 and YN144 in Vero cells.

Figure 2

The necropsies results of pigs infected with YN15 (A), YN144 (B) and the control group (C).

Figure 3

The immunohistochemical (IHC) assay of pigs infected with YN15 (A) and YN144 (B) as well as control group (C) are indicated. Arrows indicate positive signal of IHC assay in the intestinal tissue. Original magnification ×200.

Figure 4

The immunohistochemical (IHC) results were scored by the distribution or extent of positive signal of IHC assay, each data point represents an individual animal in each treatment group, and median values are indicated by horizontal bars. ** p < 0.01.

Figure 5

The change rate of non-structural and structural proteins of different passages compared with their virulent parental strain YN1. The change rate is the percentage of changed amino acid in their corresponding proteins (%). Non-structural proteins: NSP1, NSP3, NSP9, and NSP11 proteins; Structural proteins: S, ORF3, E, M, and N proteins.

Figure 6

Similarity plot of the full-genome of all Vero cell-adapted passages with parental YN1. The similarity plot was constructed using the two-parameter (Kimura) distance model with a sliding window of 200 bp and step size of 20 bp. The vertical and horizontal axes indicate the percent nucleotide similarity and nucleotide position (bp) in the graph, respectively.

Figure 7

The amino acid change in ORF1a/b, S, ORF3, E, M, and N proteins. Pair A: Virulent DR13/Vaccine DR13; Pair B: YN1/YN144.The X-axis represents the aa changes in the vaccine DR13 and YN144, and the Y-axis represents aa change position. The upper triangle represents one mutated amino acid.