Study On The I226R Gene Function Of African Swine Fever Virus

African Swine Fever（ASF）is a serious hemorrhagic infectious disease of pigs caused by African Swine Fever Virus（ASFV）.ASF has caused serious harm to China’s pig industry since it was first reported in 2018.ASFV is a double-stranded DNA virus with a full-length genome of170-193 kb,containing at least 160 reading frames,most of which are functioned unknown.So far,there has been no successful commercial vaccine.Studying the unknown gene function of ASF is the top priority in the development of ASFV.The function of I226R gene in ASFV genome is not clear yet.Natural deletions of this gene have never been found in known ASFV isolates.This gene is stable in the whole ASFV genome,so it is speculated that they may be important genes in ASFV.Moreover,DP71L,L7L-L11L,I177L and other genes near the I226R gene were proved to be related to virulence.Therefore,in order to explore the preliminary function of I226R gene in ASFV genome,the following research contents were made:1.Prokaryotic expression of p I226R protein encoded by I226R gene of ASFV genome and preparation of its polyclonal antibody.2.The effect of I226R deletion on the biological characteristics of ASFV in vitro was investigated by using single gene fragment deletion strain SY18ΔI226R,and the subcellular localization of p I226R protein was performed by using anti-PI226R polyclonal antibody.3.To study the difference of virulence between SY18ΔI226R and its parent strain ASFV SY18 in pigs,and to speculate whether I226R gene is the key gene of ASFV virulence.4.If SY18ΔI226R loses its pathogenicity,experimental animals will be challenged to evaluate its immune protection effect and study its possibility as a vaccine candidate strain.Through the above studies,the following results are obtained:1.The proliferation rate of SY18ΔI226R without I226R gene fragment was slower than that of the parent strain in vitro macrophages,but the morphology of virions and RBC adsorption characteristics were not changed.2.The expression of I226R gene was mainly located in the cytoplasmic"virus factory"by subcellular localization observation.3.When landrace pigs were inoculated with SY18ΔI226R of 10⁴TCID₅₀ and 10⁷TCID₅₀respectively to the whole observation period before challenge,all pigs remained healthy and no death occurred.The results showed that the absence of I226R resulted in the complete loss of virulence of ASFV,and I226R gene was related to the virulence of ASFV.4.After 21 days of inoculation with the SY18ΔI226R single-gene deletion strain,the immunized pigs produced high levels of antibody.After challenged with the parent strain ASFV SY18,all the immunized pigs survived.These results indicated that the pigs immunized with SY18ΔI226R single gene deletion virus could produce high levels of antibodies in vivo and resist the attack of the parent strain.In conclusion,this study is the first to show that the I226R gene in the ASFV genome is expressed in a"virus factory"in the cytoplasm.The deletion of I226R gene weakened the proliferation ability of ASFV but did not completely affect replication,and ASFV SY18 strain lost virulence in domestic swine infection,indicating that I226R gene is the key gene of ASFV virulence.The SY18ΔI226R single-gene deletion strain does not cause disease or death in pigs and provides 100%protection against the parental strain.These results provide important reference data for further study of I226R gene in ASFV,provide new genetic reference for gene deletion vaccine candidate strains,and help researchers to better understand the genome of ASFV,so as to accelerate the development of ASFV.