Genetic Variation Analysis Of Main Structural Genes Of Porcine Epidemic Diarrhea Virus Pandemic Strains In Heilongjiang Province During2012～2013Outbreaks

Porcine epidemic diarrhea virus (PEDV) is a member of Coronavirus,which can cause vomiting,diarrhea and dehydration in pigs of all ages.The morbidity and mortality of newborn piglets is very high, up to100%. The disease is widely distributed in the world, causing serious economic losses to the world’s pig industry.From the year2010,severe diarrhea(suspected to be infected PEDV), a high incidence and mortality,were observed on most swine farms in most provinces of China. Immunized farms throughout Heilongjiang province also spared.So,from March2012to December2013,porcine intestine and fecal samples were collected from10piglets with watery diarrhea and dehydration at10different farms in Heilongjiang provinces and Inner Mongolia Autonomous Region. All the samples were detected by RT-PCR.The result is that there are9samples were infected with PEDV lonely,1sample was infected with mixed infection of PoRV, there were no samples infected with TGEV.The result showed that:PEDV is dominant in the diarrhea pathogenIn this study,the S1genes of10PEDV strains were amplified by RT-PCR and cloned in the pMD18-T vector respectively,and then sequenced. The structural genes and ORF3were also amplified by RT-PCR and cloned in the pMD18-T vector respectively,and then sequenced.Sequence analysis and phylogenetic tree showed phylogenetic relationships and genetic variation of PEDV pandemic strains and reference strains.10PEDV Slgenes composed of2367nucleotides,PEDV S1gene nucleotide sequences showed homologies of86.8%-99.7%.Compared with the reference strains, there are point mutations, insertion, deletion,this phenomenon caused the change of S protein amino acid and accordingly caused PEDV genetic variation.Phylogenetic tree showed that PEDV S1genes are divided into two groups,10isolates in this study are all at Gl,they have a closely relationship with CH/SDQD/201K CH/HBQHD/201land HuN.And they have a far relationship with classic CV777and Chinese previous isolates LJB/03etc.Structural genes and ORF3sequence of PEDV HLJ-2012analysis showed that:The spike (S) protein gene, membrane (M) protein gene, envelope (E) protein gene the nucleocapsid (N) protein gene and ORF3gene of PEDV HLJ-2012were composed of4161nucleotides,681nucleotides,231nucleotides1326nucleotides and675nucleotides, respectively. Compared with classic CV777and Chinese previous isolates LJB/03etc,point mutations, insertion, deletion mainly centered5’end of S gene,caused many S protein amino acid at N end changed.The E,M,N and ORF3genes are relatively conserved,but there are many point mutations which cause amino acid changed.The194th (G-A) base of E gene changed caused the65th(R-Q) amino acid of the E protein.The567th(T-C) and632th(A-G) of M gene caused the211th(N-S) amino acid of the M protein.The132(G-A),185(G-A),231(C-T),357(A-T),708(A-C) of N gene caused the62(R-H) amino acid of the N protein.From compared with the ORF3gene,PEDV HLJ-2012has a closely relationship with Chinese wild strains after2011(except CH/BJ/2011and CH/HLJHH-1/2011) and South Korean wild strains and has a far relationship with vaccine strains.Sequence and phylogenetic analysis indicated that PEDV HLJ-2012is closely related to North Chinese field strains from recent years and genetically, differs from European strains, Korean strains, Japanese strains and early domestic strains.Immune rabbits the tissue original virus, detection of serum antibody titer by indirect ELISA is1:12800. The isolation and identification of porcine epidemic diarrhea virus (LJB/03strains) proliferated and cultivated on Africa green monkey kidney cells (Vero), virus titer reached above105TCID50/ml, then infected Vero cells by the mix of200TCID50virus and serum of different dilution. By neutralization test, determine its neutralizing effect on traditional strain LJB/03, and neutralization titer was1:53.83calculated by Reed-Muench’s method.PEDV HLJ-2012was isolated from the feces of diarrhea piglets and cultured in vero cell propagated in maintenance medium containing6μg/ml trypsin.The isolated virus was identified by stable cytopathic effects,TCID50,RT-PCR,immunofluorescence,electron microscopy,ELISA and so on.The experimental results indicated that the bland passages of20generations were undertaken until the CPE could be seen.CPE was shorten and intensified with increased passages.After40th generation in Vero cell culture,refracted reinforcement and particulate matter increaser,a great quantity of cells defluxioed,the remaining adherent cells become black slug.But the cell control still retained a pyknomonolayer and there was a small quantity of cells having defluxioed.The20,25,30,35,40,45th generation of cell culture virus were identified through RT-PCR, the results showed that all positive.Part of the M gene fragment of the45th generation cell culture virus were sequenced,the result showed that there is93.9%homologous with HLJ-2012original virus.proved that the virus has separated successfully and has adapted to the vero.By negative staining electron microscopy experiment found the virus were enveloped virus and more than100nm in diameter.The indirect immunofluorescence test showed that vero cells had specific green fluorescent response when infected the separation strains PEDV HLJ-2012after24h,but negative control had nothing obvious green fluorescence.Indirect ELISA detected that P/N was7.25.It was justified that the virus in Vero cell culture was positive.