Isolation, Identification And Molecular Evolution Characteristics Of Porcine Epidemic Diarrhea Virus Strain AJ1102

Porcine epidemic diarrhea (PED) is a kind of contagious enteric disease caused by porcine epidemic diarrhea viruse (PEDV) characterized by acute watery diarrhea, vomiting and dehydration, which was first reported in England in1971. The occurrence and prevelance were consecutively reported in other Europe countries and part of Asia countries. America never has PEDV previously, there were prevalence of PEDV in several states of United States since May,2013which was paid the high attention in porcine industry of America and the whole world.In1980s, this disease first occurred in our country. It has caused severe economic loss to swine industry due to the high morbidity and mortality to newborn piglets. Since winter in2010, epidemic disease erupted in10provinces of China with new characteristics:pandemic, high morbidity and mortality with severe loss. The pigs vaccinated previously were also infected which incidicated that the epidemic strain with strong virulence had big difference with current vaccine strain, as a result, the current vaccine strain was invalid to provide protection. It is meaningful that the genome sequence of PEDV strain isolated in Ameirica in2013shared99.5%sequence identity with the epidemic PEDV strain isolated in our country which is called Chinese variant strain. For this current porcine epidemic diarrhae virus, we established RT-PCR detection, isolation and identification method, meanwhile, we had a intensive study to genetic evolution characteristics of epidemic strain. The major studies include:1. The establishment of RT-PCR detective method diagnosis and epidemiological survey for PEDV.To the highly conservative M gene, a pair of specific primer were degined to establish the RT-PCR detective method, the amplified segment was552bp. This method is specific, sensitive and rapid which is propriate for the detection of big samples. The600clinic specimen from9provinces in central, east and south of China were detected, the result indicated the average positive rate of PEDV was58.32%, which showed the high infection rate of pandemic PEDV in China.2. Isolation and identification of AJ1102strainAccording to the research at home and abroad, the isolation of PEDV is extremely difficult although the infection rate is high. The clinic specimen of diseased piglets from Hubei pig farm was innoculated in Vero cell, after3blind passages, the apparent CPE could be observed, the specific RT-PCR decteted it positive, the further indirect immunofluorescence test veified that the isolated strain reacted with specific monoclonal antibody. Plaque assay idendified the titer of isolated strain was106.0PFU/ml. Electron microscopy after gradient centrifugation showed virion in100nm diameter with obvious envelope and spike which were the typical morphology of PEDV. All the above conformed the isolated strain PEDV named PEDV AJ1102.3. Determination and phylogenetic analysis of the full-length genomic sequences of PEDV AJ1102Refer to complete genome sequence of PEDV reported previously,16pairs of primers were designed to determinate complete genome sequence of PEDV AJ1102strain by RT-PCR segmented amplifying method. The result showed, the complete genome sequence of PEDV AJ1102strain was28044bp (GenBank accession number JX188454), arranged in5’-ORFla-ORFlb-S-ORF3-E-M-N-3’with the same sequnce as the PEDV typical strain CV777. Compared AJ1102strain with other31strains at home and aborad whose complete sequneces determined and pictured the phylogenetic tree by MEGA4, which showed the32strains of PEDV divided into two classes:group Ⅰ and group Ⅱ which had two subtypes respectively:Ⅰa、Ⅰb and Ⅱa、Ⅱb. Group Ⅰ was composed by the strains reported after2010inland and America epidemic strains in2013. AJ1102belongs to subtype Ⅱa, the neotype variant strain which is remote to classical strain CV777in Ⅰa.4. the phylogenetic analysis for major genes of AJ1102The study compared the difference in5’UTR,3’UTR, M, N, S, E and ORF3between AJ1102and classical strain CV777. AJ1102had5bases deletion,1base insertion and8bases mutation in5’UTR; there was no insertion and deletion in3’UTR except10bases mutation; the major mutation area was in S1area (1-2217nt) including3insertion areas(168nt,176-186nt,431-433nt) and1deletion area(493-498nt), and there were8amino acids mutation in COE epitope of S protein,1amino acid mutation in SS6(772LQDGQVKI779) epitope; there was no insertion and deletion in M, N, ORF3and E genes except some bases mutation. The phylogenetic analysis of M, N, S, E, ORF3and E genes showed that AJ1102was in the same branch with the other isolated strains after2010, and remote to classical strain CV777, thus proved AJ1102was the current epidemic strain and the exactly the cadidate strain for vaccine preparation of the current PEDV.5. The genetic marker for current strain of PEDVAccording to the genetic evolution analysis of complete gene and major structure proteins, we found there was comparatively big variant in SI area of S gene. In order to explore if this area had the characteristic or genetic marker of current PEDV strain, we designed a pair of specific primer to amplify the450bp segments of major variant area of S1area to do RT-PCR amplifying and sequence analysis which indicated that there was representative variation could be used as the genetic marker to distinguish between epidemic strain and classical strain to provide theoretical basis for vaccine selection.6. Analysis for the anti-PEDV effects of curcuminCurcumin is a kind of Chinese herb has not only anti-inflammation, anti-tumor, anti-oxidation and antimutagenesis effect but also anti-virus effect which can remarkably inhibit HBV, HCV, HIV and other various viruses. However the anti-virus effect of curcumin hasn’t been reported. This study utilized curcumin of lOuM,20uM and30uM to take effects on cells and then AJ1102strain was inoculated, the cells were collected after6h,12h and18h, the plague assay was used to identify the virus titer. It was found that the curcumin remarkably inhibited the proliferationof PEDV, with time and dose dependent.