Molecular Characterization and Phylogenetic Analysis of New Variants of the Porcine Epidemic Diarrhea Virus in Gansu, China in 2012

Between January 2012 and March 2012, the infection rates of porcine epidemic diarrhea virus (PEDV) increased substantially in vaccinated swine herds in many porcine farms in Gansu Province, China. The spike (S) glycoprotein is an important determinant for PEDV biological properties. To determine the distribution profile of PEDV outbreak strains, we sequenced the full-length S gene of five samples from two farms where animals exhibited severe diarrhea and high mortality rates. Five new PEDV variants were identified, and the molecular diversity, phylogenetic relationships, and antigenicity analysis of Gansu field samples with other PEDV reference strains were investigated. A series of insertions, deletions, and mutations in the S gene was found in five PEDV variants compared with classical and vaccine strains. These mutations may provide stronger pathogenicity and antigenicity to the new PEDV variants that influenced the effectiveness of the CV777-based vaccine. Our results suggest that these new PEDV variant strains in Gansu Province might be from South Korean or South China, and the effectiveness of the CV777-based vaccine needs to be evaluated.


Sequence Analysis of the S Gene
The nucleotide sequences of the S region were 4,161 bp long for JY5C, JY6C, JY7C, YJ3F, and YJ7C. Their S proteins were 1,386 aa long with a predicted Mr of 151.7 KDa. The S protein of JY5C, JY6C, JY7C, and YJ3F contained 28 Asn-Xaa-Ser/Thr sequons and 22 asparagines predicted to be N-glycosylated ( Figure 1A,B). The S protein of the YJ7C strain contained 29 Asn-Xaa-Ser/Thr sequons and 23 asparagines predicted to be N-glycosylated ( Figure 1C). However, the S protein of the CV777 vaccine strain contained 29 Asn-Xaa-Ser/Thr sequons and 22 asparagines predicted to be N-glycosylated ( Figure 1D). For JY5C, JY6C, JY7C, and YJ3F, four (from N to V at 57, from N to I at 127, from T to I at 232, and from N to S at 719) of the changes in the predicted amino acid sequence destroyed N-linked glycosylation sites, whereas another three changes (from S to N at 58, from I to T at 116, and from T to N at 1193) created three new glycosylation sites compared with the vaccine strain CV777 (Figure 2). For the YJ7C strain, three amino acid changes (from N to V at 57, from N to I at 127, and from T to I at 232) destroyed N-linked glycosylation sites and another three changes (from S to N at 58, from I to T at 116, and from T to N at 1193) created three new glycosylation sites compared with the vaccine strain CV777 (Figure 2). The changes in the N-linked glycosylation sites between the Gansu PEDV strains from our study and the vaccine strain may influence their pathogenicity and antigenicity and should be researched in the future.

Nucleotide and Deduced Amino Acid Sequence Homology
The nucleotide and deduced amino acid sequence homology results are described in Table 1. The nucleotide sequence of the five strains from our study (JY5C, JY6C, JY7C, YJ3F, and YJ7C) had 99.3% to 100% nucleotide sequence identity to one another, and their deduced amino acids had 99.0% to 100% identity to one another. The S genes' nucleotide and deduced amino acid identities of five strains from our study (JY5C, JY6C, JY7C, YJ3F, and YJ7C) with the other PEDV reference strains are described in Table 2. The PEDV strain that had the highest DNA sequence identity to our PEDV strains was CH8 (one Chinese PEDV strain), which had 98.4% identity and the deduced amino acids had more than 98.0% identity. However, the nucleotide sequence of our PEDV strains had only 93.8% to 93.9% identity to the CV777 vaccine strain, and their deduced amino acids had 93.6% to 93.7% identity to the CV777 vaccine strain. The nucleotide sequence of our PEDV strains had lower identity (93.3% to 95.7%) to the previous domestic strains (DX, LZC, LJB-03, JS-2004-2, and CHS) and their deduced amino acids had 92.6% to 95.7% identity to the previous domestic strains (DX, LZC, LJB-03, JS-2004-2, and CHS). The nucleotide sequence of our PEDV strains also had low identity (93.2% to 94.6% and 93.7% to 93.8%) to the Japanese strains (MK, NK, and KH) and the European strain (Br1-87), respectively, and their deduced amino acids had 93.1% to 94.2% and 93.4% to 93.6% identities to the Japanese strains (MK, NK, and KH) and the Europe strain (Br1-87), respectively. The nucleotide sequence of our PEDV strains had higher identity (94.7% to 97.1%) to seven South Korean strains (KNU-0801, KNU-0802, KNU-0901, KNU-0902, KNU-0903, KNU-0904, and KNU-0905), and their deduced amino acids had 94.1% to 96.8% identity.   Nucleotide identity (%) in upper triangle; Deduced amino acid identity (%) in lower triangle.

Phylogenetic Analysis of the S Gene
Phylogenetic analysis of the nucleotide sequences of the S gene revealed three major clusters, and the third group has two subgroups (3-1 and 3-2). All PEDVs isolated from China in our study belonged to subgroup 3-1 (  Figure 4). Our results indicate that the North Chinese PEDV strains from our study had a close relationship with the South Korean strains and mapped phylogenetically to the same branch. However, they differed genetically from the European strains (including the vaccine strain CV777) and the early domestic strains. Similar to the result by Li et al. [9], the appearance of strains from China similar to those from South Korea and their function in the recent PED outbreak in South China, should be further investigated.

