Development Of A Highly Attenuated African Swine Fever Virus By Combinational Deletions Of The MGF360-9L And MGF505-7R Conferred Protection Against The Virulent Challenge

African swine fever（ASF）is highly contagious animal diseases of domestic and wild boars caused by the African swine fever virus（ASFV）,posing an important threat to global pig industry.The lack of effective commercial vaccines and therapeutic options pose a major challenge for disease control and virus eradiation,further resulting in the rapid spread of ASF in China.There is an urgent need to develop an effective,cost-friendly and highly protective vaccines.Currently,existing findings demonstrate that live attenuated vaccines（LAV）are one of the most promising tools,due to its high efficiency in inducing protection in pigs against virulent challenge.ASFV encodes approximately 160-180 proteins,among which MGFs are hit for deletions.MGFs are associated with host range and cell tropism,and also play a role in virus virulence and regulating the host’s innate immune response.Previous studies have identified MGF＿360-9L and MGF＿505-7R as ASFV virulence genes that cause immune suppression in the host.The results of animal experiments showed that the deletion of these two genes resulted in decreased virulence in animals.The safety and immune protection effect after combinations deletion of MGF＿360-9L and MGF＿505-7R genes are unknown.In this study,combinational deletions of MGF＿360-9L and MGF＿505-7R were constructed using ASFV CN/GS/2018 strain as the parental strain by homologous recombination technique,resulting in ASFVΔ9L/Δ7R.Ten one-month-old Large White-Duroc crossbred pigs were obtained from a licensed livestock farm,with each pig being antigenically and serologically negative for ASFV.Animals were randomly assigned to two experimental groups in one pen in a room of the high containment facilities of Lanzhou Veterinary Research Institute（LVRI）,namely vaccination group（n=6）and contact group（n=4）,6 pigs from vaccination group received an IM（intramuscular）infection of 3 ml of cell culture medium containing 10⁴ HAD₅₀ of the ASFVΔ9L/Δ7R mutant,whereas four pigs from contact group were intramuscularly inoculated with same amount of cell culture medium only.After a 23-day observational period,four pigs from contact group and six pigs from vaccination group were housed separately,intramuscularly challenged with highly virulent ASFV CN/GS/2018 at a lethal dose(10² HAD₅₀)and monitored for extra18 days.Throughout the entire experiment,pigs were examined for ASFV-related clinical signs including rectal temperature on a daily basis.Besides,serum and EDTA samples were collected at a-two-day-interval,which was the case for oral-nasal and fecal swabs.At necropsy,tissue samples（lung,lymph nodes,heart,liver,spleen,kidney）were examined and sampled either by snap-freezing for PCR or by fixation in 4%formalin for histology and immunohistochemistry.The results of the safety experiment showed that the temperature,mental status,and appetite of pigs were normal after the intramuscular inoculation dose of 10⁴HAD₅₀ASFVΔ9L/Δ7R deletion virus,and there was no viremia in the immunized pigs.ASFV p30-specific antibodies could be detected in the immunized pigs after 13 days and maintained at a high level after 17 days.After being inoculated with 10²HAD₅₀ parental strain,it was found that all 4 pigs in the blank control group died 6-8 days after the challenge,and 83.3%（5/6）pigs in the immunized group survived.Only 2 pigs in the immunized group showed a brief rise in body temperature which then returned to normal after being inoculated with the parental strain.There were no obvious clinical symptoms of ASF and no viremia,the virus content in the tissues was low and the tissue damage was mild,indicating that ASFVΔ9L/Δ7R could play a good immune protection effect.There were no significant pathological changes in the immune-protected pigs.The results of Hematoxylin-eosin staining and tissue toxicity showed that the pathological tissue damage was less severe in immune-protected pigs,and the tissue toxicity was significantly lower in immune-protected pigs than in blank-challenged pigs.Immunohistochemistry showed that the responses of IFN-γ⁺,CD4⁺and CD163⁺in spleens of immune-protected pigs were obvious.This indicated that ASFVΔ9L/Δ7R had good immunogenicity and could induce the immunity of the organism.RNA-seq analysis showed that differentially expressed genes were significantly enriched in the RIG-I-like receptor signaling pathway,cytoplasmic DNA recognition pathway,Toll-like receptor signaling pathway,and NOD-like receptor signaling pathway at 18 h after ASFV CN/GS/2018 and ASFVΔ9L/Δ7R infection.In conclusion,the attenuated ASFVΔ9L/Δ7R strain was successfully constructed in this study,which could stimulate the immune response with high safety,and could resist the attack of the parental strain,with a good immune protective effect.This study provides theoretical support to explore the pathogenic mechanisms of ASFV and to develop a safe and effective live attenuated vaccine for ASF.