| African swine fever(ASF)is an acute,hemorrhagic and highly contagious swine disease caused by African swine fever virus(ASFV).Pigs of different breeds and ages are susceptible to the disease.So far,there is no effective treatment and no commercial vaccine.At present,the disease is prevalent in Africa,Europe and Asia threatening the world pig industry and international trade.ASF emerged in China in August 2018 and since then it has spread into many parts of the country affecting the pig industry.ASFV is a large double stranded DNA virus,which is the only member of the genus Asfivirus within the Asfarviridae family.It is an enveloped large DNA virus with a genome length of 170-194kb.At present,24 genotypes of ASFV have been identified in which two genotypes have been emerged in Europe and Asia.A safe and effective vaccine is necessary for the prevention and control of the disease.Scientists are also investing a lot of efforts to develop ASF vaccine.Inactivated,subunit,nucleic acid,live virus vector vaccines,naturally attenuated live and gene deletion vaccines have been attempted.Inactivated virions,subunit,nucleic acid and live virus vectored ASF vaccines possess very high safety,but the induced immune protection effect is less protective.Naturally attenuated and target gene-deleted ASF vaccines have better immune protection efficacy,but there are safety issue concerns.It is considered that the target gene-deleted ASF vaccines are the most promising that would be achieved in the short term.CD2like ASFV protein(CD2v)is a structural transmembrane glycoprotein mediating hemadsorption in vitro and inhibiting lymphocyte proliferation in vivo,and considered as virulence associated gene.The UK gene is a unique ASFV encoding a small protein in the right variable region of the genome.The UK gene was reported to be a significant virulence determinant of ASFV in domestic pigs.Live attenuated ASFVs generated based on CD2v,UK genes and other genes remain non applicable due to safety issues.In this study,a CD2v and UK genes-deleted ASFV(ASFV-SY18?CD2v/UK)was constructed by using homologous recombination method and purified by limiting dilution method and characterized in vitro.The virulence and protection efficacy of ASFV-SY18?CD2v/UK were evaluated in five SPF pigs.The results showed that ASFV-SY18-?CD2v/UK has no longer the characteristics of hemadsorption,and the deletion of the CD2v and UK genes has no significant effect on the replication of African swine fever virus in vitro.After inoculating ASFV-SY18-?CD2v/UK at the dose of 104 TCID50,none of the pigs showed fever and ASF associated clinical signs,and no viral nucleic acid was detected in the blood,nasal swab and tissue samples of the inoculated pigs.In addition,ASFV specific antibodies were induced.At 28 days post inoculation,all pigs were challenged with 104 TCID50 of ASFV-SY18.The results during the 21 day observation period showed that,all the pigs inoculated with ASFV-SY18-?CD2v/UK had no fever and ASF associated clinical signs after challenge,and all of them remained healthy.At necropsy,no pathological changes were observed in the tissues and organs of the inoculated pigs.Only low levels of ASFV-SY18 nucleic acid were detected in blood,nasal swab and anal tissue samples.In comparison all the control pigs developed fever and ASF associated clinical signs and died between 6-and 11-days post challenge.All the pigs from the control group developed the major ASF associated pathological lesions.Additionally,a high level of ASFV-SY18 nucleic acid was detected in the blood,nasal swab and tissue samples from control pigs.Taken together,we successfully constructed a CD2v and UK genes-deleted ASFV-SY 18.This double gene-deleted ASFV has similar in vitro replication characteristics with its parent ASFV-SY18.The virulence of the gene-deleted ASFV-SY18 in pigs was significantly attenuated at the given dose and provides 100%protection against the virulent ASFV challenge. |
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