Construction and immunogenicity of a trypsin-independent porcine epidemic diarrhea virus variant

Abstract

Porcine epidemic diarrhea virus (PEDV) is a re-emerging enteropathogenic coronavirus that causes high mortality in neonatal piglets. The addition of trypsin plays a crucial role in the propagation of PEDV, but also increases the complexity of vaccine production and increases its cost. Previous studies have suggested that the S2' site and Y976/977 of the PEDV spike (S) protein might be the determinants of PEDV trypsin independence. In this study, to achieve a recombinant trypsin-independent PEDV strain, we used trypsin-dependent genotype 2 (G2) PEDV variant AJ1102 to generate three recombinant PEDVs with mutations in S (S2' site R894G and/or Y976H). The three recombinant PEDVs were still trypsin dependent, suggesting that the S2' site R894 and Y976 of AJ1102 S are not key sites for PEDV trypsin dependence. Therefore, we used AJ1102 and the classical trypsin-independent genotype 1 (G1) PEDV strain JS2008 to generate a recombinant PEDV carrying a chimeric S protein, and successfully obtained trypsin-independent PEDV strain rAJ1102-S2'_JS2008, in which the S2 (amino acids 894-1386) domain was replaced with the corresponding JS2008 sequence. Importantly, immunization with rAJ1102-S2'_JS2008 induced neutralizing antibodies against both AJ1102 and JS2008. Collectively, these results suggest that rAJ1102-S2'_JS2008 is a novel vaccine candidate with significant advantages, including no trypsin requirement for viral propagation to high titers and the potential provision of protection for pigs against G1 and G2 PEDV infections.

Keywords: chimeric virus; immunogenicity; neutralizing antibodies; porcine epidemic diarrhea virus (PEDV); trypsin-independent.

Figure 1

Sites R894 and Y976 in S protein are not determinants of trypsin dependence of PEDV strain AJ1102. (A) Amino acid alignment of S2 domains of AJ1102 and JS2008. Dotted green and purple boxes represent the S2′ site R894 and Y976, respectively. Black underline indicates the sequence of the fusion peptide (FP). (B) Schematic overview of the construction of recombinant BAC plasmids with a single point mutation (R894G or Y976H) or a double mutation (R894G/Y976H) in AJ1102 S protein. (C) Agarose gel electrophoresis of sgRNA-a and sgRNA-b and linearized pBAC-AJ1102 in vitro. (D) Identification of the recombinant BAC plasmids with amino acid mutations (pBAC-AJ1102-S-R894G, pBAC-AJ1102-S-Y976H, and pBAC-AJ1102-S-R894G/Y976H) with DNA sequencing. Red dotted boxes indicate amino acid mutation sites. (E) Observation of CPEs in Vero cells infected with different recombinant PEDVs at 24 hpi in the presence of trypsin. Scale bars, 100 μm. (F) Immunofluorescent assay of Vero cells infected with different recombinant PEDVs (MOI = 0.1) in the presence or absence of trypsin. Scale bars, 100 μm.

Figure 2

Growth characteristics of strains AJ1102 and JS2008 with and without trypsin. (A) Vero cells were infected with AJ1102 or JS2008 at a multiplicity of infection of 0.1 in the presence or absence of trypsin for 24 h, followed by IFA to detect viral infection. Scale bars, 100 μm. (B, C) Growth kinetics of AJ1102 (B) and JS2008 (C) in Vero cells in the presence or absence of trypsin. ns, not significant; **P < 0.01, ***P < 0.001.

Figure 3

Determination of the subdomains of the S2 protein crucial for trypsin dependence of strain AJ1102. (A) Construction strategy of three recombinant BAC plasmids, pBAC-AJ1102-S2(aa 894-993)_JS2008, pBAC-AJ1102-S2(aa 994-1386)_JS2008, and pBAC-AJ1102-S2(aa 894-1386)_JS2008, in which individual S2 subdomains of the AJ1102 S gene were replaced with the corresponding domains of JS2008. Red and black background frames represent the S2 gene sequences of strains AJ1102 and JS2008, respectively. (B) Schematic diagram of CRISPR/Cas9-mediated cleavage of pBAC-AJ1102 plasmid in vitro and homologous recombination between linearized plasmid pBAC-AJ1102 and DNA fragments of interest. (C) Identification of three recombinant BAC plasmids with DNA sequencing. (D) IFA of Vero cells infected with rAJ1102 or rAJ1102-S2(aa 894-1386)_JS2008, designated rAJ1102-S2′_JS2008, with or without trypsin for 24 h. Scale bars, 100 μm.

Figure 4

Growth characteristics of rAJ1102 and rAJ1102-S2′_JS2008. (A) Plaque assays of rAJ1102 and rAJ1102-S2′_JS2008 in Vero cells. (B, C) Growth kinetics of rAJ1102 (B) and rAJ1102-S2′_JS2008 (C) in Vero cells with or without trypsin. **P < 0.01, ***P < 0.001.

Figure 5

Evaluation of immunogenicity of recombinant PEDVs in piglets. (A) Schematic overview of animal experiments, including immune procedures and specimen collection schedule. (B, C) Detection of specific neutralizing antibody levels against strain AJ1102 (B) or JS2008 (C) in the sera of piglets immunized with wild-type or recombinant PEDVs on different days postvaccination. ns: not significant; *P < 0.05, **P < 0.01, ***P < 0.001.