Cloning And Seqencing Of Fractional Gene Of Avian Paramyxovius Isolates And Production Of Ndv-F/Hn Pseudotyped Humanimmunodeficiency Virus-1

Newcastle disease, also known as Asian avian plague, is caused by Newcastle disease virus （NDV） and one of the most disastrous diseases in poultry industry.It is classified as a list A contagious disease of poultry by the World Organisation for Animal Health （OIE） and the first category disease in China. NDV belongs to the genus Rubulavirus of the family Paramyxoviridae and is enveloped, single-stranded negative-sense RNA virus. NDV, the second category of pathogenic microorganisms, is easy to be mutant and has a broad host-infection range and the new-emerging pathotypes. Thus, it is very difficult for the present criteria to discriminate the significant deviation of virulence of different strains and pathogenicity of different host. In this proposed study, we plan to clone five main genes encoding NDV（WF00C, WF00D, WF00G strains） F、HN、NP、Pand M genes and sequecing, and to explore classification, virulence, characteristics and evolutionary relationships of the past and provide a basis to explain the evolution of NDV at the molecular level.Construct the pseudotype virus of NDV with the ability of one-time infection,in order to study relationship between of Genovariation of F-HN proteins and fusion activity of NDV.1. Cloning and sequencing of Fand HN gene of Avian paramyxovirus isolatesIn order to replicate and determinate the sequence of F gene of the newcastle disease virus （WF00C、WF00D、WF00G） isolated from chicken、ducks and goose resources by RT-PCR, then analyze their sequence of nucleotide and amino acid. The results showed that F gene of different virus included one complete open reading frame （ORF） and its full length were1223bp, which encoded553amino acids and had6glycolation region. The deduced amino acid sequence of the cleavage site region had the same amino acids pattern as velogenic strain was112R-R-Q-K-R-F117, which had the same character as genetype Ⅶ were lysine in one hundred first region and Valine in one hundred twenty third region. The WF00C strain had13Cysteine residues, but the WF00D and WF00G strains had12Cysteine residues. In the distribution of restriction endonuclease （nt334-1682）,the three strains existed particular Rsa I region at973nt and1249nt of a majority of goose resources. To analyze the homology of F gene encoding region among WF00D、WF00C、WF00G and15representative strains downloaded from GeneBank, their homology of nucleotide sequence was84.7～99.3%, and their homology of amino acid sequence was88.4～98.9%. The HN gene of different virus included one complete ORF and its full length were1716bp, which encoded571amino acids. To analyze the homology of HN gene encoding region among WF00D、WF00C、WF00G and15representative strains downloaded from GeneBank, their homology of nucleotide sequence was84.7～100.0%, and their homology of amino acid sequence was88.1～100.0%. The homology of three isolated strains was high, especially the sequence of HN gene from ducks and goose was the same. The difference of homology between three isolated strains and goose resources such as ZJ1、SF02、NA₁strains and so on was small, but the difference was significant in comparison with Lasota、 F48E9and so on. Their genetype belonged to VII according to the basis of phylogenetie analysis of NDV, the analysis of restriction site and the results of genetype.2. Cloning and sequencing of NP、P and M gene of Avian paramyxovirus isolatesIn order to replicate the sequence of P gene isolated from chicken、ducks and goose resources strains（WF00C、WF00D、WFooG）by RT-PCR according to the sequence downloaded from GeneBank then designed a pair of primer. To purify the replication production then insert into pMD18-T carriers, we had restriction、PCR and sequence determination, then analyzed the nucleotide and amino acid sequences, the basis of phylogenetie and amino acid sequences from three Avian resources. The results showed that NP、P and M gene of different virus included one complete open reading frame （ORF） and their full length were1470bp、1188bp、1095bp, which encoded489、395、364amino acids respectively. To analyze the homology of NP、P and M gene encoding region from ducks resources strains and representative strains downloaded from GeneBank, their homology of nucleotide sequence was85.2～99.4%、81.5～99.7%、84.1～99.7%, and their homology of amino acid sequence was90.6～99.4%、880.1～99.7%、86.1～99.7%, respectively. The homology of three isolated strains was high, contained6potential asparagine_linked glycosylation sitesand13cysteine（Cys） residues.The difference of homology among three isolated strains and goose resources such as ZJ1、SF02、NA₁strains and bucks resources such as PX2/03、Duck/1/05、FP1/02were small, which belonged to genetype Ⅶ,but the difference was significant in comparison with Lasota、 F48E9and so on, which showed the NP、P and M gene of this strain existed vast variation. Their differences of amino acid in comparison with nucleotide were small. The results of comparing the NP、P and M gene from different Avian resources strains showed that vast similarity, as revealed the co-infection variation from different Avian resources existed, and this variation was synchronous. The P gene in NDV VII existed particular amino acid sequences.3. Production of NDV-F/HN pseudotyped human immunodeficiency virus-1and analys is on its ability of infectionA pair of PCR primers for F and HN gene of including Flag tag sequence with EcoRI/SalI restriction enzyme sites was designed according to the sequence in GeneBank. Then the F and HN gene of NDV was amplified from the plasmid pMDC-F and pMDC-HN using PCR.Subsequently, amplified gene fragments were digested and cloned into pcDNA3.1（+） vector to create recombinant eukaryotic expression plasmid pcDNA-JF-Flag and pcDNA-JHN-Flag. HEK293T cells were infected, which was harvested from HEK293T cells cotransfected by pcDNA-JF-Flag、pcDNA-JHN-Flag and pNL4-3.Luc.R-E-.Then HEK293T cells were lysed to calculate the relative luciferase unit （RLU）. The full length of F and H gene of NDV were1662bp and1716bp respectively, and the homology in comparison with pMD-F and pMD-HN was100%. The activity value of Luciferase increased significantly HEK293T cell infected by NDV pseudotyped virus than controls. Construct the eukaryotic expression vector of including Flag tag sequence of envelope glycoprotein of NDV, and prepared the NDV pseudotyped virus.