Preliminary Establishment And Application Of ELISA Detection Method For Jingmen Tick Virus

Objective:Jingmen tick virus(JMTV)belonging to the Flaviviridae family is a kind of segmented single-stranded RNA viruses with an envelope.JMTV forms an independent branch which separates from Flavivirus,Hepacivirus,Pestivirus and Pegivirus in phylogenetic tree.JMTV is transmitted to humans and a variety of animals(e.g.,bats,cattle,sheep and rodents)by ticks.The clinic features of JMTV infection are nonspecific fever,persistent moderate headache,accompanied by other symptoms such as fatigue,nausea,cough and pharyngeal discomfort.At present,most scientists at home and abroad have focused on the study of JMTV diversity in animal hosts based on highthroughput sequencing or specific RT-PCR primers.However,there are few studies on the status of human infection with JMTV.In this paper,JMTV NS3 protein was expressed and purified in E.coli.A preliminarily ELISA for detection of JMTV specific antibody was established.Samples of human and domestic animals in Hubei Province in 2022 were collected,specifically the serum of low-risk population(healthy people in central hospital of wuhan)and the serum and blood clots of animal hosts(cattle and sheep).This preliminary study on the JMTV infection will provide basic data for the infection status of JMTV in human and animals.Methods:(1)The prokaryotic expression plasmid of JMTV NS3 was constructed and induced by IPTG in E.coli.NS3 proteins were purified by Ni-column affinity chromatography and imidazole gradient elution,refolded with PBS buffer,and stored in aliquots for subsequent experiments.(2)The purified NS3 protein was used as antigen to immunize white rabbits subcutaneously on the back to prepare polyclonal antibody.One week after the fourth dose of immunization,blood was collected to detect antibody titer.Five weeks after immunization,blood was collected to isolate serum and purify rabbit polyclonal antibody.(3)Purified NS3 protein was used as coating antigen,rabbit polyclonal antiserum was used as antibody,and the optimal coating concentration of antigen in ELISA method was determined by checkboard titration.Meanwhile,the coating buffer,blocking solution and color substrate were optimized.(4)Serum and blood clot samples of cattle and sheep from several farms in Hubei Province were collected from May to June 2022.In October 2022,serum samples from physical examination people in Wuhan Central Hospital,Hubei Province,were collected as lowrisk population samples.(5)The average OD value of samples plus 3 times SD was set as the cut-off value to determine the cut-off value of human,cattle and sheep respectively,and the JMTV antibody screening in human and animal serum was carried out.(6)RNA was extracted from all blood samples,and all samples were tested by RTPCR using the self-designed merger primers and the JMTV screening primers published in the published literature to screen JMTV nucleic acid.Results:(1)Through epitope analysis and codon optimization,the prokaryotic expression plasmid p ET-22b-JMTV-NS3(148aa-808aa)containing NS3 gene was constructed.The molecular weight of the inclusion body NS3 recombinant protein obtained by IPTG induction was about 75 k Da.(2)The rabbit polyclonal antibody against JMTV NS3 was prepared,and the titer was about 106 by ELISA.Western blot showed that the antibody could react specifically with NS3 protein.(3)The ELISA method was preliminarily established,and the optimal coating concentration of NS3 recombinant protein was determined to be 0.375ug/ml by checkerboard titration.The coating buffer,blocking solution and color substrate were optimized,and the best coating solution was phosphate coating buffer,the best blocking solution was Tris.HCl+0.5% skim milk powder,and the best color substrate was ED company’s two-component color substrate.(4)A total of 187 human sera were collected in Hubei Province in 2022,including 174 sera from low-risk people and 13 sera from high-risk people.There were 44 bovine serum samples and 44 corresponding blood clot samples.There were 193 sheep serum samples and 105 corresponding blood clot samples.The serum of forest residents in Hubei Province preserved in our laboratory is set as the serum of high-risk population.(5)The established ELISA method was used to screen the JMTV specific antibodies in the above sera.The results showed that 174 sera from low-risk people and 13 sera from high-risk people were negative for ELISA.Two of the bovine serum samples and seven of the sheep serum samples were positive,with seropositive rates of 2.27% and 4.15%,respectively.(6)RT-PCR results showed that all samples were negative.Conclusion:JMTV NS3 protein has good immunogenicity,and laboratory animal immunization can stimulate white rabbits to produce JMTV-specific antibodies.JMTVspecific antibodies were detected in the serum of cattle and sheep,suggesting that attention should be paid to the risk of virus spread caused by cattle and sheep as tick carriers.