The Establishment Of Minigenome And Full-Length Genome For NDV Of Duck Origin Of NDV08-004 Strain

Newcastle disease (ND) is a serious avian disease that can cause substantial economic losses and remain a major threat to the poultry industry. The aetiological agent of the disease, Newcastle disease virus (NDV), is a member of the genus Avulavirus in the family Paramyxovidae. The genome of NDV is a non-segmented, single-stranded, negative-sense RNA of 15186, 15192 or 15198 nucleotides in length. According to the characteristics of the genome, NDV can be divided into Class I and Class II. World rarely finds Class I NDVs, most of which are demonstrated avirulence or low virulence expect a velogenic strain found in Ireland, and that are only isolated from waterfowl, shorebirds and live bird markets. Class I NDVs have been isolated from China since first reported in 2008.A lentogen strain named NDV08-004 in the duck flocks was isolated in 2008, which of the complete genome sequence was determined to be 15198nts that is 12 nts longer than the Class II NDV in P gene non-coding region (NCR). Phylogenetic analysis of Class I NDVs isolates revealed that the isolate is similar to the viruses isolated from live bird markets in Hong Kong in recent years, and is classified into the genotype 3 in Class I. For elucidating the biological properties of this additional 12 nts in the NCR of P gene, functions of genes and the molecular mechanisms of interspecies transmission of duck original NDV, a transcription vector containing the full-length cDNA from NDV08-004 genome and the Minigenome of NDV08-004, and three helper plasmids for NP,P and L genes were constructed to establish the reverse genetics system for NDV08-004 strain. 1. Establishment of Minigenome and helper plasmids for NP,P genesProducts of NP and P genes were amplified and cloned into pGEM-T Easy Vector with the designed primers, and then subcloned into mammalian expression vector pCI-neo. Plasmids pCI-NP and pCI-P were identified by digested with restriction enzymes and PCR; Plasmids pCI-NP and pCI-P were transfected into cells and using an indirect immunofluorescence(IIF) assay, yellow and green fluorescence then was observed. This is told that helper plasmids pCI-NP and pCI-P could automatically replicate and transcribe in eukaryocyte.Three fragments were amplified with the designed primers Leader,GFP,Trailer. A fragment named LGT, which is 1032bp in length followed"rule of six", was overlapped by overlap PCR with three fragments Leader,GFP,Trailer. The Minigenome of NDV08-004 strain was constructed by cloning the fragment LGT into the transcription vector pOLTV5.2. Establishment of full-length genome and helper plasmid for L geneNine fragments were amplified and cloned into pGEM-T Easy Vector with the designed primers. The fragments, amplified with primers LP,TP,A,B,C,D,E,F,G, were respectively subcloned into pGEM-T Easy Vector to construct the plasmids LTP-T,ABC-T,DE-T,FG-T which continued sequentially to be subcloned into a vector called LGT-04 digested with Mfe I and Bsu36 I. The resultant plasmid ND08004-pO that contained the full-length cDNA of NDV08-004 strain was constructed.A fragment E'was amplified with the specific primers and cloned into pGEM-T Easy Vector. Plasmid E'-T was digested with Afl II and Sal I, and then the fragment of interest was ligated into plasmid FG-T digested with the same enzymes, to constructed the plasmid L-T which was then subcloned into pCI-neo vector to construct the expression plasmid pCI-L.