Generation Of An EP402R-Deleted African Swine Fever Virus Mutant Expressing EGFP And Its Application In Evaluation Of Disinfectants

African swine fever(ASF),caused by african swine fever virus(ASFV),is a highly contagious infectious disease characterized by fever and extensive bleeding in pigs with a fatality rate reaching100%.Since August 2018,the first ASF outbreak in China,ASF has spread through the country within a year,which causes enormous economic losses.As there are no commercially available vaccines and effective therapeutic drugs,comprehensive measures based on biosafety are the main means to prevent and control ASF.Effective disinfection of environmental surfaces contaminated by pathogenic microorganisms is the key to prevent and control infectious diseases.Therefore,it is vital to screen out disinfectants that can effectively kill ASFV.Evaluating disinfectants in vitro requires determination of the virus titer before and after inactivation in order to determine the average logarithmic value of inactivation for judgment.If the ASFV wild strain is used for disinfectant evaluation,it is necessary to determine the virus titer through red blood cell adsorption or indirect immunofluorescence test,which is cumbersome.It is more convenient and quicker to use the fluorescent reporter virus that can determine the virus titer by directly observing the fluorescent.ASFV isolate ASFV HLJ/18 was used as the parent strain in this study,the EP402 R gene deletion strain ASFV HLJ/18-?EP402R expressing green fluorescent protein were constructed by using homologous recombination technology and limiting dilution method.The biological characteristics of ASFV HLJ/18-?EP402R were preliminarily evaluated.The results showed that porcine alveolar macrophages(PAM)infected with the ASFV HLJ/18-?EP402R strain lost their red blood cell adsorption;The growth kinetics of ASFV HLJ/18-?EP402R strain and ASFV HLJ/18 strain was not significantly different,indicating that deletion of EP402 R gene will not affect the level of ASFV replication in vitro.After that,the gene-deleted virus ASFV HLJ/18-?EP402R and parent strain ASFV HLJ/18 were used to systematically evaluate and compare the efficacy of povidone-iodine solution in killing ASFV.Under the condition of 21°C,the povidone-iodine solution of different concentrations reacted with the two strains for 5 min,30 min and 1 h respectively,and the neutralizer was added to stop the reaction.The reaction product was diluted by a 10-fold gradient and inoculated to PAM.Then,the virus titer was measured and the log inactivation was calculated.The results showed that there is no difference between inactivation ability of povidone-iodine solution of different concentration when they react with the same strain at 5 min,30 min or 1 h respectively;the active ingredient concentration of the povidone-iodine solution was more than or equal to 0.1% povidone-iodine can effectively inactivate ASFV;ASFV HLJ/18-?EP402R can replace the parent strain in the screening and evaluating ASFV effective disinfectants.In summary,an EP402R-deleted african swine fever virus mutant expressing EGFP was constructed by using homologous recombination technology.This ASFV mutant can replace the parent strain ASFV HLJ/18 in the rapid screening and evaluating ASFV effective disinfectants.This study lays the foundation for screening ASFV effective disinfectants and the in-depth study of EP402 R gene functions.