Discussion
China is the largest country for pig farming in the world, with an annual pig slaughter in 2020 about 600 million. The re-emergence of PEDV especially the variant strains since 2010 has brought a great threat to Chinese pig industry. PEDV was the major causative agent that lead to the diarrheal disease and death in piglets in a comparison with other coronaviruses, such as TGEV, PDCoV, and SADS-CoV (Su et al., 2020). Although a variety of commercial vaccines based on classical GI strain CV777 and the variant GII strain AJ1102 have been employed to control the outbreak of PEDV, their efficacies have been always poor. The immunity failure was associated with the constant variation of virus and the an insufficient mucosal immunity induced by the vaccines (Guo et al., 2019). Therefore, understanding the prevalence and variation characterization of PEDV genome is of importance in preventing PEDV prevalence. In a recent study, 49 complete S genes were sequenced in China from 2014 to 2016, and novel insertions, deletions, and multiple S gene recombination events were found in PEDV variant strains (Fan et al., 2020). Lei et al. collected 184 specimens from pig farms in China in 2017–2018 and detected an average PEDV-positive rate of 38.04% (Tan et al., 2020). Zhang et al. made a survey of molecular characteristics of PEDV in Henan province, China in 2015–2019 and found PEDV existed widely in both PEDV-vaccine immunized (25.00%) and non-immunized swine herd (62.29%). Sixteen of the sequenced PEDV Henan strains were located in the GII clade (H. Zhang et al., 2021). However, there is no nationwide epidemiological survey of PEDV in the past two years. Furthermore, African swine fever (ASF) outbreaks were declared in China since 2018 and quickly swept through the whole country (Bao, Qiu, Luo, Rodriguez, & Qiu, 2021). Management and prevention measures of epidemic diseases have been altered in pig farms. It is still unknown about if the prevalence characterization of PEDV has changed under the background of ASF. In this study, 115 PEDV positive samples were collected in 17 different provinces throughout China, including large swine-raising provinces, such as Sichuan, Henan, Hunan, and Shandong province. Our data showed that the positive rate of PEDV was from 10.77% to 25.10% from March 2020 to April 2021, indicating that PEDV was still one of the major pathogenic agents in Chinese swine herd. Our data also showed that the positive rate of PEDV was the highest in October and December. Thus, we should pay more attention on the prevention of PEDV when the weather turns cold, such as a turn of autumn and winter and the winter.
The full-length nucleotide sequences of S genes of 91 field strains were successfully sequenced. Based on the phylogenetic analysis for the S genes, all of the PEDV strains could be classified into two main genotypes and five subgroups, including GI-a (classical strain), GI-b (attenuated classical strain), GI-c (S-INDEL strain), GII-a, and GII-b (variant strain). All strains detected in the study shared 94.2%–100% homology with each other. A majority of them belonged to GII genotype (84 out of 91) which represented the pandemic strains of PEDV in recent years. GII genotype strains shared 93.2%–93.8% and 96.7%–99.1% homology with attenuated Chinese vaccine strains CV777 and AJ1102, respectively. Vaccines based on CV777 strain was widely used and contributed to a good control of PEDV in China before 2010 (Yang et al., 2013). However, the variant strains which were clustered into different subgroup (GII) shared lower homology with CV777 (GI). The different genotypes between CV777 and PEDV epidemic strains may lead to an incomplete protection provided by the vaccines in China since 2010.
The detected strains CH/JSXZ/202012, CH/SCYB/202012, CH/SCGA/202103, CH/SCST/202004, CH/HNBR/202101, CH/JXXG/202101, and CH/HNSQ/202102 were more related to S-INDEL strains. They were obtained from four provinces of China and classified into GI-c subgroup, thus suggesting that S-INDEL like strains have spread and circulated in China. Seven of S-INDEL like strains shared a high homology of 98.6%–99.3% with each other. Although the presence of S-INDEL strains of PEDV have been described to induce less severe symptoms and low fatality rate as compared with non-INDEL strains (Lin et al., 2015; Vlasova et al., 2014), the pathogenicity of this subgroup is still controversial. Severe diarrhea and vomiting, along with high mortality rates were associated with the S-INDEL PEDV strains in some European countries (Stadler et al., 2015). Therefore, the pathogenicity and infection rate of S-INDEL like strains in China should be constantly monitored.
