Identification Of L7L-L11L Gene Fragment Of African Swine Fever Virus And Related Vaccine Candidate Strains

African Swine Fever(ASF),an acute fever hemorrhagic infectious disease of domestic pigs or wild boars caused by African Swine Fever Virus(ASFV),has spread into China since 2018 and brought great damage to the swine-breeding industry.Its pathogen,ASFV,is an encapsulated double-stranded DNA virus with a 170kb-190kb genome,containing 160-175 open reading frames(ORFs)and encoding 150-200 proteins,most of which is unclear on function.Not one ASF vaccine is available currently.Elucidation of the function of unknown genes and screening immune-related genes are the keys of ASF vaccine development.In this study,L7L-L11L(containing L7L,L8L,L9R,L10L,L11L),a gene fragment located at the right terminal of ASFV genome,was taken as the research object:1.The conservation of the whole L7L-L11L gene fragment among different strains was analyzed,and the L7L-L11L gene-deletion strain SY18?L7-11 was constructed,following by the identification of its in-vitro biological characteristics;2.The virulence and immune response induction of SY18?L7-11 on pigs was identified,compared with its parent strain ASFV SY18,and the possible effect of L7L-L11L fragment on ASFV was analyzed;3.Five single-gene-deletion(L7L,L8L,L9R,L10L,L11L)strains were constructed,following by the research of their biological characteristics,to identify the key virulence-related gene(s)of L7L-L11L fragment;4.Based on the L7L-L11L gene-deletion strain SY18?L7-11,two double-gene deletion strains,combined with the deletion of virulence-related genes CD2v or MGF360/505,were constructed respectively.The safety and immune protection effect of these two strains were evaluated,and the possibility of using them as vaccine candidate strains was explored.Through the above experiments,the following results were obtained:1.The L7L-L11L gene fragment was highly conserved among the strains with same genotype.The morphology,hemosorption characteristics and the proliferation of SY18?L7-11 in vitro were same as that of the parent strain after the deletion of L7L-L11L gene fragment.2.Of the 12 pigs inoculated with low dose(103 TCID50)or high dose(106 TCID50)of SY18AL7-11,only 1 pig in the low dose group(n=6)died.On the 28dpi,the rest 11 pigs were challenged with parental strain ASFV SY18,then all survived.Compared with the parental strain,the gene-deletion strain could induce the production of ASFV specific antibody and IFN-?.3.The in vitro growth curves of single-gene-deletion strain,namely SY18AL7L,SY18AL8L,SY18?L9R,SY18?L10L,SY18?L11L,were consistent with the parental strain.The survival rate of SY18?L7L infected pigs was 25%(1/4),and that of SY18?L11L infected pigs was 100%(4/4).,while the survival rate of SY18?L8L,SY18?L9R and SY18?L10L were O.These results suggested that L7L and L11L might be virulence related genes(s)of ASFV.4.After combined deletion of virulence-related gene CD2v or MGF360/505 with SY18?L7-11,the two double-gene deletion strains SY18?L7-11?CD2v and SY18?L7-11?MGF constructed were proved safer than SY18?L7-11,for the animals inoculated with the double-gene deleted strains did not develop fever or any other clinical symptoms within 28 dpi.And the protection rate of SY18?L7-11?MGF and SY18 ?L7-11 ?CD2v immunization group was 40%(2/5)and 80%(4/5)respectively,after challenge with parental ASFV SY18.The results showed that combinate deletion with the CD2v gene improved the safety of SY18?L7-11 and did not impair the immune protection efficiency,which is more suitable as a vaccine candidate than combinate deletion with MGF360/505.In summary,this study(1)demonstrated for the first time that deletion of the L7L-L11L gene fragment of ASFV did not change the proliferation,morphology and hemadsorption characteristics of ASFV in vitro,but significantly reduced the virulence to swine;(2)L7L and L11L were identified as virulence related genes of ASFV for the first time;(3)the strain combinate deletion of CD2v and L7L-L11L genes was proved safe and high effective as a vaccine candidate,which could produce 80%protection.These results provide important reference data for the elucidation of virulence-related genes and the pathogenesis of ASFV,and also lay an important foundation for the construction of a safe and effective vaccine based on L7L-L11L gene deletion.