| Rabbit hemorrhagic disease(RHD)is an acute,highly lethal infectious disease caused by Rabbit hemorrhagic disease virus(RHDV).The disease has brought huge economic losses to the rabbit breeding industry and seriously threatened the ecological balance.To understand the prevalence of RHDV in Linyi,Shandong Province,the following studies were carried out.According to the VP60 gene sequence of RHDV reported in Gen Bank,a pair of universal primers P1-F/P1-R were designed.157 rabbit samples collected in Linyi,Shandong Province were detected by RT-PCR method,and the corresponding gene fragments were sequenced.The results showed that 16 samples were positive for RHDV,with a positive rate of 10.2%(16/157).The strains in the positive samples with correct sequencing results were named RHDV-LY01,RHDV-LY02,RHDV-LY03,RHDV-LY04,RHDV-LY05,RHDVLY06,RHDV-LY07,RHDV-LY08,RHDV-LY09,RHDV-LY10,RHDV-LY11,RHDVLY12,RHDV-LY13,RHDV-LY14,RHDV-LY15 and RHDV-LY16,respectively.The sequence analysis demonstrated the 16 RHDVs were RHDV GI.1a type.To rapidly detect RHDV GI.1 with high throughput,a pair of specific primers P2-F/P2-R were designed for the VP60 gene sequence of RHDV GI.1.By optimizing the reaction conditions,the RHDV GI.1RT-qPCR assay with high sensitivity and good stability was established.The minimum detection concentration of the method was 13.1 copies/μL,which was 10 times more sensitive than the common RT-PCR.To clarify the genetic variation of RHDV,RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDV-LY16 selected were amplified and sequenced.As a result,the total genome lengths of the four RHDVs were all 7353 bp.Sequence analysis of the total genome of the RHDVs was performed by DNAstar.The results showed that there were 154 nucleotide mutations in RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDV-LY16,among which there were 25 missense mutations.The nucleotide homology of RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDV-LY16 was99.7%-99.8%,the nucleotide homology of P16 genes was 99.5%-99.8%,P23 genes 99.4%-99.8%.2C-like genes 99.9%-100%,VPg genes 99.5%-99.7%,3C-like genes 100%,Rd Rp genes 99.9%-100%,VP60 genes 99.8%-99.9%,and VP10 genes 99.5%-100%.The nucleotide homology of RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDV-LY16 with reference strains was 88.6%-97.3%,and the nucleotide homology of P16 genes was 87.4%-97.8%,P23 genes 86.9%-99.1%,2C-like genes 87.3%-97.5%,VPg genes 86.5%-97%,and 3C-like genes88.6%-97.1%,Rd Rp genes 87.3%-97.2%,VP60 genes 90.2%-97.9%,and VP10 genes 92.3%-98.4%.RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDV-LY16 all showed S121 L mutation on P16 protein.In addition,L62 S mutation was found on P16 proteins of RHDVLY01 and RHDV-LY11.RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDV-LY16 all showed V209 I mutation on P23 protein.2C-like proteins of RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDV-LY16 showed five mutations including F30 L,H38Y,G189 R,C254R and R309 G.There were 6 mutations on VPg proteins of RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDV-LY16,including H131 Y,C198R,C228 R,C242R,H300 Y and W356 R.In addition,RHDV-LY01 and RHDV-LY11 showed L162 R mutation on VPg protein.RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDV-LY16 showed T40 A mutation on the 3C-like protein.Rd RP proteins of RHDV-LY01,RHDV-LY05,RHDVLY11 and RHDV-LY16 showed three mutations,including V133 I,I264T and L282 P.RHDV-LY01 showed I329 V mutation on VP60 protein,and RHDV-LY11 showed I329 V,I496K mutations on VP60 protein.RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDVLY16 showed four mutations on VP10 protein,including C22 R,C24R,R71 G and H103 Y.Phylogenetic analysis showed that RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDVLY16 had the closest genetic relationship,belonging to the branch of GI.1a.In conclusion,RHDV GI.1a was the dominant endemic strain of RHDV in Linyi,Shandong Province.The sequence analysis of RHDV-LY01,RHDV-LY05,RHDV-LY11 and RHDV-LY16 showed that some mutations were observed in the genes,implying that RHDV continued to evolve.This study enriched the epidemiological data of RHDV and provided a theoretical basis for the prevention and control of RHDV. |
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