Establishment Of Real-Time PCR For Detecting The Broad Spectrum Genotypes Of ASFV And Development Of Monoclonal Antibodies To P72 And P11.5 Proteins As Well As Their Interaction

African swine fever(ASF)is an acute hemorrhagic disease caused by African swine fever virus(ASFV),with a mortality rate of up to 100%in domestic pigs.ASF is currently epidemic in many countries and threatening the global swine industry.Although vaccine candidates for ASF are under development,especially a commercialized live attenuated viruses(LAV)vaccine with the deletion of I177L gene was approved for use in Vietnam,the safety and efficacy of this vaccine remains to be further verified.Therefore,the global pig industry is still faced with the threat of ASF epidemic.The major capsid protein(MCP)P72 among the ASFV proteins is the most important structural component of the virion,accounting for about onethird of the total weight of the virus particle.It is considered to be one of the most immunogenic proteins and the main target of nucleic acid detection.The genotype of the virus is also determined based on the variation of P72.P72 as the target gene of the virus may affect the accuracy of real-time PCR detection methods.So,it is necessary to find new detection targets in time and update the current detection methods.P72 protein has complex structure.However,there is relatively little information on the exact epitope of P72.In fact,the nonstructural protein B602L has been described as a chaperone facilitating the correct folding of the P72.Conformational misexpression of recombinant P72 protein may obscure some important epitopes and limit monoclonal antibody production.Previous studies have shown that P72 interacts with H240R and B602L respectively.Inhibition of H240R or B602L protein expression affects viral replication and decreases viral titers.In addition to the two proteins mentioned above,are there any other proteins that interact with P72 and participate in virus replication and pathogenesis?In order to solve those problems,this study carried out several researches from the establishment of detection method,the preparation of monoclonal antibodies,the mapping of the linear B cell epitopes of P72 protein and the identification of proteins interacting with P72.The research is described in details as follows:1.Development of Real-Time PCR Based on A137R Gene for the Detection of African Swine Fever VirusTo date,no safe and effective vaccines are available for ASF.Therefore,early diagnosis of ASFV is important for the control and eradication of ASF.New virus isolates have shown new genetic variation,and mismatches have occurred between primers recommended by OIE and multiple ASFV isolates.Therefore,it is necessary to update the current real-time PCR method to be able to detect all genotypes of currently prevalent ASFV.Previous proteomic and transcriptomic analysis showed that P11.5 protein encoded by A137R gene was highly expressed in ASFV-infected cells,suggesting that A13 7R gene may play an important role in the virus replication cycle,which makes it an interesting candidate detection target.In this study,a SYBR Green-based real-time PCR assay targeting the A137R gene was established for the detection of ASFV.Compared with the primers recommended by OIE,the primers designed in this study are more conservative and suitable for universal detection of ASFV.For futher evaluation of this method,34 clinical samples were assessed by both the A137R genebased real-time PCR and OIE-recommended TaqMan PCR.The results showed that the total number of positive samples was 29(29/34)detected by A137R gene-based real-time PCR,while only 27(27/34)positive using OIE-recommended TaqMan PCR.Moreover,no crossreaction with other common swine pathogens was found in the A137R gene-based real-time PCR.The sensitivity of this method is superior to the TaqMan PCR method recommended by OIE,which is 1000 times higher than the conventional PCR method.The repeatability test results showed that the coefficient of variation value of intra-assay and inter-assay was<1%,indicating that the method has good repeatability.These results indicate that the real-time PCR method established in this study has a promising application prospect in the clinical detection ofASFV.2.Development and biological characteristics analysis of monoclonal antibodies against P72 protein,B602L protein and P11.5 protein.As one of the most immunogenic proteins of ASFV,P72 protein plays an important role in the development of the diagnosis and vaccines.However,the epitope of the protein is not clear.In this study,B602L protein promotes the production and assembly of P72 protein in Sf9 cells.A total of ten monoclonal antibodies(mAbs)specific to P72 protein and two mAbs specific to B602L protein were developed by fusions between SP2/0 cells and spleen cells of mice immunized with the recombinant-P72&B602L proteins expressed in Sf9 cells.In this study,three mAbs to P72(ASFV-P72-7F10、ASFV-P72-7C12 and ASFV-P72-7D1)were found to recognize conformational epitopes of P72 protein which were formed only in the presence of B602L.Seven mAbs specific to P11.5 protein were developed using the recombinant P11.5 protein with GST tag expressed in prokaryotic expression system as immunogen.The above studies provide reliable biomaterials for the future study of protein interaction.A total of 50 truncated fragments were designed and screened.According to IFA and WB results,four linear B cell epitopes in P72 protein 31 SNIKNVNKSY 40,41 GKPDP 45,56 HLVHFNAH 63 and 185 ERLYE 189 were identified.Biological information analysis illustrated that epitopes 31 SNIKNVNKSY 40,41 GKPDP 45 and 185 ERLYE 189 were highly conserved within different ASFV strains.These findings may lead to a better understanding of the antibody-antigen interaction and provide new insights into the vaccine research and serological diagnosis of ASF.3.Exploring the interaction between ASFV P72 protein and P11.5 proteinThe P72 protein,which is critical in the formation of major components of the viral capsid,is involved in viral entry and plays an important role in the virus replication.Previous studies have shown that P72 interacts with H240R and B602L,respectively,and inhibits the expression of both genes,affecting viral replication.In order to further identify other proteins interacting with P72 and explore their roles in ASFV infection,monoclonal antibodies to P72 were used to co-immunoprecipitate IPAM and PK-15 cell lysates infected with ASFV,and then the silver stain,mass spectrum and western blot analysis were carried out.The results showed that P72 protein could effectively interact with P11.5 protein.The 1-216 amino acids of P72 protein and the 1-68 amino acids of P11.5 protein are the key structural domains of their interaction.The above research provides a reference basis for the functional research of P72 and P11.5 proteins,as well as the development of new antiviral drugs.Firstly,overexpression of P11.5 had no significant effect on the replication of ASFV.Next,we deleted the A137R gene on the basis of ASFV△MGF100-1R,constructed the recombinant virus ASFV△MGF100-1R△A137R,and further explored the impact of deletion P11.5 on ASFV replication.By analyzing the virus growth curve,it was found that the virus titer of ASFV△MGF100-1R△A137R was significantly lower than that of ASFV△MGF100-1R,which proved that A137R has important effect on ASFV replication.After P11.5 protein was supplemented,the virus titer was significantly incresed than that of the control group.In addition,the virus titer supplemented with 1-100 amino acids was higher than that of virus titer supplemented with 68-138 amino acids,indicating that 1-100 amino acids encoded by A137R gene may be the key region affecting virus replication.This study established the A137R gene-based real-time PCR method,which has good sensitivity,specificity,and repeatability,and can be used for the detection of different genotypes of ASF V.At the same time,monoclonal antibodies against ASFV P72,B602L,and P11.5 proteins were prepared using baculovirus expression system or prokaryotic expression system,and four new linear B cell epitopes of P72 protein were identified.The interaction between P72 and P11.5 was proved by using the monoclonal antibodies prepared above.Deletion and complement tests showed that A137R gene had an important effect on ASFV replication.The above results provide reference for the prevention and control,structural analysis,and vaccine development of ASFV.