Developing Next-Generation Live Attenuated Vaccines for Porcine Epidemic Diarrhea Using Reverse Genetic Techniques

Abstract

Porcine epidemic diarrhea virus (PEDV) is the etiology of porcine epidemic diarrhea (PED), a highly contagious digestive disease in pigs and especially in neonatal piglets, in which a mortality rate of up to 100% will be induced. Immunizing pregnant sows remains the most promising and effective strategy for protecting their neonatal offspring from PEDV. Although half a century has passed since its first report in Europe and several prophylactic vaccines (inactivated or live attenuated) have been developed, PED still poses a significant economic concern to the swine industry worldwide. Hence, there is an urgent need for novel vaccines in clinical practice, especially live attenuated vaccines (LAVs) that can induce a strong protective lactogenic immune response in pregnant sows. Reverse genetic techniques provide a robust tool for virological research from the function of viral proteins to the generation of rationally designed vaccines. In this review, after systematically summarizing the research progress on virulence-related viral proteins, we reviewed reverse genetics techniques for PEDV and their application in the development of PED LAVs. Then, we probed into the potential methods for generating safe, effective, and genetically stable PED LAV candidates, aiming to provide new ideas for the rational design of PED LAVs.

Keywords: PEDV; live attenuated vaccine; rational design; reverse genetic techniques; virulent protein.

Figure 1

Schematic representation of the PEDV virion (a) and its genome organization (b). The red and green bars in (b) represent the leader transcriptional regulatory sequence (TRS) and body TRS, respectively. The expression of ORF1a and ORF1b yields two known polyproteins (pp1a and pp1ab) through a -1 programmed reading frameshift (RFS). These polyproteins are then processed into 16 distinct nonstructural proteins (nsp1–16), with the numbers for pp1a and pplab indicating nsp1–16. Abbreviations: RNP, ribonucleoprotein; S, spike; ORF3, open reading frame (ORF) 3; E, envelope; M, membrane; N, nucleocapsid; 5′UTR, 5′ untranslated region; 3′UTR, 3′ untranslated region; A(n), polyadenylated tail; PLpro, papain-like cysteine protease; 3CLpro, 3C-like cysteine protease; RdRp, RNA-dependent RNA polymerase; Hel, helicase; ExoN, 3′-5′ exonuclease; N7-MTase, N7-methyltransferase; EndoU, endoribonuclease; 2′-O-MTase, ribose-2′-O-methyltransferase. Adapted from Niu et al. [13] and Jang et al. [6].

Figure 2

A diagrammatic representation of the development and design of next-generation live attenuated vaccines for PEDV using various reverse genetic systems (RGSs). (a) Targeted RNA recombination scheme for creating recombinant PEDV with ORF3 gene deletion/substitution. This scheme can be used to produce the interspecies chimeric virus mPEDV (Step 1) or recombinant PEDV variants lacking the ORF3 gene (Step 2). Synthetic RNAs transcribed from transfer vectors are introduced into PEDV- (Step 1) or mPEDV (Step 2)-infected cells through electroporation. A single recombination event within the 3′ region of ORF1b in the donor RNA and viral genome results in the generation of a recombinant genome. Selection of recombinant progeny viruses is based on their ability to form plaques in murine cell (LR7) monolayers (Step 1) or infect Vero cells while losing the ability to infect LR7 cells (Step 2). (b) BAC-derived PEDV reverse genetic system. The diagram displays the genome structure of PEDV and the genome fragments used for cloning the entire viral genome into a BAC plasmid (pBeloBAC11). The complete viral genome is divided into twelve fragments, with the CMV promoter at the 5′ end and HDVr and BGH sequences at the 3′ end. Transfection of the PEDV BAC plasmid into Vero cells leads to the rescue of PEDV. (c) Vaccinia virus vector-based reverse genetics system for PEDV. The PEDV genome is divided into multiple fragments, which are used to construct intermediate plasmids. These plasmids cover the entire PEDV genome and are introduced into the vaccinia virus genome through homologous recombination with GPT as a positive or negative selection marker. After linearization of the vaccinia virus genome by endonuclease digestion, the infectious mRNA is transcribed and electroporated into Vero cells to rescue recombinant PEDV. (d) An in vitro ligation/transcription approach for generating recombinant PEDV. The diagram shows the structure of the PEDV genome with the in vitro ligation method. The full-length cDNA of PEDV is assembled directionally using in vitro ligation, with a T7 promoter at the 5′ end and a poly (A) tail at the 3′ end. The assembled full-length cDNA is transcribed into genomic RNA in vitro and then introduced into Vero cells through electroporation to rescue recombinant PEDV. (e) Yeast vector-based PEDV reverse genetic system. The diagram shows the genome structure of PEDV and the overlapping DNA fragments used to clone the PEDV genome into a YAC vector (pYES1L). The first and last fragments have overlapping sequences for the YAC vector, with the CMV promoter fused to the 5′ end of the viral genome and HDVr and BGH added after the poly (A) sequence at the 3′ end. All cDNA fragments are transformed with a linearized YAC vector (pYES1L) into yeast competent cells for assembly through transformation-associated recombination. Positive clones are identified and extracted, and the full-length cDNA clones are transfected into Vero cells for virus recovery. In this figure, five reverse genetic systems (RGSs) utilized in the development of PEDV vaccines are depicted with distinct colored backgrounds (This schematic diagram is the author’s own creation).