To date, S-INDEL strains have been detected widely in the world including America, Asia, and Europe. The S-INDEL strains could be separated into two distant clades (INDEL 1 and INDEL 2) based on the characteristics of the S-INDEL genotype (de Nova et al., 2020). INDEL 2 contained prototype American (OH851) and Europe strains (FR/001/2014), as well as the first identified Chinese S-INDEL strain ZL29. Some other Asia S-INDEL strains belonged to the INDEL 2 clade. A further phylogenetic analysis using the seven S-INDEL like strains and reference S-INDEL strains retrieved from the NCBI nucleotide database showed that all of the detected S-INDEL like strains were categorized into INDEL 2. However, they were clearly clustered within a new cluster in INDEL 2 clade (Figure 5). Although the identified seven S-INDEL like strains had the common four aa deletions (58QGVN61) in line with reference S-INDEL strains, they had novel three amino acid mutations and one deletion located at the positions 156-160 aa. As previously described, INDEL 2 clade might originate from virulent strains DR13, Italy/7239/2009 or other field NON-INDEL strains, whereas INDEL 1 showed a common ancestor including CV777 or other PEDV strains detected in China before 2010 (Guo et al., 2019; J. Zhang et al., 2018). According to our results, the novel S-INDEL like strains detected in this study were originated from FR/001/2014 in INDEL 2 clade and vaccine strain AJ1102. This indicated that natural recombinant events might exist among variant strains and vaccine strains in China. Therefore, the use of live vaccine strains should be carefully considered especially in PEDV positive pig farms.
Spike protein is the most variable protein of PEDV. The variation of S protein is considered to be responsible for changes of viral antigenicity, determination of the genetic diversity for PEDV and affected the viral virulence (Suzuki, Terada, Enjuanes, Ohashi, & Kamitani, 2018; Van Diep et al., 2020). To evaluate whether the antigenicity of PEDV field strains has changed as compared with vaccine strains used in China, the neutralizing antigenic epitopes on S protein were analyzed. Several single nucleotide polymorphisms (SNPs) were observed on the S1A,SS6,2C10 epitopes and COE domain. Five main serine substitution (520A-S, 552T-S, 597G-S, 764L-S, 766D-S) and one Glutamate substitution (636Q-E) were found in the field strains as compared with the vaccine strain CV777. One common substitution in S1A (479S-A) and multiple mutations (499I-T, 502V-I, 524H-S/L/Y, 539F-L, 566K-N, 569D-A/N, 606Y-H, 612G-V/S and 634P-S) in individual sequences were detected in the field stains as compared with the vaccine strain AJ1102. These mutations might involve in an immune escape and the antigenicity of virus, resulting in the less effective immune protection provided by the commercial vaccines CV777 and AJ1102. Notably, five PEDV samples with new insertion in the S protein sequences were detected. CH/GDMM/202012 had a continuous 5 aa insertion (360QGRKS364) located in S1 domain while the other four (CH/GXDX/202010, CH/AHBZ/202010, CH/AHLA/202010 and CH/HNLY/202003) had three or four amino acid insertions (1279DVF1281 or 1278VDVF1281) located in S2 domain. To the best of our knowledge, this is the first report for these novel S-insertion variants. The S-INDEL-variants were proved to have decreased virulence in host, whereas one strain with a novel four-amino-acid insertion in the COE domain was highly pathogenic to neonatal pigs (Ji et al., 2021). Although we did not obtain the isolated novel S-insertion strains, it could be predicted that the conformational structure of S protein might have changed, resulting in a change for the pathogenicity of these strains.
Collectively, this study described the nationwide investigation of the PEDV prevalence in China in recent years. The major causative virus strains were GII genotype variants with the ratio of 92.3%. Notably, seven S-INDEL like strains were detected in four provinces in China. Alignment of S deduced aa sequences revealed novel mutations and deletion compared with prototype S-INDEL strain. Based on recombination analyses, the novel S-INDEL like strains were originated from FR/001/2014 in INDEL 2 clade and vaccine strain AJ1102. Moreover, variant PEDV strains with novel insertions (360QGRKS364 and 1278VDVF1281) in S protein sequences were detected and needed to be addressed on the specific function of insertions. In addition, we also identified multiple mutations in the aa sequences of S proteins of the variant strains compared to those of the vaccine strains. These PEDV mutants derived from genetic mutations, deletions, insertions, and recombination of the S genes might be the major cause of antigenic drift and immune failure. The molecular characterization of S protein should be investigated continuously and would work in the control and prevention of PED in China.