Phylogenetic Analysis Of PEDV Henan Strains And Study On Recombinant PRV Expressing The Neutralizing Epitope Region Of PEDV S Gene

Porcine epidemic diarrhea virus(PEDV)is one of the main pathogens that causes pig epidemic diarrhea(PED).Since the year of 2010,PEDV has continued to cause outbreaks that mainly threatened sucking piglets with high mortality rate in China and was subsequently reported in many other countries including Asia,America and Europe,causing significant welfare and economic problems.Although periodic vaccination strategies have been applied nationwide to control the disease,PEDV outbreaks have continued to present high morbidity and mortality rate among pig farms.To better understand the genetic diversity,as well as the epidemiological and molecular characteristics of PEDVs from Henan province,20 pairs of oligonucleotide primers based on the classic CV777 strain were designed for amplifying the complete genome of CH-HNKF-15 strain.The whole genome was identified as 28,038 nt in length excluding the poly(A)tail by piecewise amplified and sequenced.The CH-HNKF-15 strain was then subjected to homology and phylogenetic analysis together with the reference strains available in Gene Bank.The genetic evolution result demonstrated that each of the protein-encoding gene of CH-HNKF-15 has a distant genetic relationship with the Korean strain(attenuated DR13),earlier Chinese strains(CH-S,LZC)and CV777 strain except ORF3 gene.Meanwhile,the homology analysis indicated that the S gene region exhibited high sequence variation between PEDVs and the complete genome sequence of CH-HNKF-15 shared a nucleotide homology of 96.7% with the classical strain CV777.According to aforesaid work,a total of 30 suspected PEDV clinical samples collected during 2014~2015 were identified by reverse transcription-polymerase chain reaction(RT-PCR),resulting in the positive rate of 73.3%(22/30).Then,the S genes of 22 positive samples were further sequenced and analyzed.Sequence analysis showed that the S genes obtained in this study shared 93.%~94.0% nucleotide identity and 92.7%~93.5% amino acid identity when compared with the CV777 strain respectively.Meanwhile,all the PEDV strains in this study had discontinuous 5-aa insertions(59QGVN62 and 140N)and 1-aa deletions(160N)at the N-terminalregion of the S gene,implying that the relatively stable variation have been existed among PEDV field isolates since 2010.Phylogenetic analysis based on PEDV S genes revealed that 22 Henan PEDVs were clustered into separate branches and were very distant from the attenuated DR13 strain,earlier Chinese strains and vaccine development strain CV777,which might be the reason why the vaccine was inefficient to control the disease in clinical treatment.Previous research has indicated that truncated S1 D gene(S1636-789 gene)of PEDV could induce the production of neutralizing antibodies,and the preparation of immune serum could neutralize the PEDV S natural protein.In this study,the amplified PEDV S1 D gene was cloned into prokaryotic expression vector p ET-28 a and followed by its expression in E.coil Rossetta.The 16 k Da target protein was successfully expressed and identified by Western blot analysis.After the optimization of protein induced conditions and Ni-sepharose purification,the recombinant protein was used as immunogen to generate rabbit anti-S1 D polyclonal antibody,and then the bioactivity of antiserum was identified by Western blot analysis.In conclusion,PEDV S1 D protein was successfully truncated expressed and its polyclonal antibody was also prepared,which layed a foundation for further establishment of rapid immunology detection kit of PEDV,and provided a reference for the study of S protein function and development of genetic engineering subunit vaccine.Pseudorabies virus(PRV)is another significant causative agent that encountered by chinese swine industry on its process of development,resulting in nervous disorders,stillbirth,abortion and boar infertility as clinical symptoms.The genome of PRV is a linear DNA molecule of approximately 150 kb,which includes several nonessential genes that foreign genes can be stably inserted,making the attenuated PRV a promising candidate for the development of a live vaccine vector to confer protection against both PRV and other diseases.In order to develop the recombinant attenuated PRV expressing PEDV S1 D,the target S1 D gene was amplified using specific primers with the restriction enzyme cutting site Bam HI and was further inserted into the downstream close to the g G-pro promoter within the p G vector to generate the recombinant plasmid p GS1.The genome of PRV attenuated vaccine and the transfer plasmid p GS1 were transfered into ST cells with lipofectin transfection to generate the recombinant r PRV-p GS1 strain that carry GFP gene and foreign antigenic gene S1 D.The recombinant virus r PRV-p GS1 was subsequentlysubjected to five cycles of plaque purification and was subsequently identified by fluorescence assay as well as PCR analysis.The PEDV S1 D protein was successfully expressed within the recombinant virus genome and was identified by RT-PCR and Western blot analysis.These results suggest that the r PRV-p GS1 strain in this study might be an attractive candidate vaccine,which provide the effective theoretical basis and technical support agianst PEDV and PRV infections in pigs.