Antigenicity Analysis of the S Gene
The PEDV S protein is a type I glycoprotein. Its neutralizing epitopes contain COE (499-638 aa), SS2 (748-755 aa), SS6 (764-771 aa), and 2C10 (1,368-1,374 aa) [3][4][5], and regions of the alignment sequences ( Figure 5) correspond to these regions are COE (504-643 aa), SS2 (753-760 aa), SS6 (769-776 aa), and 2C10 (1,373-1,379 aa). In our study, compared with the vaccine strain CV777, eight mutations (A→S at 517, S→G at 523, V→I at 527, T→S at 549, G→S at 594, A→E at 605, L→F at 612, and I→V at 635) were found in all the five PEDV strains from our study (JY5C, JY6C, JY7C, YJ3F, and YJ7C) and one mutation (L→H at 521) was found in JY5C, JY6C, and JY7C in the neutralizing epitope COE. Compared with the vaccine strain CV777, one mutation (Y→S at 766) was also found in all the five PEDV strains from our study (JY5C, JY6C, JY7C, YJ3F, and YJ7C) in the neutralizing epitope SS6. However, compared with the vaccine strain CV777, no mutations were found in the five PEDV strains from our study (JY5C, JY6C, JY7C, YJ3F, and YJ7C) in the neutralizing epitopes SS2 and 2C10 ( Figure 5). Similar to other coronavirus S proteins, the PEDV S protein is a glycoprotein peplomer (surface antigen) on the viral surface, with a pivotal function in stimulating induction of neutralizing antibodies in the natural host. Thus, the S glycoprotein is a primary target for the development of effective vaccines against PEDV. Further research is needed to determine whether the amino acid changes in the Gansu strains from our study result in any antigenicity changes.

Discussion
RT-PCR amplification and sequencing analysis of the full-length PEDV S genes were used to investigate the isolates from diarrhea samples from local pig farms with severe diarrhea in piglets. The variant strains were detected in this study, implying an identical distribution profile for PEDV on pig farms in Gansu, China. The sequence insertions and deletions in the S gene and mutations in the antigenic regions found in variant strains possibly provided stronger pathogenicity and antigenicity to the new PEDV variants that influenced the effectiveness of the CV777-based vaccine, ultimately causing the 2012 outbreak of severe diarrhea in the porcine farms of Gansu Province, China. Future studies should investigate the biological functions of these particular insertions, deletions, and mutations. Figure 5. Alignment of amino acid sequences of S proteins of Gansu PEDV strains and reference strains. Ellipses represent the consensus amino acids. Boxes indicate substitution amino acids compared with CV777. Shadows indicate the neutralizing epitopes (COE, SS2, SS6, and 2C10 motif).

Sample Collection
A total of 17 samples (fecal and intestinal) were collected from seven dead piglets showing signs of watery diarrhea and dehydration from two farms in Gansu Province, China (Table 3). These samples were collected individually and placed in separate sterile specimen containers. Samples were homogenized with PBS. The suspensions were vortexed and centrifuged for 10 min at 10,000 × g. The supernatants were stored at −80 °C before use.

RNA Extraction and Reverse Transcription
All samples were evaluated by reverse transcription PCR (RT-PCR) using PEDV special primers (Table 4). In brief, viral RNA was extracted from the supernatants of the homogenized samples with TRIzol LS (Invitrogen Co., Carlsbad, CA, USA) according to the manufacturer's instructions. RT was carried out using random hexamer primers (TaKaRa Bio Inc., Otsu, Japan), and the cDNA was immediately amplified with primers, which were designed based on the sequences of PEDV reference strains (Table 4) under the following conditions: denaturation at 95 °C for 5 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 90 s. The RT-PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized by ultraviolet illumination after ethidium bromide staining.

Sequence Analysis
Five of 17 pig samples were positive for PEDV by RT-PCR. Sequencing analysis of the full-length S gene was performed for the five samples designated as JY5C, JY6C, JY7C, YJ3F, and YJ7C. In brief, bands of the corresponding size of the gene were excised, and the synthesized DNA was purified using a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and sequenced by BGI Company (Peking, China). The five PEDV S gene sequences were aligned with the sequences of 32 previously published PEDV S genes (Table 5) using the DNASTAR, DNAMAN, and MegAlign version 5.0 (DNAStar Inc., Madison, WI, USA) software packages [17]. To investigate their molecular and epidemiological characteristics and to determine their profile of genetic

Conclusions
There were few positives (5) by RT-PCR when 17 piglets with water diarrhea and dehydration were sampled. There may be two reasons for this phenomenon. Firstly, inability to amplify virus from all piglets might impact our results. Secondly, the piglets maybe have coinfections that might skew our results.
Our study of the full-length S gene revealed a more comprehensive distribution profile that reflects the current PEDV status in pig farms in Gansu Province, China, including the presence of strains similar to those from South Korea. These data indicate that the new variant PEDV strains in Gansu Province, which were first found in 2011, may have originated from South China. Thus, certain actions must be taken to prevent the continued transmission of this virus, including the development of novel vaccines for prevention.