A Rapid Detection Method And Immune Escape Mechanism Of The African Swine Fever Virus

The global pig industry and national economies are at risk from the lethal acute infectious disease called African swine fever(ASF),which is caused by the African swine fever virus(ASFV).ASF is severe and spreads quickly.The disease originated in Africa in1921,later,it spread to Europe,South America,and Southeast Asia,before being detected in China in 2018.ASFV is the only known DNA arbovirus and a member of the swine fever virus family.ASFV is mostly spread by soft ticks,African wild boars,wild boars,and domestic pigs.African wild boars do not show clinical signs of ASFV infection,however,ASFV causes fatal disease in domestic pigs and wild boars.ASFV is a large DNA virus with an icosahedral structure,which has a complex interaction mechanism with the host and is able to evade the host immune system.No safe and efficient vaccines or drugs are available,and the prevention and control of ASF mainly depends on the isolation,eradication,and strict biosafety control of infected animals.Rapid and accurate monitoring of pathogens is the most important intervention to prevent the outbreak of African swine fever.Therefore,establishing a rapid and accurate detection method for ASFV and elucidating the mechanism of ASFV regulation of the host immune response will lay the foundation for effective control of ASF and development of safe and effective vaccines.【Objective】(1)At present,the Taq Man probe real-time PCR detection method is widely used for the early detection of ASFV,however,the probe is expensive and the detection sensitivity is limited.Therefore,this study aimed to establish an economical,rapid,accurate,and sensitive TB Green q PCR detection method for the early screening of ASFV.(2)The complexity of ASFV’s genetic structure and the incomplete understanding of virus genes interactions with hosts limit the ability to build an ASF vaccine.Therefore,to design safe and effective vaccines,this study investigated how viral genes interact with hosts and examined the molecular mechanism by which ASFV evades the host immune response.【Methods】(1)Establishment of TB Green q PCR detection method for African swine fever virusMultiple sequence alignments were performed for the P72 genes derived from the 30ASFV strains,and two pairs of distinct primers were created according to their conserved areas.Using the standard plasmid PUC57-P72 as a template,the fluorescent dye TB Green q PCR method was used to draw a standard curve and screen for the best primer pairs.The detection sensitivity of the best primer pair was improved by optimizing the TB Green q PCR reaction system and conditions,and a repeatability test was performed.The nucleic acids of common swine viruses,such as swine influenza virus H1N1,porcine circovirus type 2(PCV2),porcine pseudorabies virus(PRV),porcine parvovirus(PPV),and ASFV standard plasmid,were used as templates for specific tests.The detection coincidence rate of the method was evaluated by detecting known ASFV-positive and ASFV-negative reference samples.(2)Bioinformatics analysis of the effects of ASFV B119L and MGF360-13L genes expression in 3D4/21 cellsThe RNA-Seq transcriptome data of porcine whole blood infected with ASFV low pathogenic OURT88/3 and high pathogenic Georgia 2007/1 were analyzed.ASFV B119L and MGF360-13L overexpression plasmids were constructed and transfected into 3D4/21 cells.Gene expression was detected by RT-q PCR.Transcriptome sequencing was used to analyze the changes in differential genes in host cells after plasmid transfection.GO enrichment was used to analyze the functional regulation involved in differential genes,and RT-q PCR was used to verify the changes in related gene expression.(3)Molecular mechanism of ASFV MGF360-13L and ZDHHC1 affecting type I interferon signaling pathway in 3D4/21 cellsThe distribution of ZDHHC1 in tissues and the expression of ZDHHC1 in cells were analyzed using the HPA database.ZDHHC1 was silenced by si RNA and its inhibitory effect was verified by q PCR and western blotting.MGF360-13L overexpression plasmid and ZDHHC1 si RNA(si ZDHHC1)were transfected into 3D4/21 cells,respectively,and transfected cells were stimulated with poly(I:C)to detect interferon-βand transcript levels of the downstream key interferon-stimulated genes MX1,OAS1,and ISG15.【Results】(1)The optimized specific primer pairs and reaction system based on the P72 gene amplified the PUC57-P72 standard plasmid at 1.0-1.0×10~6copies/?L.The established standard curve R~2 reached 0.9954,and a target gene of at least 1 copies/?L was detected.Because the TB Green q PCR amplification reaction used a two-step method,the detection time was reduced to within 1 h.The results of the repeatability test revealed that both the intra-and inter-batch coefficients of variation were under 2%.This method had strong specificity and no cross-reaction with other four common pig pathogens.The coincidence rate of clinical samples was 100%.(2)The differential genes of pathogenic pigs infected with the ASFV low strain were enriched in the type I IFN signaling pathway,but there was no significant change in these pathway genes after high strain infection.The ASFV B119L and MGF360-13L overexpression plasmids were highly expressed in 3D4/21 cells after transfection.Bioinformatics analysis has screened ZDHHC1 as a key target gene for MGF360-13L to regulate the innate immune response of host cells by inhibiting ZDHHC1 m RNA transcription.(3)HPA database analysis showed that ZDHHC1 is widely distributed in tissues and highly expressed in macrophages.si ZDHHC1 effectively inhibited ZDHHC1 expression at both the molecular and protein levels.MGF360-13L and si ZDHHC1 inhibited IFN-βand interferon-stimulated m RNA expression of genes such as MX1,OAS1,and ISG15.【Main conclusions】(1)The established TB Green q PCR detection method for African swine fever virus has the advantages of short duration,high specificity,and strong sensitivity.(2)The highly virulent ASFV strain inhibits the enrichment of type I IFN signaling pathway.ASFV MGF360-13L is involved in the regulation of host innate immune response and effectively targets ZDHHC1.MGF360-13L and si ZDHHC1 inhibit m RNA transcription of IFN-βand its downstream genes.【Innovation and research significance】(1)A procedure for detecting the African swine fever virus with high sensitivity,strong specificity,and good repeatability was established,which will be useful for the rapid diagnosis and population screening of African swine fever virus infection.(2)The study demonstrated that MGF360-13L of the ASFV multigene family inhibited the expression of ZDHHC1 in host cells and regulated the innate immune response of the host.These results provide a new strategy to further elucidate the immune escape mechanism of the virus and to develop a safe and effective African swine fever vaccine.