| Porcine epidemic diarrhea(PED), caused by porcine epidemic diarrhea virus(PEDV), broke out all the country in the winter of 2010, which is characterized by sever diarrhea, vomit, dehydration and significant mortality in piglet, resulting in sever economic losse is in swine industry. To analysise molecular characteristics, epidemic situation of PEDV in Henan Province, Its genetic variation analysis, serological epidemiology and culture characteristics was studied in this research. It can provide a basis for comprehensive prevention and control of PEDV.1. To investigate the variation of M and ORF3 genes of porcine epidemic diarrhea viruses(PEDV) in Henan during 2012~2015, M and ORF3 genes of 16 samples were cloned and sequenced, their phylogenetic trees were analysised. The results showed that there is no nucleotide sequences and amino acid sequences insertion or deletion. Amino acid homologies of M genes among the 16 isolates were 96.9%~99.6%; Compared with classic strain CV777, Korean strains and the most pandemic domestic strains after 2010, the amino acid homologies were 97.4%~99.1%, 96.9%~99.6% and 97.8%~99.6%, respectively. Compared with classic strain CV777, the position of 13 occurred mutation(E-Q/L)in 15 epidemic strains excepted CHHeN ZZ-2012(ZZ) strain. There is a 49 bp nucleotide sequence deletion in ORF3 genes of pandemic strains CHHeN DF2-2012(DF2) and CHHeN ZZ-2012(ZZ). The ORF3 genes of two deletion strains had 100% nucleotide suquence identities with vaccine strain CV777. Compared with classic strain CV777, There are 13 nucleotide and 5 amino acid mutations in the same position of two deletion strains; The amino acid of the other 14 isolates had 98.8%~100% similar with each other, they respectively had 95.1%%~96.0%, 90.2%%~91.1% and 97.3%~99.1% amino acid identities with classic strain CV777, vaccine strain CV777 and Korean strains; With the most pandemic domestic strains after 2010, the amino acid homology was up to 97.3%~99.6%. The 19 nucleotide and 8 amino acid mutations existed in other 14 strains, compared with classic strain CV777. Phylogenetic analyses of the M genes revealed that all PEDV strains in this study could be separated into four groups, Most of the PEDV isolates in Henan belonged to groups 3, but ZZ was groups 2. Based on phylogenetic analyses of the ORF3 nucleotide sequences, all the PEDV strains could be divided into three groups, DF2 and ZZ belonged to groups 2, the other 14 isolates belonged to groups 3. It suggested that M and ORF3 genes of PEDV isolated in Henan shared high homology with the most pandemic domestic strains after 2010 and had a close phylogenetic relationship with Korean strains. In addition, compared with the classical and vaccine strain, PEDV in Henan exhibited variation.2. The prokaryotic expression of purified N protein of PEDV as envelope antigen, through the optimization of ELISA composition and reaction conditions, indirect ELSIA antibody detection method was established, the reaction condition was: envelope antigen concentration was 0.5ug/mL, 0.5% Casein 4 ℃ and closed for the whole night, optimum diluted multiples of serum was 1:100, 37℃ 60 miniutes, the anti-antibody concentration was 1:20000, 37℃ for 60 miniutes, serum samples ratios for S/P more than or equal to 0.262 could be jailed for positive, less than0.247 as negative, and the rest was suspicious. Testing the swine fever, porcine reproductive and respiratory syndrome, pig pseudo rabies and pig foot-and-mouth disease positive serum, the results were negative. The intra-assay and inter-assay variation coefficient were vary from 2.22% to 4.21% and from 3.06% to 10.81%. Compared with the commercial kits, the sensitivity, specificity and corresponding rate of ELISA was 95.00%, 97.22% and 95.83%. To test the clinical 200 serum samples with indirect ELISA, the results showed that the positive rate of PEDV was 66.00% and the positive rate of oneset pig farms was 70.00%, the positive rate of immune pig farms was 85%, the positive rate of unimmunized pig farms was 15.00%; the antibody positive rate of sow was 68.33%, the antibody positive rate of piglets and fattening pigs was 33.33% and 56.67% respectively. The experimental results showed that the established indirect ELISA method had better specificity, sensitivity and repeatability, It can be used for antibody detection and epidemiological investigations of PEDV. 200 clinical serum detection results showed that the positive antibody rate of PEDV in henan province is higher, but lower positive antibody rate in piglets.3. A PEDV strain was isolated in Vero cell cultures from the small intestine of a piglet experimentally infected with PED. The isolation of the PEDV was propagated in Vero cell maintained in the maintenance medium containing 10ug/m L trypsin without fetal bovine serum. then used PT-PCR, IFA(indirect immunofluorescence assay) and gene sequencing to detect the PEDV. Determined its characteristics of culture in cell, Viral multiplication rule and passage stability. Cross antibody-neutralization test was detected with serums from different clinical backgrounds. Results showed 6 blind passages were performed until the CPE appeaered on 7 passages, with the passages increased, the time of CPE appeaered became shorter, the change was regularity. The result of RT-PCR shows PEDV positive and the detection of IFA shows obvious fluorescence, gene sequencing showed the S1,M and ORF3 genes of isolated consistented with the PEDV gene fragment. It is confirmed that isolated a strain of PEDV, named, CHHe N-ZZ. Through FQ-PCR, the growth curve of virus in intracellular and extracellular was determined, intracellular virus reached the peak at 12 h, then decreased gradually. Extracellular virus rapid growth during 24h-72 h, the peak was at 72 h, then gradually decreased. Serial passage between 20 and 50 generation, TCID50 of each generation is stable at 10-5.5~10-6.2/0.1 mL, titer of the virus was 10-6.11/0.1 m L at 50 passages; Analysis sequence of M gene and S1 gene in different generations of isolated strains, the nucleotide homology were 99.6%~99.9%, 99.8%~99.9%. The S1 gene of isolated and CV777 vaccine strain belong to the group I. Cross antibody-neutralization test showed that neutralizing antibody titers of serum from no immunity but infected PEDV group III swine was almost zero; the serum from the pig farm that vaccine immunited but no PED disease history, its neutralizing antibody titers was 54.1~74.9. The results showed that: A PEDV strains was successful isolated, which had good growth characteristics and stability cultured in cells. The cross antibody-neutralization test proved that the poor cross protection between the antibody of CV777 vaccine strain immunited and the viruses of PEDV strains belonged to group III. the pathogenic characteristics of PEDV was studied through the isolation and culture of HeN-ZZ strain which would provide the references for the immunogenicity and cross protection of PEDV. |
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