Detection and phylogenetic analysis of porcine epidemic diarrhea virus in central China based on the ORF3 gene and the S1 gene

Porcine epidemic diarrhoea (PED) is an acute, highly contagious disease of swine caused by the PED virus (PEDV), which leads to severe vomiting and diarrhea along with dehydration and high mortality in new-born piglets [1]. PEDV belongs to the family Coronaviridae, genus Alphacoronavirus and was first reported in England [2]. PEDV has now been reported worldwide, including in Belgium, France, Japan, Korea, Italy, Thailand, USA, Canada and Mexico [1‐5] and poses severe economic burdens. Since 2010 serious PED has been detected in China [6]. PEDV is an enveloped ssRNA coronavirus with a 28 kb genome, including seven open reading frames (ORFs), a 5′ untranslated region (UTR), and a 3′ UTR with a polyadenylated tail. The seven ORFs encode four structural proteins, spike (S), envelope (E), membrane (M) and nucleocapsid (N) and three non-structural proteins, replicases 1a, 1b and ORF3 [7, 8]. The S protein contains a specific receptor binding site that is important for cell membrane fusion and virus entry and is an antigenic target for neutralising antibodies [9]. The M protein is the most abundant surface protein, and coexpression with E protein to form pseudo-particles results in interfering genic activity [10]. The N protein is highly conserved and binds to virion RNA to provide a structural basis for the helical nucleocapsid, and it is used for early diagnosis [11]. For non-structural proteins, replicases 1a and 1b are multi-functional and associated with viral genome replication [12], and the accessory ORF3 protein is thought to influence virulence [13].

The S1 domain (amino acids (aa) 21–793) contains two subdomains: NTD (aa 21–324) and CTD (aa 253–638) [14], with the latter binding to porcine aminopeptidase N (pAPN) which is important for cell membrane fusion and virus entry and it is the antigenic target of neutralising antibodies [15]. Thus, the variable S1 gene has been widely used for studies of PEDV genetic evolution and diversity [16, 17]. PEDV CV777 has three main neutralising epitopes: aa 498–637 (CO-26 K equivalent, COE gene) [18], aa 747–754 (YSNIGVCK) and aa 763–770 (LQDGQVKI) [19]. Sequence analyses indicated that the latest PEDV isolated strains in China were different from attenuated CV777 [16, 20, 21]. Multiple mutations of the S protein resulted in two PEDV genotypes, G1 and G2 (classical strains and variant strains) [16], and a recent study suggested that antigenic variation exists between G1 and G2 [22]. The variable S1 gene has been widely used for studies of PEDV genetic evolution and diversity [16, 17]. The accessory ORF3 gene is highly relevant to the virulence of PEDV as previously stated [13], and reduction in virulence is produced through cell culture adaptation [23, 24]. The ORF3 gene of attenuated vaccine isolates has a continuous deletion of 17 amino acids (aa 82–99), thus distinguishing the vaccine isolates from variant PEDV [24]. Therefore, the ORF3 gene has been the focus of molecular epidemiology PEDV studies [16, 20, 21, 25]. As aforementioned, we chose the S1 and ORF3 genes as the target genes for phylogenetic analysis.

Phylogenetic analysis of variations in the S1 gene of isolates collected in China distinguished the PEDV genotypes G1 and G2, including 1a classical PEDV, 1b classical PEDV, 2a circulating PEDV and 2b circulating PEDV subgroups [26]. Based on the ORF3 gene, the phylogenetic analysis of PEDV isolates in China manifested in genotypes G1 and G2, the G2 genotype including 2a PEDV and 2b PEDV subgroups [27]. To investigate further infectivity, genetic diversity and molecular epidemiology of PEDV, we performed phylogenetic analyses based on the S1 and ORF3 genes of the latest Chinese isolates collected. In this study, 129 samples were obtained from 67 farms in 17 cities of central China, and the S1 and ORF3 genes of 21 representative field strains from 21 farms in 11 cities were sequenced and analysed.