Analysis Of Complete Genome Sequences And Construction Of An Infectious CDNA Clone Of Porcine Epidemic Diaririea Virus

Porcine epidemic diarrhea virus (PEDV) is a member of Coronavirus which can cause vomiting,diarrhea and dehydration in pigs of all ages, especially newborn piglets within1week. The morbidityand mortality of newborn piglets is very high, up to100%. The virus causes severe economic losses toChina's swine-breeding industry.The complete genome sequences of PEDV Vero E6cell-adapted strain CV777-P125, virulent strainCH/S and field strain CH/FJND-3/2011in2011are27953nucleotides (nt),28026nt,28038nt in length,respectively. The sequence homology of the complete genome sequences of CV777-P125, CH/S andCH/FJND-3/2011with that of PEDV reference strain CV777is97.6%,97.2%and96.9%, respectively.The sequence homology of CH/S, CH/FJND-3/2011with CV777-P125is97.7%,97.2%, respectively.The sequence homology of CH/S with CH/FJND-3/2011is97.4%. The5' untranslated regions (UTRs)of CV777-P125, CH/S and CH/FJND-3/2011comprise of292nt and are4nt shorter than that ofCV777. Compared to the complete sequence of CV777, there is a24nt-deletion region in the replicasegene, a3nt-deletion region in S gene and a49nt-deletion region in ORF3of CV777-P125, respectively;there is a3nt-deletion region in the replicase gene of CH/S; and the S gene of CH/FJND-3/2011hasnucleotide deletion and insertion regions which lead to the S gene is9nt longer than that of CV777.TheS genes of27field strains (including CH/FJND-3/2011) in2011are composed of4146~4170nt, ofwhich,17are4161nt in length. Based on the phylogenetic tree of S gene,27field strains and33reference strains are divided into two large groups (G1, G2), and each group is divided into twosubgroups (G1-1, G1-2, G2-1, G2-2). Of27field strains,17in G1-1have a closely relationship withKorean field strains in2009;3in G2-1have a closely relationship with three Chinese field strainsJS-2004-2, DX, LJB/03;7in G2-2have a closely relationship with the attenuated strains. The analysisof S genes indicated17field strains in G1-1have nucleotide deletion region, nucleotide insertion region,generous nucleotide point mutations, which are mainly clustered in the1100nt region of the N-terminalof the S genes. The field strains in2011in G2-2have the highest sequence homology with PEDVattenuated strains, the field strains in2011in G2-1have the lower sequence homology with PEDVattenuated strains and those in G1-1have the lowest sequence homology with PEDV attenuated strains.The epitope region (COE) and the antigen epitopes (S1D5, S1D6,2C10) inducing neutralizingantibodies against PEDV of S proteins of CV777-P125, CH/S, field strains in2011were compared tothose of S protein of CV777. The results indicated that there is no amino acid deletion and insertion inthe COEs of29strains, but there are amino acid point mutations, and the number of amino acidmutations is7to13. The number of amino acid point mutations between field strains in2011andCV777-P125is0to8, and the mutation rate is0%-5.7%. The antigen epitope S1D5has an amino acidpoint mutation in CH/HLJHG/2011, while conserved in26field strains in2011. The antigen epitopeS1D6is conserved in CV777-P125, CH/FJND-4/2011, CH/GXNN/2011, CH/GXWM/2011,CH/HNZZ/2011and CH/JL/2011, while has2or3amino acid point mutations in other field strains in2011. The antigen epitope2C10is conserved in all the strains. The intermediate plasmid pBAC-PEDV-5'-3' was constructed based on the vector pBeloBACll inwhich the sequences of cytomegalovirus (CMV) immediate-early promoter, PEDV5' end (402bp),multiple clone sites (MCS), PEDV3' end (2434bp, including polyA tail), hepatitis delta virus (HDV)ribozyme, bovine growth hormone (BGH) termination and polyadenylation signals were sequentiallyadded between ApalI and HindIII sites in the MCS of pBeloBACll. Four overlapping cDNA fragments(AB, C, D and E) covering the full-length genome of PEDV were amplified by overlapping PCR andsequentially cloned into the intermediate plasmid pBAC-PEDV-5'-3' to generate the recombinantplasmid pBAC-PEDV-FULL.Transfection of Vero E6cells with the recombinant plasmidpBAC-PEDV-FULL resulted in cytopathic effects (CPE) in Vero E6cells. Positive and negative strandRNAs were detected in the transfectant after two cell passages by nested RT-PCR. PEDV particles weredetected in the cell culture of the rescued virus of passage5by electron microscope. The sequence of5'end of passage5has99.9%and99.6%homologies with those of the recombinant plasmidpBAC-PEDV-FULL and the parent virus, respectively. The molecular tag introduced into the5' end ofthe recombinant virus was confirmed which enables differentiation from the parent virus. These resultsindicated that an infectious cDNA clone of PEDV was successfully constructed.