Genetic Variation Analysis Of RHDV And Establishment And Application Of Detecting Methods For Viruses And Antibodies

Four RHDV strains, SQ-1, SQ-2, DB-1and BJ-1, were isolated by animalchallenge test. The SQ-1isolate could not agglutinate human type “O” erythrocytes.The VP60protein genes of these strains were cloned and then the gene phylogeneticanalysis was performed. The results showed that the four strains were all subdividedinto the RHDVa variant group according to the phylogenetic tree based on nucleotidesequences, but SQ-2was the only member in a solo branch which was except fromthe RHDVa and the original RHDV subtypes based on amino acid sequences. Thenucleotide sequence homology of SQ-1, SQ-2, DB-1and BJ-1with the reportedviruses were89.5%-98.0%,91.4%-95.6%,90.2%-98.7%and89.7%-97.6%,respectively, while the amino acid sequence homology were94.1%-99.3%,94.8%-96.5%,94.4%-99.5%and94.0%-99.3%, respectively. The mutation at432and480amino acid residues of VP60protein of the SQ-1isolate maybe resulted in losing thehemagglutinating properties. The genetic variations occurred, including amino acid304,305,414,425and432, and the variation trend during the past40years, includingamino acid13,237,299,309,324,346,348and351, were clarified,12amino acidsin the RHDVa strains were totally defferent from the original RHDVs, severalbilateral revertable amino acids did exist in the two subtypes.According to the sequences that published in the GenBank and these four isolates,the different combined primers were designed to establish the real-time RT-PCRdetecting method which was based on SYBR Green I fluorescent dye. This methodwas much more sensitive, reproducible and specific than ordinary RT-PCR, and aminimum of101copies of viral cDNA or more could be detected. The clinicalapplication results showed that the positive detectable ratio of this method was74.8%,but only65.5%with the RT-PCR. Another detecting method, RT-LAMP with highsensitivity and specificity, was also established, the amplification could be finishedwith no more than30min, and a minimum of102copies of viral cDNA or more couldbe detected. A total of30clinical samples were detected with RT-LAMP, the positive rate was higher than RT-PCR. Animal challenge test showed that all the RHDVpositive samples that detected with the two methods could lead to100%morbidityand mortality.The recombinant truncated VP60protein was expressed by using the prokaryoticexpression system, the recombinant purified proteins were coated to develop anindirect ELISA method to detect the antibodies againt RHDV. The highest positiveserum antibody dilution titer was1:6400with high specificity, the intra coefficient ofvariation(CV) was less than4.9%, and the inter coefficient of variation(CV) was lessthan6.9%. The compliance rate between ELISA and hemagglutination inhibition (HI)test was as high as94.4%. A total of1710serum samples from Henan, Shandong andHebei provinces were detected with the ELISA method, and the positive rate was81.5%. The RHDV antibody colloidal gold rapid detecting method, which is based onthe colloidal gold labeling technique and immunochromatography, was established.Firstly, the full length of VP60protein were expressed in prokaryotic expressionsystem, and the soluble portion of recombiant proteins were purified, then thecolloidal gold labeled SPA (Staphylococcus aureus protein A) was used as a probe tocapture the rabbit IgG which was regarded as quality control line, furthermore, thelabeled SPA with rabbit anti-RHDV antibodies would conjugate with the recombinantVP60protein, which was regarded as the detection line. The sensitivity of this methodwas not only4times higher than the hemagglutination inhibition test, but also withhigh specificity. There was no differences among the inter or intra CV. Clinicalapplication showed that the antibody positive rate of the colloidal gold detectingmethod was90.0%, but only68.3%by using the hemagglutination inhibition test.In summary, the genetic variation and evolution trend analysis of the fourisolated strains of RHDV were performed, and the reasons which resulted in losingthe property of hemagglutinating the human “O” type erythrocytes of SQ-1strainwere also analyzed. The established methods for RHDV antigen or antibody detectingwere potential for clinical purpose with applicable prospects. The researchachievements in this study provide a theoretical basis and technical support fordisease prevention and control.