Biological Characteristics and Pathogenicity Analysis of a Low Virulence G2a Porcine Epidemic Diarrhea Virus.

Since the outbreak caused by a porcine epidemic diarrhea virus (PEDV) variant in 2010, the current epidemic of PEDV genotype 2 (G2) has caused huge economic losses to the pig industry in China. In order to better understand the biological characteristics and pathogenicity of the current PEDV field strains, 12 PEDV isolates were collected and plaque purified during 2017 to 2018 in Guangxi, China. The neutralizing epitopes of the spike proteins and the ORF3 proteins were analyzed to evaluate genetic variations, and they were compared with the reported G2a and G2b strains. Phylogenetic analysis of the S protein showed that the 12 isolates were clustered into the G2 subgroup (with 5 and 7 strains in G2a and G2b, respectively) and that they shared 97.4 to 99.9% amino acid identities. Among them, one of the G2a strains, CH/GXNN-1/2018, which had a titer of 106.15 PFU/mL, was selected for pathogenicity analysis. Although piglets infected with the CH/GXNN-1/2018 strain exhibited severe clinical signs and the highest level of virus shedding within 24 h postinfection (hpi), recovery and decreased virus shedding were seen after 48 hpi, and no piglets died during the whole process. Thus, the CH/GXNN-1/2018 strain had low virulence in suckling piglets. Virus neutralizing antibody analysis showed that the CH/GXNN-1/2018 strain induced cross-protection against both homologous G2a and heterologous G2b PEDV strains as early as 72 hpi. These results are of great significance for understanding PEDV in Guangxi, China, and they provide a promising naturally occurring low-virulent vaccine candidate for further study. IMPORTANCE The current epidemic of porcine epidemic diarrhea virus (PEDV) G2 has caused huge economic losses to the pig industry. Evaluation for low virulence of the PEDV strains of subgroup G2a would be useful for the future development of effective vaccines. In this study, 12 field strains of PEDV were obtained successfully, and they were characterized from Guangxi, China. The neutralizing epitopes of the spike proteins and the ORF3 proteins were analyzed to evaluate antigenic variations. One of the G2a strains, CH/GXNN-1/2018, was selected for pathogenicity analysis, and it showed that the CH/GXNN-1/2018 strain had low virulence in suckling piglets. These results provide a promising naturally occurring low-virulent vaccine candidate for further study.

Phylogenetic and Spatiotemporal Analyses of Porcine Epidemic Diarrhea Virus in Guangxi, China during 2017-2022.

Since 2010, porcine epidemic diarrhea virus (PEDV) has swept across China and spread throughout the country, causing huge economic losses. In this study, 673 diarrhea samples from 143 pig farms in Guangxi during 2017-2022 were collected and detected for PEDV. Ninety-eight strains were selected for S1 gene analyses and these strains were classified into four subgroups (G1b, G2a, G2b and G2c), accounting for 1.02 (1/98), 75.51 (74/98), 16.33 (16/98) and 7.14% (7/98) of the total, respectively. Importantly, an increased number of strains in the G2c subgroup was found from 2019 onwards. Bayesian analysis revealed that Guigang may have been the epicenter of PEDVs in Guangxi. In addition, Guigang was identified as the primary hub from which PEDVs spread via two routes, namely Guigang-Wuzhou and Guigang-Laibin. Moreover, several coinfections of novel PEDV variants bearing large deletions in the partial S1 protein and PEDVs possessing an intact partial S1 protein were found in pigs. Further recombination analyses indicated that two of the strains, 18-GXNN-6 and 19-GXBH-2, originated from intra-genogroup recombination. Together, our data revealed a new profile of PEDV in Guangxi, China, which enhances our understanding of the distribution, genetic characteristics and evolutionary profile of the circulating PEDV strains in China.

Comparative Characterization and Pathogenicity of a Novel Porcine Epidemic Diarrhea Virus (PEDV) with a Naturally Occurring Truncated ORF3 Gene Coinfected with PEDVs Possessing an Intact ORF3 Gene in Piglets.

Coinfection caused by various genotypes of porcine epidemic diarrhea virus (PEDV) is a new disease situation. We previously reported the coexistence of PEDV strains containing different ORF3 genotypes in China. In this study, the PEDV strains 17GXCZ-1ORF3d and 17GXCZ-1ORF3c were isolated and plaque-purified from the same piglet, which had a natural large deletion at the 172-554 bp position of the ORF3 gene or possessed a complete ORF3 gene, respectively. Meanwhile, 17GXCZ-1ORF3d had >99% nt identity with 17GXCZ-1ORF3c in the 5'UTR, ORF1a/1b, S, E, M, N and 3'UTR regions but only demonstrated low nucleotide identities (80.5%) in the ORF3 gene. To elucidate the pathogenicity, 7-day-old piglets were infected. Piglets infected with these two PEDV strains exhibited severe clinical signs and shed the virus at the highest level within 96 hpi. Compared with the piglets inoculated with the 17GXCZ-1ORF3c strain, the piglets inoculated with the 17GXCZ-1ORF3d strain had higher mortality rates (75% vs. 50%), an earlier onset of clinical signs with a significantly higher diarrhea score, lower VH:CD ratios and a higher percentage of PEDV-positive enterocytes. This study is the first to report PEDV coinfections with different ORF3 genotypes, and a PEDV strain with a large deletion in the ORF3 gene might have the advantage of a potential genetic marker, which would be useful during vaccine development.

Genetic Diversity of Porcine Epidemic Diarrhea Virus With a Naturally Occurring Truncated ORF3 Gene Found in Guangxi, China.

Porcine epidemic diarrhea virus (PEDV) is one of the major enteric pathogens, causing severe enteric disease, resulting in enormous economic losses. The ORF3 gene encodes an accessory protein which is related to the infectivity and virulence of PEDV. In this study, 33 PEDV positive field samples were collected from Guangxi, from 2017 to 2019, and the genetic diversity of ORF3 was investigated. Thirty-eight strains of ORF3 were obtained, and these were composed of five strains of ORF3 named Guangxi naturally truncated strains that were 293 bp in length, with continuous deletions from 172 to 554 bp. The Guangxi naturally truncated strains encoded a truncated protein of 89 amino acids, which had clustered into a new group referred to as Group 3, and these might be involved in the variations of virulence. Three genotypes (G1-1 subgroup, G1-3 subgroup, and Group 3) existed simultaneously in Guangxi based on the genetic and evolutionary analysis of the ORF3 gene. The sequence information in the current study will hopefully facilitate the establishment of a diagnostic method that can differentiate the PEDV field stains. Continued surveillance will be useful for monitoring PEDV transmission. Differentiation of the ORF3 genes in PEDV field strains can help us to choose an appropriate PEDV vaccine candidate in the future and prevent outbreaks of PED more effectively.