The Full Length Sequencing Of Guangxi-9Strain And The Rescue Of Mukteswar Strain From CDNA Genome Of Newcasstle Disease Virus

Newcastle disease(ND) is a serious avian disease, which with caused severeeconomic losses in the poultry worldwide and were classified as list A diseases by theOffice Internationale des Epizooties(OIE).The ND has been attracted by publicity. In1935, the ND was reported in Henan province of our contry. The causative agent ofthe disease, Newcastle disease virus (NDV) is a member of the genus Avulavirusin thefamily Paramyxoviridaeand which contains is three pathotypes depending on theseverity of the disease in birds: lentogenic, mesogenic, and velogenic. There are threetoxicity index indexs, inclonding MDT,ICPI and IVPI. The genome of NDV is anonsegmented, negative-sense, single-stranded RNA that encodes six major structuralproteins, including nucleoprotein, phosphoprotein, matrix protein, fusionprotein,haemagglutinin-neuraminidase and the large RNA-dependent RNApolymerase. HN and F glycoproteins,which are important for most studied andpathogenicity.Now, reverse genetics has been widely applied to molecular studies of NDV.The first reports of NDV rescue from cDNA were in1999. Both groups used thelentogenic NDV strain LaSota, which was further attenuated by modifying the RNAediting site in the P gene. The generation of a chimeric recombinant NDV wasreported. To construct the lab system of homologous recombination in NDV, thereverse genetics system of NDV is necessary, which includes cloning of full-lengthcDNA of NDV strains, cloning and expression of NP, P and L genes.Moreover,theconstruction of a cell lines was necessary, which could stably express T7RNApolymerase and generation of infectious NDV from full-length cDNA.The role of thehelper plasmids is necessary in the replication of NDV. The helical nucleocapsid is the template in the replication of RNA, and the core structure are constituted with thenucleocapsid protein NP and genomic RNA. Phosphoprotein P and protein L attachedto the core structure.In this study, we sequenced the genome of Guangxi-9stain which layed afoundation to study whether there are recombiations between the Guangxi-9strainand other strains. Because of its genome is too long, we cloned8segments to besequenced. We successfully obtained the full-length sequence of Guangxi-9.To rescue NDV Mukteswar (M) strain, we constructed helper plasmidsrespectively expressing NP, P, L. The T7RNA polymerase promoter was added at thebeginning of the full-length cDNA and the autocatalytic hepatitis delta virus ribozymewas added at the end. The helper plasmids NP and P were cloned to pCDNA3.0, andL to pMC18-CMV. The cell strain BHK-21was co-infected with the five plasmidsand cultured until cytopathogenicity occurred to rescue M strain. The rescued viruswas identified with RT-PCR and sequencing.