Genetic variations in S gene of porcine epidemic diarrhoea virus from 2018 in Sichuan Province, China

Abstract Porcine epidemic diarrhoea virus (PEDV) belongs to the family Coronavirus, a genus of coronavirus, a highly contact‐infectious intestinal disease pathogen. In this study, we downloaded 62 PEDV S gene sequences uploaded to GenBank, including 10 uploaded by our laboratory from 2018, and constructed a PEDV S gene evolution tree using MEGA V7.0 software. Phylogenetic tree analysis indicated that the genogroup of PEDV in Sichuan Province was divided into three coexisting genogroups (GII‐a, GII‐b and GI‐a), of them, GII‐a has become the main genogroup in the province due to its prevalence and range of spread. Amino acid sequence analysis showed that there were amino acid insertions and deletions in the S protein encoded by the amplified S gene, and there were amino acid mutations in the COE and SS6 of the epitope in the amplified S protein. These results provide a basic research theory for understanding the prevalence of PEDV variation and controlling PED in Sichuan.


| Samples
Naturally infected pigs suspected of having PEDV were collected in various parts of Sichuan Province and the intestinal tissues and contents of the affected pigs were stored at -80°C after grinding with liquid nitrogen.

| Primer design and synthesis
Four pairs of specific primers were designed according to the conserved regions of CV777 (GenBank:AF353511.1) published in GenBank and PEDV mutants published in recent years, and the S gene was divided into four fragments (S1, S2, S3 and S4). During amplification, there was partial overlap between each segment, the primers are synthesized by Sangon Biotech. Primer information is shown in Table 1.

| Complete genome extraction and sequencing
Samples were diluted with five volumes of 0.9% saline (w/v), frozen and thawed with liquid nitrogen three times and then clarified by centrifugation for 5 min at 3,000 rpm. Three hundred microlitres of the supernatants were used for RNA extraction using TRIzol (Total RNA Extractor)(Solarbio Co), according to the manufacturer's instructions. cDNA was synthesized by PrimeScript TM RT Reagent Kit (TaKaRa Co), according to the manufacturer's instructions. The S gene fragment was amplified by 2 X High Fidelity PCR Master Mix (Sangon Biotech Co). Briefly, the reaction was carried out at 95°C for 3 min (for denaturation), 35 cycles of 95°C for 15 s (for denaturation), 52°C/54°C for 15 s (for annealing) and 72°C for 90 s (for extension), followed by 72°C for 5 min (for final extension). The products were examined by electrophoresis using a 1.0% agarose gel (Sangon Biotech Co), the product recovered by gel recovery kit (Sangon Biotech Co) and the recovered product used to add adenine at the end, according to the manufacturer's instructions. The amplified products were purified and cloned into pMD-19T. The cloned products were then sequenced (Sangon Biotech Co). For each of the amplified fragments, three clones were sequenced.
The sequencing results were assembled by Geneious V4.8.4 software and the flanking sequence removed by sequence matching. The complete S gene was obtained and submitted to the NCBI database.

| Phylogenetic analysis
Sequences of PEDV S gene Sichuan isolates were compared with other representative PEDV S gene sequences using MEGA V7.0 software.

TA B L E 1 Sequences of amplified PEDV S gene
The phylogenetic tree was calculated using the neighbour-joining (NJ) method. Bootstrap values were calculated based on 1,000 repeats of the alignment. The phylogenetic tree was used to analyse the genogroup evolution of PEDV isolates in Sichuan Province from 2018.
The amino acid/nucleotide sequence homology of the S gene of 25 strains and the amino acid differences between isolates and CV777 and CH/ZMDZY/11 were analysed by Clustal W with MegAlign software.

| PEDV S gene amplification sequence
The clinical data of 10 PEDV samples and the size of S gene amplification fragments are shown in Table 2.

Distribution of isolates in
Sichuan Province is shown in Figure 1.

| Phylogenetic analysis of the S gene
According to the phylogenetic analysis of the S gene, 9 of the 10 PEDV strains in this study were subtype GII and one was of subtype GI, distributed in three subgroups: GII-a, GII-b and GI-a ( Figure 2).
In terms of the number of infections, GII-a has become the main genogroup in Sichuan. Our GII subtype isolates showed a close relationship to CH/HNAY/2015, PEDV-WS and PEDV-10F isolates. Only one GI subtype isolate was closely related to CV777, CH/S and LZC isolates.
The neutralizing epitopes of amplified S gene fragments were compared with the CV777. The results showed that the sequences at SS2 (748-755aa) and 2C10 (1368-1374aa) were conserved between the latest Sichuan PEDV isolates and CV777 strain, however, the sequences at positions COE (499-638aa) and SS6 (764-771aa) were variable (Table 3).

| D ISCUSS I ON
At present, PEDV remains prevalent in China with several reports implicating the PEDV GII genogroup as the specific causative agent in many recent outbreaks (Daesub & Bongkyun, 2012;Douglas et al., 2013). Using the DNA sequence of the S gene fragment, we analysed the genetic variation and phylogenesis of PEDV strains collected in 2018 from farms in various regions of Sichuan Province. Our results revealed that eight of the amplified S genes were closely related to the isolates of the GII-a genogroup, which were previously identified after the 2011 outbreaks in China and the United States. Moreover, our phylogenetic analysis showed that the PEDV strains in Sichuan Province were very similar to those previously reported (Wang, Fang, Fang, & Xiao, 2016), indicating that the currently prevalent PEDV strains in Sichuan are of GII genogroup.
In terms of PEDV vaccination, the current attenuated vaccines produced using the classical strains CV777 and ZJ08, which both belong to the GI genogroup, may not be sufficient to confer complete protection against the emerging PEDV strains of GII genogroup (Daesub & Bongkyun, 2012;Douglas et al., 2013). This partial immunoprotection has been demonstrated in clinical practice where pigs vaccinated against the classical strains (GI genogroup) remained vulnerable to PEDV infection (Luo et al., 2012;Sun et al., 2012;Tian et al., 2013). Researchers from China and the United States have also confirmed that all available commercial vaccines (GI genogroup) do not provide adequate immune protection against the currently prevalent strains (GII genogroup) (Lin et al., 2016;Opriessnig et al., 2017). After PED outbreaks in the United States in 2013, an inactivated vaccine was developed by Collin et al. (2015) based on the isolated US PEDV strain (GII genogroup) and was used to vaccinate a group of 4-week-old piglets.
The inactivated vaccine was shown to trigger sufficient humoral immunity against PEDV thereby preventing subsequent infections (Collin et al., 2015). Baek et al. (2016)