Isolation And Identification Of African Swine Fever Virus And Development Of A Vaccine Candidate Strain SY18ΔI226R

African swine fever（ASF）is a hemorrhagic and highly lethal disease caused by African swine fever virus（ASFV）infection in domestic pigs.As of March 2022,there are ASF outbreaks in at least 70 countries in the five world regions of Africa,Europe,Asia,the Americas and Oceania.ASFV virions have a complex structure and a huge genome.The complex virus structure and immune evasion mechanisms have hindered current vaccine research.ASFV mutates to varying degrees during the epidemic,resulting in the emergence of virus strains with different genotypes,serotypes and virulence.ASFV genetic variation mostly occurs in the number and sequence changes of left and right MGFs,such as OURT88/3,NH/P68,Estonia/WB/2014 and BA71V strains all have MGFs changes.In addition,the EP153R and EP402R（CD2v）genes of the NH/P68 strain and Lv17/WB/Rie1 strain were all frameshifted,and the EP402R gene（CD2v）mutation in the domestically isolated mutant strains,such as China/GD/2019 strain,Hu B20 strains,HLJ/HRB1/20 and He B/Q3/20,all had no erythrocyte adsorption capacity,and their virulence decreased to varying degrees.In addition,ASFV type I He N/ZZ-P1/21 and SD/DY-I/21 strains that are highly similar to NH/P68 have also been isolated in China.The prevalence of mutant strains is one of the biggest problems and challenges facing the prevention and control of ASF in our country.Inactivated vaccines,natural attenuated strain vaccines,subunit vaccines,DNA vaccines,gene deletion vaccines and viral vector vaccines have been continuously tried.At present,breakthrough research progress has been made in gene deletion vaccines.Combined deletion of multiple genes such as 9GL/UK,MGF505/360/CD2v,L7L-L11L or single deletion of B119L（9GL）,I177L and A137R alone can completely attenuates ASFV and resists challenge with lethal doses of their respective parental strains.ASFV encodes more than 150 genes,and the functions of more than half of the genes are currently unknown and need to be studied.The purpose of this study is to:firstly,to determine the prevalence of ASFV and the characteristics of genomic variation through the detection of ASFV in pork samples from many regions in China;Secondly,to screen new ASFV virulence genes,construct gene deletion strains,and finally obtain ASFV gene deleted vaccine candidate strains with safety and immune efficacy.Research Methods and Results1.ASFV isolation and analysisDuring the period from 2018 to 2020,our laboratory collected a total of 2914tissue samples such as pork,ribs,lungs,and livers from 8 provinces and cities in China.Among them,97 ASFV-positive tissues were detected by q PCR method,36 ASFV strains were isolated by inoculating BMDM cells,and and 2 natural variant strains Hu B20 and LYG18 without hemosorption characteristics were obtained after the common variant genes p72,MGF505/360 and CD2v were amplified by PCR and sequenced.Compared with the earlier reported strains in China,the two strains showed different growth characteristics and virulence.2.The characterization of I226RThe homology analysis of nucleotide and amino acid sequence of 21 ASFV I226R from 11 genotypes showed that I226R gene was highly conserved in different ASFV strains.Subcellular localization analysis showed that p I226R protein was as localized to the virus factory as P54 protein.The relative m RNA expression levels of CP204L,B646L and I226R genes of ASFV at different infection times were detected by q PCR,and combined with the expression characteristics of ASFV genes,p I226R was mainly expressed in the middle and late stages of ASFV infection.The SY18δI226R strain with I226R gene deletion was constructed by homologous recombination method.The strain could be cloned in vitro,indicating that I226R was a nonessential gene for ASFV replication.3.SY18ΔI226R safety testPiglets were infected with high dose（107.0 TCID50）SY18ΔI226R intramuscular injection and observed for 35 days.The survival rate of piglets was 100%,and there were no abnormal clinical symptoms such as fever and depression,and no histopathological and pathological damage.The tissues with high copy viral load in immunized piglets were selected for virus isolation in vitro and animals infection in vivo.Results showed that SY18ΔI226 decreased or lost the ability to infect in vivo and in vitro.The results showed that I226R was an important virulence related gene.SY18ΔI226R completely lost virulence and had an excellent safety profile.4.Immune-protective effect of SY18ΔI226RPiglets were immunized with a single dose(10^3.5,10^4.5,10^5.5,and 10^6.5TCID₅₀)of SY18ΔI226R intramuscular,and challenged with a lethal dose(10^2.5TCID₅₀)of the parent strain after 21 days.The results showed that the survival rate of immunized piglets was 100%,and there were no abnormal clinical symptoms such as fever and depression.SY18ΔI226R promoted the immune and inflammation response.After challenge,the piglets with 10^4.5TCID₅₀≤single dose immunization≤10^5.5TCID₅₀achieved partial protection and the survival rate was 100%,while the piglets with single dose immunization≥10^5.5TCID₅₀achieved complete protection and the survival rate was 100%.The results showed that SY18ΔI226 was a candidate ASFV single gene deletion vaccine with an excellent safety profile.5.The construction and immune-protective effect of SY18ΔI226R/CD2vThe SY18ΔI226R/CD2v strain was constructed by homologous recombination method with further deletion of CD2v gene in SY18ΔI226R strain.Piglets were immunized with intramuscular injection of 10^6.5TCID₅₀dose and challenged with intramuscular injection of SY18 strain(10^2.5TCID₅₀)21 days later.It was found that piglets infected with SY18ΔI226R/CD2v strain did not have viremia,but all piglets developed fever lasting about 5 days after challenge with virulent parent virus.The results showed that the deletion of CD2v gene reduced the immunogenicity of SY18ΔI226R strain.Conclusion1.LYG18 and Hu B20 were the earliest variant ASFV strain and the first natural attenuated ASFV strain discovered in China,respectively.There were significant changes in genome sequence,erythrocyte adsorption,virulence and replication ability in both of them.2.I226R gene is highly conserved in ASFV and is transcribed in the middle and late stage of infection.p I226R is located in the virus factory and is a non-key gene for ASFV replication.3.The SY18ΔI226R was constructed for the first time.The high dose(10^7.0TCID₅₀)immunization of this strain did not cause damage,but a single dose(≥10^5.5TCID₅₀)could achieve complete protection.The SY18ΔI226R deletion strain is the first ASFV vaccine candidate with excellent safety profile and immunoefficacy.4.Animals immunized with SY18ΔI226R/CD2v decreased viraemia and immunoprotection...