Establishment Of Double Antigen Sandwich CLEIA And ELISA For Detecting The Antibody Of Novel Akabane Virus And Preliminary Investigation Of Its Infection Spectrum

Akabane virus(AKAV)mainly infects ruminant animals such as cattle and sheep,causing abortion,premature delivery,stillbirth,fetal malformation,and can also cause hydranencephalic syndrome and joint curvature of the fetus through vertical transmission.Akabane disease is listed by the World Organization for Animal Health as a key quarantine object for statutory reporting of animal diseases and international animal trade,and is also a Class II infectious disease specified in China’s“List of Imported Animal Quarantine Diseases”.AKAV can infect various animals.In 2016,a novel AKAV(GXLCH16-70 strain)was isolated from bamboo rats,which was the first confirmation that the virus can infect rodents.To investigate the animal infection spectrum of the novel AKAV,this study prepared polyclonal antibody against the N protein of novel AKAV,established double antigen sandwich chemiluminescence enzyme immunoassay(CLEIA)and ELISA antibody detection methods,and used the established methods and RT-qPCR technology to conduct serological and pathogenic investigations of the novel AKAV.The AKAV genome is composed of three gene segments:L(large),M(medium),and S(small),encoding four structural proteins and two non structural proteins.N protein is the most conserved gene among AKAV structural proteins,and its main function is to participate in viral genome transcription and replication.N protein has high immunogenicity and can produce antibodies 3 to 5 days after virus infection,which can be used for early diagnosis after virus infection.Therefore,this study used a prokaryotic expression system to express N protein of novel AKAV,synthesized primers based on the gene sequence of AKAV GXLCH16-70 strain,amplified the target gene,and connected it to the p ET-32a(+)expression vector to construct a recombinant plasmid p ET-AKAV-N.After inducing expression at 37℃and IPTG concentration of 0.5 mmol/L for 6 hours,the soluble protein was obtained and purified.After identifying the recombinant protein by SDS-PAGE and Western blot,the concentrated recombinant N protein was immunized with New Zealand white rabbits to successfully prepare polyclonal antibody.The antibody titer detected by indirect ELISA method reaches 1:8.192×10~7;Western blot and IFA confirmed that the prepared polyclonal antibody can specifically recognize AKAV N protein.Subsequently,the recombinant N protein was used as the capture antigen,and the enzyme labeled antigen HRP-AKAV-N prepared by horseradish peroxidase was used as the detection antigen.By screening the optimal reaction conditions for the two detection methods,the double antigen sandwich CLEIA and ELISA antibody detection methods were successfully established.The established method had no reaction with the positive serums of BTV,EHDV,BEV,and PRV,so they were highly specific;The sensitivity was 32and 16 times higher than that of commercialized ELISA kits,respectively;The coefficient of variation within and between batches was lower than 10%,with good repeatability;The coincidence rates with the detection results of commercialized ELISA kits were 91.16%and 93.33%,respectively,with a high coincidence rate,which can be used for the detection of clinical serum samples.To investigate the animal infection spectrum of the novel AKAV,two antibody detection methods established in this study were used to detect antibodies in clinical serum samples;Simultaneously applied RT-qPCR and RT-nPCR for antigen detection of animal tissue samples.Double antigen sandwich CLEIA and ELISA methods were used to detect429 serum samples and 430 serum samples,involving the sera of cynomolgus monkeys,black leaf monkeys,gibbon,chickens,ducks,geese,pigs and humans.Results of both methods detected positive antibodies in the sera of primates such as cynomolgus monkeys,black leaf monkeys and gibbon.At the same time,a total of 251 animal tissue samples were tested,including crab monkey feces,brown backed viscera,vole viscera,forest mouse viscera,pangolin viscera,and other animal tissue samples.Among them,28 positive samples were detected in 37 samples of wild rodents.By cloning the AKAV S gene and sequence alignment,it was found that the AKAV S gene in the positive samples mentioned above has the closest genetic relationship with the novel AKAV strain GXLJ13-80,with homology of 98.9%～99.7%.In summary,this study has prepared a polyclonal antibody against N protein of novel AKAV isolated from bamboo rats,and established double antigen sandwich CLEIA and ELISA method.Combined with pathogenic detection,it indicates that the animal infection spectrum of novel AKAV isolated from bamboo rats is expanding,and more attention should be paid to its epidemic trend.