Construction Of Ehv Type1Infectious Clone And Recombinant Virus Expressing Subunits Of SS2

Equine herpesvirus type1（EHV-1） is a major cause of respiratory and reproductive disease in horses worldwide.The genome of EHV-1strain438/77isolated from aborted equine fetus was cloned as bacterial artificial chromosomes （BAC） in E. coli without any gene deletion. Mini-F sequence was inserted in the middle of ORF19and20via homologous recombination upon co-transfection of viral DNA and plasmid pE19₂0/HA into RK13cells. Circular viral DNA was extracted from RK13cells infected with purified recombinant virus expressing green fluorescent protein （GFP） and electrophorated into E. coli DH10B cells. The clone harboring the BAC was screened and analyzed by PCR and RFLP. Reconstitution of the recombinant virus was achieved successfully by transfection of the BAC DNA into RK13cells. The mini-F sequence in the reconstituted virus was further removed by homologous recombination between virus DNA and plasmid pE1920XM, inducing a point mutation in the Xbal site in ORF19. Comparison of RFLP profiles of the rescued, recovered and the wild type viral genome demonstrated that no unexpected change occurred during mutagenesis. In vitro replication assay showed that the BAC-reconstituted virus mutant was able to grow with plaque size and kinetics indistinguishable with those of wild type.Streptococcus suis serotype2（SS2） is a zoonotic pathogen causing diseases in swine and streptococcal toxic shock syndrome in humans.An effective swine or human SS2vaccinewithout side effects does not exist. Suilysin （SLY） is an extracellularly secreted exotoxin produced by SS2that has been shown to protect vaccinated pigs challenged with SS2. The aim of this study was to construct an avirulentSLY mutant （SLYm）, keeping the immunogenicity of wild type SLY, as a subunit vaccine to protect againstSS2infection. Recombinant SLY and SLYm, with Trp-463and Trp-464substituted with Ala residues, were made and reacted with anti-SLY monoclonal antibody by westernblot analysis. The hemolytic and cytopathogenic activities and toxicity of SLYm and SLY were examined, confirming that SLYm lostits perforationability towards eukaryotic cellswhile wild type SLY did not. Both anti-serum of mice vaccinated with SLYm and SLY inhibited the hemolytic activity of wild type SLY.The hemolytic activities were indistinguishable from each other （p>0.05）. Both mice vaccinated with SLY and SLYm were protected from the challenge of a lethal dose ofwild type SLY（p>0.05）. Most importantly, mice vaccinated with SLYm gained body weight similar to control mice, while those vaccinated with SLY gained less weight （p<0.001）. Additionally, SLYmlacked perforation activity towards eukaryotic cellsand was verified to be avirulent to the host cell and animal but kept the immunogenicity of SLY that would make the subunit vaccine effective against the pathogen andsafer to the host.Application of vaccine is one of the effective methods to control the SS2, killed vaccines are limited torepeated immunizationand whether these vaccines confer protection against challenges with heterologous strains has not been evaluated. An effective SS2vaccine without side effects that is convenient to use in swine or humans should be developed. Based on the BAC p438/77, mrpfragment and sly were introduced to the genome of EHV-1by the two-step Red E/T, respectively. The recombinant virus were comfired by western blot and could express SLY and MRP. The virus4M and4S strains were generated by homologous recombination with the removal of mini-F sequence harbouring CAM and gfp. The MRP antibody titers of miace inoculated with4M strain were assayed and the4S strain could protect the mice against the lethal dosage of recombinant SLY.