Genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central China from 2010 to 2011

Porcine epidemic diarrhea has re-emerged with devastating impact in central China since October 2010. To investigate and analyze the reason of this outbreak, the M and ORF3 genes of 15 porcine epidemic diarrhea viruses (PEDV), which were collected from different areas of central China during October 2010 and December 2011, were amplified by reverse transcriptase polymerase chain reaction, cloned, sequenced, and analyzed. Sequence analyses showed that the nucleotides and amino acids were changed at some sites in the M and ORF3 genes of the 15 PEDV strains compared with those genes of CV777 reference strain. Based on the phylogenetic analyses, PEDVs in central China and reference strains could be separated into three groups: G1, G2, and G3. The 15 PEDV strains belonged to G3 group and showed a close relationship with Korean strains (2007), Thai strains (2007–2008), and partial other Chinese strains (2010–2011), but differed genetically from European strains (Br1/87) and the vaccine strain (CV777 vs) being used in China. Furthermore, all 15 PEDV strains from central China and some other isolates in China from 2003 to 2007 (LJB-03, QH, and LZC) belonged to different group. Therefore, PEDV exhibits rapid variation and genetic evolution, and the currently prevailing PEDV strains in central China are a new genotype.

PEDV can cause porcine epidemic diarrhea (PED), an acute and highly contagious enteric disease characterized by severe enteritis, vomiting, watery diarrhea, and dehydration with high mortality in suckling piglets. PED was first reported in Belgium [3] and the United Kingdom [4] in 1978. It has subsequently been reported in many other swine-producing countries throughout the world. The presence of PED in China was confirmed by the immunofluorescence assay and serum neutralization test in 1984 [5]. PED has spread and become widely prevalent in some countries and regions of Asia in recent years [6][7][8][9][10]. It also outbroke in China during 2010-2011 [11][12][13]. Pigs of all ages exhibited especially severe diarrhea and dehydration with up to 100 % mortality among suckling piglets. The pigs of Henan, Shanxi, Anhui, and Hebei provinces located in central China, of which breeding stock accounts for 25 % of the total pig population in China, were seriously affected and sustained great losses. Porcine intestinal tissues and fecal specimens were collected between October 2010 and December 2011 from 82 swine farms in the four provinces in central China. Among those farms, 51 samples were confirmed to be positive for PEDV by reverse transcriptase polymerase chain reaction (RT-PCR). In the present study, 15 representative samples from different area at different time were selected from 51 positive samples to investigate the molecular epidemiology and gene variation of PEDV in central China using sequence analyses of the M and ORF3 genes so as to determine the reason for the recurrence of this disease and provide recommendations for its prevention.

Materials and methods
Positive samples 15 intestinal tissues and fecal specimens were collected from four provinces located in central China from October 2010 to December 2011, which were determined to be positive for PEDV by RT-PCR and excluded the contaminant of extraneous transmissible gastroenteritis virus, rotavirus A, kobuvirus, porcine circovirus-2, and suid herpesvirus 1 by PCR or RT-PCR assay. The detailed informations of PEDV strains are shown in Table 1.

Primers
Two sets of primers were designed and synthesized (Sangon biotech, China) to amplify the M and ORF3 genes, respectively (Table 2), based on the genome of PEDV CV777 (GenBank No. AF353511).

Sample preparation
Intestinal tract and fecal specimens of sick pigs were diluted five times in phosphate buffered saline. The samples were ground and then centrifuged at 8,0009g for 10 min after freezing and thawing three times. The supernatants were stored at -20°C.

RNA extraction
PEDV RNA was extracted and dissolved in 30 ll 1 % diethyl pyrocarbonate water, as described in the Trizol reagent introduction (Invitrogen), then stored at -70°C.
The PCR mixtures consisted of 5 ll 109 Taq DNA polymerase buffer (10 mM Tris-HCl, 50 mM KCl), 2.5 mmol MgCl 2 , 5 ll cDNA, 10 mmol dNTP, 2.5 U Taq DNA polymerase, and 20 pmol each sense and antisense specific primer of M or ORF3 gene. Sterile distilled H 2 O was added to reach a total volume of 50 ll. Amplification was performed as follows: one cycle at 95°C for 5 min followed by 32 cycles at 95°C for 1 min, 55°C for 1 min, 72°C for 1.5 min, and a final extension at 72°C for 10 min. The products were stored at 4°C.
Cloning and sequencing of PCR products PCR products were identified by electrophoresis on 1 % agarose gel and cloned into pMD18-T. The recombinant vector was identified by PCR and enzyme digestion. The positive clones were sent to Beijing Genomics Institute (BGI) for sequencing. All sequencing reactions were performed in duplicate. The sizes of PCR products should be 873 and 964 bp, respectively. The former contained the M gene and its flanking sequences, while the latter contained ORF3 gene and its flanking sequences.

