Study On Infection Status Of DBSV In Ticks,Humans And Animals In Liaoning Province

Hantavirus(HV)is an RNA virus of the Bunyaviridae,which is the pathogen of hemorrhagic fever with human renal syndrome and pulmonary syndrome caused by Hantavirus.Dabieshan virus(DBSV)is a novel tick-borne virus,belonging to the Bunyavirus family and hantavirus genus.It was first isolated from rodents in Dabie Mountain area,Anhui Province,China in 2000,and is closely related to Hantaan virus(HTNV).Originally thought to be a subtype of Hantaan virus,it was officially named by the International Committee on Classification of Viruses(ICTV)in 2015.In recent years,novel tick-borne viruses have attracted widespread attention because of their potential to cause serious human diseases.Although the pathogenicity of DBSV to humans and animals is unknown,hantavirus can spread across species and DBSV is closely related to HTNV,which is suspected to be involved in human febrile diseases.Therefore,it is urgent to establish molecular biological and serological detection methods to investigate the prevalence of DBSV in ticks and human and animal populations.Metagenomic sequencing in 2020 found that DBSV already existed in Liaoning.Therefore,this paper conducted a preliminary study on the infection status of DBSV in Liaoning.The specific research contents are as follows:1.Establishment of DBSV PCR and pathogen detection in ticks,humans and wild animals in some areas of Liaoning ProvinceFrom May to September 2020,tick samples were collected in most areas of Liaoning Province.After regional classification(divided into eastern Liaoning,western Liaoning,southern Liaoning and central and southern Liaoning),macrogenome sequencing was carried out.The sequencing results showed that there was widespread DBSV infection in ticks in eastern and Western Liaoning.In this chapter,a PCR method was established to detect DBSV in ticks,humans and wild animals in Liaoning Province.Results: the positive rate of DBSV in ticks in Liaoning was 13.80%,and the positive rate of DBSV in human and wild animals ranged from 0 to 100%.2.Biological characteristics analysis,prokaryotic expression and purification of DBSV N proteinAccording to the gene sequence analyzed by metagenomic sequencing,a pair of primers were designed for the N gene of DBSV,and the recombinant protein p ET-28a-DBSV-NP was obtained by the E.coli prokaryotic expression The recombinant protein was purified after identification by SDS-PAGE and Western Blot,and a large amount of high-purity recombinant protein was obtained.3.Preparation of DBSV N Protein Polyclonal AntibodyNew Zealand white rabbits were immunized with purified DBSV recombinant N protein,and a polyclonal antibody with a titer of 1:32000 was obtained,which was verified by Western blot to have good specificity.This experiment successfully prepared anti-DBSV N recombinant protein rabbit polyclonal antibody.4.Establishment and preliminary application of indirect ELISA detection method for DBSV N proteinUsing recombinant N protein as the coating antigen,DBSV standard positive serum as the primary antibody,and HRP-labeled goat anti-rabbit Ig G as the secondary antibody,an indirect ELISA method for detecting DBSV antibody was established by optimizing the reaction conditions.The concentration of recombinant N protein as coating antigen is 2.5μg/m L,coating at 37°C for 2h,the optimal blocking condition is 2% BSA at 37°C for 1h,serum(1000-fold dilution)at 37°C for 15 min,secondary antibody(2000 double dilution)at 37°C for 15 min,and the substrate TMB was developed at 37°C for 20 min.The established indirect ELISA method was used to detect clinical(human,bovine,sheep,and pig)serum,and 50 serums with lower OD450 values were selected,and the mean plus 3times the standard deviation was used as The critical value of negative and positive serum.The specificity test confirmed that the method has good specificity and does not cross-react with antibodies of other viruses(Alongshan virus,Neobunya virus).The coefficients of variation of the intra-assay and inter-assay replicate experiments were both less than 5%,indicating that the established indirect ELISA method has good repeatability and stability.The established indirect ELISA method was used to detect clinical human,bovine,sheep,and pig serum from all over Liaoning.A total of 75 samples were positive for DBSV antibody,and the positive rate was7.4%.In summary,the PCR method established in this experiment can be used for the detection of DBSV pathogens,and the indirect ELISA method can be used for the detection of DBSV antibodies in serum,and a large amount of high-purity DBSV N recombinant protein has been obtained,and rabbit anti-polyclonal antibodies have been prepared,which are clinically useful.The research on the current status of DBSV infection in human and animal groups provides a certain basis.