Screening And Molecular Characteristics Of Novel Genotype PEDV Vaccine Candidate Strain

Porcine epidemic diarrhea virus?PEDV?,as a main etiological agent responsible for porcine viral diarrhea,has spread widely in the world.Phylogenetic analysis show that PEDV can be divided into group 1?G1?and group 2?G2?,classical vaccine strain was isolated from G1,while currently prevalent isolates belong to G2.Though a lot of work has been done for the control of PEDV epidemic via classical PEDV attenuated and inactivated vaccine,it is not high efficiency,and PEDV also threatens the pig industry.Thus,the development of a new type of PEDV attenuated vaccine is extremely urgent.In this project,a variant PEDV strain,named LNCT2,which belongs to G2 was studied.Phylogenic analysis indicated that LNCT2 strain has close relationship to the field prevalent strains,a single clone strain with good antigenicity and immunogenicity was obtained by plaque purification and serum antibody neutralizing?SN?.We intended to attenuate LNCT2 strain by serial passage on the Vero E6 cells and characterized for 100 passages.In order to identify the influence of temperature on the mutation rate of PEDV,we implemented two schemes:low-temperature adaptation?CA?and normal temperature adaptation?NA?.Genome analysis showed that low temperature can promote the mutation rate of PEDV;virus titers of both NA and CA was increasing with the viral passage,especially after passage 30,it ranked 10⁶.Median tissue culture infective dose(TCID₅₀)/mL at passage 100?P100?.In order to detect genome mutation in the process of serial passage,complete genome was sequenced,compared,and analyzed.The results indicated that the mutation rate was increasing with the serial passage in vitro,ORF1ab and S protein were two high mutational sites,five continuous amino acids deletion appeared in the N-terminal domain of S protein,and ORF3 protein had no deletion mutation in P100;genomic comparison of LNCT2-NA and LNCT2-CA showed that N protein had no mutation,M protein shared the same mutation,indicated that N protein was unrelated to Vero E6 cells adaptation or sensitive to low temperature adaptation;M protein was not sensitive to low temperature adaptation.Genomic comparison with the attenuation process of CV777 strain showed that the mutation rate was higher than LNCT2 strain.In order to identify the virulence of P100,we selected LNCT2 strain which adapted in normal temperature?LNCT2-NA-P100?and LNCT2-P10 to perform infection experiment,each piglet in different group was orally administrated with 2×10⁴TCID₅₀ and 2 mL DMEM,Piglets infected with LNCT2-P10 showed severe vomiting and diarrhea,the thin-walled intestine filled with yellow liquid;histopathology?HE?and immunohistochemistry?IHC?detection showed that intestinal villi fell away;the viral load in intestine could be detected by RT-qPCR.Piglets infected with LNCT2-P100 were as normal as negative control group during the experiment.In conclusion,we confirmed that piglets inoculated with PEDV LNCT2-P100 did not show clinical symptoms;and early termination did not appear in ORF3,and PEDV attenuation could occur through multiple molecular mechanisms;low temperature adaptation could promote the mutation rate of PEDV.The present research work paves the way to the development of novel type and high effective attenuated vaccine,for a better understanding of the virulence difference of PEDV.