Development Of A Recombinase Polymerase Amplification Assay And An Indirect ELISA Antibody Detection Technique For Rabbit Haemorrhagic Disease Virus

Rabbit hemorrhagic disease(RHD)caused by Rabbit hemorrhagic disease virus(RHDV)is characterized by an acute course,hemorrhagic septicema and a high mortality in adult rabbits.RHDV isolated belonged to one of the six identified genotypes(GI~GVI),among which the GVI is an antigenic subtype(RHDVa).In 2010,an additional RHDV was identified,phylogenetically and antigenically distinct from RHDV and provisionally called RHDV2 or RHDVb.A similar disease,termed European brown hare syndrome(EBHS),had been described in the hare(Lepus europaeus).In the study,recombinase polymerase amplification(RPA)detection method was established by RHDV pathogen.By bioinformatics analysis three encoding regions of RHDV were sub-cloned into pET32 a for expression in E.coli respectively.This study was divided into the following four parts:1 The isolation and genetic variation analysis of RHDVThree RHDV strains were named BZ/2015,LW/2015 and MY/2015.They were isolated by animal challenge test in different regions of Shandong Province.The VP60 protein genes of these strains were cloned and then the gene phylogenetic analysis was performed.The results showed that the three strains were all subdivided into the RHDVa variant group according to the phylogenetic tree based on nucleotide sequences.The nucleotide sequence homology of BZ/2015,LW/2015,and MY/2015 with the reported VP60 gene of RHDV were90.2%~98.6%,90.5%~99.9% and 89.4%~99.5%,The nucleotide sequence homology of VP60 gene of three RHDV strains with those of RHDV2 were 82.6% ~ 82.9%,82.5% ~82.8% and 81.5%~81.9%.HB Strain was the isolation of Hebei Province in 2014 belonged to the second gene group.In this group.The nucleotide sequence homology of HB Strain and WX84 Strain was very close,it probably come from the same strain WX84.Exception WX84 Strain and HB Strain,all other Chinese RHDV isolates belonged to fifth and sixth gene group.The hereditary distance between the strains isolated in recent years and WX84 Strain was variant.The result indicated that the VP60 gene sequences were very conservative,and the homology of the nucleotide among different isolates exceeded 90%,thus the gene group and region had negative connection.2 The establishment of recombinase polymerase amplification(RPA)detection for RHDVAccording to the sequences of VP60 gene of BZ/2015 strain,the specific primers were designed to establish recombinase polymerase amplification detection method.Through the Specificity detection,sensitivity of artificial infection experiment and clinical application,the method could detected RHDV.The virus could be detected a minimum of 0.1 LD50 of viral cDNA by RPA and one step of RT-PCR.RPA detecting method with high sensitivity and specificity,the amplification could be finished with no more than 30 min,Animal challenge test showed that all the RHDV positive samples that detected with the RPA and one step of RT-PCR could lead to 100% morbidity and mortality.3 Prokaryotic expression and antigenicity analysis of three encoding regions of VP60 geneTo establish a serological detection method for rabbit haemorrhagic disease virus(RHDV),three encoding regions of RHDV VP60-1(2 ~208aa),VP60-2(174 ~ 425aa)and VP60-3(342~579aa)were analyzed through bioinformatics and sub-cloned into pET32a(+)for expression in E.coli respectively.VP60 were sub-cloned into pCold? for expression in E.coli respectively.They are named pET-VP60-1,pET-VP60-2,pET-VP60-3 and pCold-VP60.The VP60-1 gene,VP60-2 gene,VP60-3 gene and VP60 gene were expressed steadily and higly in E.coli by IPTG.SDS-PAGE show that three truncated recombinant proteins of 42 ku,47.5 ku and 45.6 ku were gained and could be recognized by anti-serum of RHDV.Western blot show that recombinant proteins VP60-1,VP60-2,VP60-3 can and RHDV positive serum specificity reaction?4 The establishment of the indirect ELISA method for detecting antibody to RHDVBase on the expressed protein an indirect ELISA was established to detect RHDV antibody in rabbit serum.After immunized 7d,14 d,21d,28 d,35d and 42 d,blood was collected from each rabbit in seven groups to test the specific antibody level by indirect ELISA and HI assay.These data suggested that the two methods showed significantcorrelation(r2=0.9547).These results simply that VP60-1 and VP60 detection antibody levels change rules,and the indirect ELISA method with recombinant protein VP60-1 could be suitable for immunological evaluation of RHDV vaccine.