Primary Study On The RT-PCR Dectetion Methods Of Rabbit Hemorrhagic Disease Virus (RHDV) And Rabbit Hemorrhagic Disease Virus Type2 (RHDV2)

Rabbit hemorrhagic disease (RHD). also named rabbit plague, is a highly contagious and fatal disease for domestic and wild rabbit(Oryctolagus cuniculus) aged more than 2 months, which is caused by rabbit hemorrhagic disease virus (RHDV) and was first reported in China in 1984. RHD is associated with hepatic necrosis, hemorrhages and congestion in several organs, including the upper respiratory tract and lungs, and splenomegaly, and the acute form of RHD can cause up to 80%-100% of mortality within 1-3 days. The disease has become a disease in the OIE list. Rabbit hemorrhagic disease virus type 2 (RHDV2), which emerged in France in 2010 and rapidly spread to several European countries, such as Italy. Spain, Germany. Portugal mainland, England. Wales. Scotland and several islands of the Azores, is responsible for causing a new rabbit hemorrhagic disease (nRHD). It can cause mortality in less than two months old rabbits and vaccinated rabbits that are typically not susceptible to classical RHDVstrains. There are no RHDV2 and associated research reports in China. In this study, the sequence information of RHDV and RHDV2 were collected and analysed, then the RT-PCR methods were established for RHDV2 and RHDV detection on the basis of synthesis of RHDV2 VP60 sequence by overlapping PCR. All these maybe summarized as follows:In order to develop a rapid method for the detection RHDV2,4 specific primers and 10 overlapping oligo primers were designed to amplify the conserved RHDV2 specific DNA fragment according to the genome sequences of RHDV and RHDV2 published in GenBank. Based on the synthesis of a conserved part of the RHDV2 sequence using overlap extension PCR and the recombinant plasmid construction, RT-PCR assay was first preliminary established and evaluated in China after a series of tests, including, optimization of reaction conditions, the sensitivity and specificity tests, and the application tests of 35 samples. The results showed that a 435 bp specific DNA fragment of the RHDV2 capsid protein (VP60) gene was synthesized in vitro and the recombinant plasmid pMD-19T-RHDV2 was constructed, and the RT-PCR method for RHDV2 rapid detection had good specificity and sensitivity. The sensitivity of the RT-PCR could reach about 230 copies of cloned viral genomic fragments of RHDV2, and there was no amplification for RHDV, pGM-T-EBHSV, Pasteurella multocida, E.coli and Salmonella from rabbits detection by this method. And the application tests showed that there were not RHDV2 in the experimentally infected samples and clinical samples.To develop a rapid method for the detection and differentiation of rabbit hemorrhagic disease virus (RHDV) and rabbit hemorrhagic disease virus 2(RHDV2),6 specific primers and 10 overlapping oligo primers were designed according to the VP60 gene sequences of RHDV and RHDV2 published in GenBank. A 435 bp DNA fragment of the RHDV2 capsid protein (VP60) gene was synthesized in vitro by using overlap extension PCR and two DNA fragments about 441 bp and 294 bp of the RHDV capsid VP60 gene were amplified by using reverse transcriptase polymerase chain reaction (RT-PCR) to construct the recombinant plasmids pMD-19T-RHDV2, pMD-19T-RHDV441 and pMD-19T-RHDV294. Then a complex RT-PCR assay for rapid detection and differentiation of RHDV and RHDV2 was developed and optimized after a series of tests, including, optimization of reaction conditions, the sensitivity and specificity tests, and the application tests of 35 samples. The results showed the established complex RT-PCR method for the detection of RHDV and RHDV2 amplified the specific products size of RHDV and RHDV2 were separately 294 bp and 435 bp, and it had good specificity and sensitivity. The complex RT-PCR detection limits of RHDV and RHDV2 were separately 160 copies and 230 copies of the cloned viral genomic fragments, and no amplification for pGM-T-EBHSV, Pasteurella multocida, E.coli and Salmonella from rabbits detection by this approach. And the application tests results showed the RHDV detection positive rate of the experimentally infected samples was 100%, and there were no RHDV or RHDV2 in the tested clinical samples.This paper set basis for the research on the RHDV2 control study and supplied a back-up technique for the rapid detection and prevention of RHDV2 in China, and supplied a new tehnology for the differentiation of RHDV and RHDV2.