The Isolation And Identification And The Cloning Of Capsid Protein VP60 Gene Of RHDV Strains And RT-PCR Detection For RHDV

Rabbit hemorrhagic disease (RHD) ,which is caused by Rabbit hemorrhagic disease Virus (RHDV) , is a rather acute,fatal and contagious disease,and causes enormous economy loss in rabbit farming. To control the disease, it's necessary to isolate and identify the virus strains, and study their molecular structure characteristics, as well as develop a sensitive and accurate detecting method for RHDV.In this research,three strains of RHDV were isolated and identified from clicinal materials,which were named RHDV-whn-1,2,3. The RHDV strains displayed stable pathogenicity after 3 passages in vivo.The RHDV-whn-2 shows high hemagglutinin valence of 1:640, but whn-1 and whn-3 show low hemagglutinin valence of 1:5,1:10 respectively. Limpid precipitation manifest can be seen during identification of 3 RHDV isolations by AGP and countercurrent immunoelectrophoresis, as well virion with a diameter of about 30nm can be observed in the electron microscope.Using a pair of 21 bp primers to amplify 496bp fragment situating the interior of conserved RHDV capsid protein VP60 gene sequence, the RT-PCR method was set up to test the dynamic distribution of RHDV RNA in different samples of rabbits killed by RHDV-whn-2 immediately after optimizing experiment conditions. The specific fragment about 500 bp in length could be amplified by this method.The RT-PCR method is specific, rapid and sensitive rate is 215 ng.mL^-1 RHDV template cDNA, therefore, this method is useful for clinic diagnose and molecular epidemiology investigation of RHDV.Furthermore, The particles of RHDV-whn-1,2,3 strains were extracted from liver samplesof rabbits.The capsid protein VP60 gene of 3 strains were cloned into pMD18-T vector by RT-PCR and sequenced. VP60 gene of whn-1,2,3 strains were in size of 1740nt and encoding 579aa respectively, the 3 sequences were submitted to GenBank with the accession number: DQ069280, DQ069281, DQ069282.Alignment with other 16 VP60 gene sequences of RHDV, RCV and EBHSV isolates in the world registered in GenBank showed that, with regard to nucleotide sequence ,whn-1 and whn-2 share the 96.1% homology, whn-1 and whn-3 share the 96.5% homology, whn-2 and whn-3 share the 99.4% homology. All the 17 RHDV strains(including whn-1,2,3) and RCV share the 84.7%-86.4% nucleotide homology, and share the 69.0%⁷0.5% amino acids homology, but the RHDV isolates themselves share the high homology of 89.7%-99.4% for nucleotide sequence, so VP60 gene of RHDVwas a rather conserved gene. For the Phylogenetic tree, all the RHDV strains trend to 4 genealogies on the level of nucleotides, while 3 genealogies on the level of amino acids. There are not noticeable Physical and temporal characteristic, such as RHDV-whn-1,2,3 strains situate 2 different genealogies.The other two members of Lagovirus of Caliciviridae family, RCV and EBHSV, parallel to RHDV and form 2 different genealogies both on the level of nucleotides and...