| African Swine Fever Virus（ASFV）is a large double-stranded DNA virus that can be transmitted by insect vectors.It has the ability to infect both domestic and wild pigs,leading to a range of clinical symptoms including high fever,multiorgan hemorrhages,coagulation disorders and splenomegaly.In 2018,African Swine Fever（ASF）was introduced into China and rapidly spread in several countries in the Asia-Pacific region,and high pathogenicity strains cause nearly 100%mortality in swine,making it one of the most serious animal diseases affecting the pig industry and even the livestock industry.Variation in gene families plays an important role in the genetic evolution of large DNA viruses.ASFV contains five multi-gene families（MGFs）that contain more than 50genes.The gains or losses of members from multigene families led to ASFV variations.These complex gene insertions,deletions,substitutions,and recombination have caused genetic diversity in ASFV and also altered the biology of ASFV,leading to repeated failures in vaccine studies.An approach that is increasingly gaining interest is the development of antiviral drugs.Therefore,this study aimed to isolate,identify and biologically characterize ASFV,as well as screen and validate drugs against it.1.Isolation,identification and biological characterization of ASFVIn this study,ASFV-positive samples were collected from both wild boars and domestic pigs in Hubei Province for virus isolation.The study results indicated that two strains of African swine fever virus（ASFV）,namely pig/Hu B1/2019 and wild boar/SNJ/2020,were successfully isolated.It was observed that pig/Hu B1/2019 could replicate at low levels in continuous cell lines such as Vero and WSL.Furthermore,phylogenetic analysis based on the B646L（p72）and EP402R（CD2v）genes revealed that both strains belonged to genotype II and serogroup 8.Based on the analysis of complete genome sequences,wild boar/SNJ/2020 and the Chinese high virulence strain pig/HLJ/2018 exhibit high sequence identity but differ in four genes.Pig/Hu B1/2019differs from pig/HLJ/2018 in 21 genes,including MGF in the variable region on the left side of the genome and large natural deletions in large segments of 17 genes in ASFV＿G＿ACD.In this study,the infection model of Chinese Bama miniature pigs was successfully established using the ASFV isolates,and the model effectively replicated the clinical symptoms of ASFV infection.The pathogenicity of two ASFV isolates was evaluated using this model,revealing that both strains induced acute infections in piglets with a fatality rate exceeding 100%and a half lethal dose below 1 HAD50.Infected piglets exhibited severe viremia,clinical manifestations,and organ damage.The establishment of this model is of great significance for investigating the virulence of ASFV and evaluating vaccines and drugs.Furthermore,this study investigated the disparities in host immunity levels induced by the ASFV isolates and demonstrated that pig/Hu B1/2019 was capable of eliciting elevated levels of interferon production compared to wild boar/SNJ/2020,while there was no significant difference observed in the impact on SLA-I expression.The diversity of ASFV strains and alterations in their biological properties have contributed to the intricacy and challenge associated with preventing and controlling ASF.In this study,two ASFV strains were successfully isolated and their genomic features,biological characteristics,virulence and pathogenicity were analyzed,thus providing important clues and data for the in-depth study of the pathogenic mechanism of ASFV.2.Screening of anti-ASFV compoundsIn this study,an antiviral compound screening model was developed based on primary porcine alveolar macrophages（PAM）,and the inhibition rate of compounds against ASFV was calculated by measuring the copy number of the viral genome under compound treatment.We screened a kinase inhibitor library including 298 kinase inhibitors using this model.Six candidate compounds with>90%inhibition of ASFV were identified,including Brincidofovir,Niclosamide,Ciclopirox,Ivermectin,Tenofovir（Disoproxil Fumarate）and Salinomycin.By calculating and comparing the selective index of the candidate compounds,it was found that Brincidofovir showed a better potential application.The above study provides a new direction for the development of anti-ASFV drugs and new drug candidates for the prevention and control of ASFV.3.Validation of the effect of Brincidofovir in inhibiting ASFVIn this study,we systematically evaluated the effect of brincidofovir on the inhibition of ASFV in vitro and in vivo.Brincidofovir reduces ASFV replication in a dose-dependent manner without cytotoxicity in PAM,Vero and WSL cells,the 50%inhibitory concentration of approximately 2.76 nmol/L for ASFV and a 50%cytotoxic concentration of 58μmol/L in PAMs,that significantly inhibits the viral titer,and early and late protein expression.Brincidofovir inhibited the post-entry stage of ASFV and viral replication in this study.We have molecularly docked the binding activity of brincidofovir to ASFV DNA polymerase,the result showed that brincidofovir competitively binds to the 42ARG domain of viral DNA polymerases through covalent bonds.In animal infection experiments,brincidofovir was effective in inhibiting the replication and horizontal transmission of ASFV in vivo.After administration of brincidofovir to ASFV-infected piglets,the survival rate was significantly increased,the hyperthermia caused by infection was significantly improved,the peripheral blood viral load was significantly reduced,the duration of viremia was significantly shortened,the virus shedding was significantly shortened,the amount of virus shedding was significantly reduced,and the degree of histopathological damage was significantly reduced.This study is the first to identify and validate the inhibitory activity of brincidofovir against ASFV infection in vitro and in vivo.These findings provide new ideas for the prevention and control of ASF,which is of great value in the absence of commercial vaccines or antiviral drugs against ASFV at present. |
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