Sequence analysis of M and ORF3 genes
Nucleotide and the deduced amino acid sequences of M and ORF3 genes of 15 strains were aligned and analyzed by the MegAlign software (DNAstar). The reference strains used for sequence alignment and analysis with the 15 PEDV strains are presented in Table 1.
Phylogenetic analysis of PEDV Phylogenetic trees were constructed by Molecular Evolutionary Genetics Analysis (MEGA) software (version 4.0) with the neighbor-joining method based on amino acid sequence of M and ORF3 proteins, respectively. To assess the relative support for each clade, bootstrap values were calculated from 1,000 replicate analyses. The reference strains used for phylogenetic analysis with the 15 PEDV strains are presented in Table 1.

Results
Sequencing of the M and ORF3 genes All the M and ORF3 genes of the PEDV strains from 15 farms were amplified and sequenced. The sequences were submitted to GenBank. PEDV strains sequenced in this study and their accession numbers are shown in bold text in Table 1.     Phylogenetic analysis based on the ORF3 protein Phylogenetic analysis showed that the PEDVs fell into three groups (Fig. 2). The 15 PEDV strains from central China belonged to group 3, together with nine isolates of other domestic strains from 1986 to 2011 and seven Korean strains from 1999 to 2007. Group 2 comprised a south China strain (CHGD-01) and two European strains (CV777  and Br1/87). Group 1 included a Korean strains (DBI865), CV777 vaccine strain (CV777 vs), and a Chinese strains (CH/GSJIII/07). They had distant relationship with CV777 vs.

Discussion
The M protein encoded by the ORF5 gene is a membrane glycoprotein. In addition to playing an important role in the viral assembly process [14], the M protein induces antibodies that can neutralize virus in the presence of complement. The nucleotide sequences of M gene possess low identity with other coronavirus, but are highly conserved among PEDV strains. Nonstructural proteins encoded by the ORF3 gene of coronavirus closely relate to its pathogenicity. Highly adapted transmissible gastroenteritis coronavirus (TGEV) and PEDV have sequence variations in the ORF3 gene and exhibit reduced pathogenicity [15,16]. Wang et al. [17] has shown that the virulence to cells of PEDV was reduced by RNA silencing to inhibit the expression of ORF3 gene. Therefore, the ORF3 gene of PEDV has been suggested as an important virulence gene. The differences of ORF3 genes between the wild-type and attenuated type can serve as a marker of the adaption to cells of PEDV and as a potential tool to study molecular epidemiology [16]. Therefore, the M and ORF3 genes are often used to analyze the molecular epidemiology of PEDV [6,10,18]. A dual-combination, attenuated vaccine against TGEV and PEDV has been used in China since 1995 [19,20]. This vaccine has significantly reduced the losses in the pigbreeding industry from these two diseases. However, according to the report of Chen et al. [6], PEDV became epidemic in immunized swine after 2006. Furthermore, as reported by Sun et al. [13], PED broke out in more than 10 provinces in South China and had caused more than one million piglets deaths since October 2010. Suspected PEDV-infected samples of feces and intestinal tissues, which were collected from 82 farms in Henan, Shanxi, Anhui, and Hebei provinces, were detected by duplex RT-PCR detecting TGEV and PEDV, which was developed in our laboratory. The result showed that the positive rate of PEDV was 62.2 % (51/82) and the positive rate of TGEV was 1.22 % (1/82). Furthermore, there was no coinfection of PEDV and TGEV (issued in another article). From these results, it can be deduced that the epidemic piglet diarrhea was mainly caused by single infection of PEDV, which was different from previous co-infections.
The M and ORF3 genes of 15 PEDV strains were amplified by RT-PCR, cloned, sequenced, and analyzed to study the gene variation and molecular epidemiology of the PEDV that reappeared in central China and to determine the reason that the disease became epidemic again. Our study shows that there is a conserved ATAAAC sequence at the 11th nucleotide upstream of the initiation codon (ATG) of the M gene, and a conserved CTAGAC sequence at the 46th nucleotide upstream of the initiation codon (ATG) of ORF3 gene which is consistent with previous reports [21]. Both sequences are hexameric motifs, similar to the six-dimerization motifs XUA (A/G) AC of other ORFs, which have been proven to be the transcription start sites of PEDV subgenomic mRNAs [22]. Most of the samples in this study were selected from the farms where pigs were immunized with PED inactivated vaccine or attenuated vaccine, which were manufactured with CV777 vs. However, the morbidity and mortality of suckling piglets suffering from the PED outbreak were still high. The sample taken from a farm where severe diarrhea epidemic broke out in October 2011 was confirmed to be positive for PEDV. The infection at the farm subsided gradually by vaccine immunization and artificial infection of all the sows using the feces of sick pigs. But, PED broke out again at the same farm after only 2 months. PEDVs isolated from the farm at different time were named as CH/ HBXX2/11 in October and CH/HBXX3/11 in December. The M and ORF3 genes of the two PEDV strains were analyzed. Homology results show that the two PEDV strains have 100 % (100 %) nucleotide (deduced amino acid) sequence identities for the M gene and 99.9 % (100 %) nucleotide (deduced amino acid) sequence identities for the ORF3 gene, which show that the two PEDV strains are the same strain. This result indicates that the current PEDV has strong immune escape capability or enhanced virulence, which can cause immunity failure. It is urgent to study the biologic characteristics, antigenicity, and mechanism of virulence variation of these strains further. Better vaccine strains must be developed for the prevention and control of the disease.