| Background:Paramyxovirus is single-stranded RNA virus which has two subfamilies, Paramyxovirinae and Pneumovirinae. The members of Paramyxovirinae include Paramyxovirus, and Rubulavirus. The representatives of Paramyxovirinae are parainfluenza virus (PIV), Newcastle disease virus (NDV) and Sendai virus (SeV). PIV and NDV are the most typical viruses of paramyxovirus. Measles virus (MV) is the only member of Morbillivirus and human is the only host of measles virus. Mumps virus (MuV) is the main member of Rubulavirus. The Pneumovirinae include respiratory syncytial virus (RSV) and avian pneumovirus (APV). In recent years, new members of paramyxoviruses have been found including Nipah virus (NiV), Hendra virus (HeV) and human metapneumovirus (hMPV). Paramyxovirus can cause respiratory system diseases of human, other mammals and avians. It can also cause reproductive system disease, digestive system disease and nervous system disease.The virus particle of paramyxovirus is similar with influenza virus but the size was 100-150nm, bigger than that of influenza virus. The general form of the virus is sphere and it could have many shapes some times, such as filiform. The genome of paramyxovirus is 13-16 kb with 6 open reading frame coding NP, P, M, F, HN and L proteins, successively. RNA dependent RNA polymerase (RDRP) of paramyxovirus is constructed by NP, P and L proteins. The RDRP can synthesis mRNA of paramyxovirus post-infection. M protein can combine with NP, HN and F proteins, playing an important role in virus assembling and budding procedure. Attachment protein and F protein are two glycoproteins inserting in the virus membrane. Attachment protein has different names in different viruses, in NDV and hPIV called HN protein with adsorption hemagglutination ability and neuraminidase activity, in measles virus called H protein only having adsorption hemagglutination ability without neuraminidase activity, in Nipha virus and Hendra virus called G protein neither having hemagglutination ability nor neuraminidase activity. F protein is an important protein which can mediate cell-cell and virus-cell membrane fusion. In most members of Paramyxoviruses, only the homologous HN and F proteins can promote the fusion process.The previously research indicates that bonding with sialic acid receptor of HN transmits a signal to its stalk region and interacts with F protein, subsequently. This interaction induces rearrangement of the disulfide bond in F protein, the fusion peptide is exposure and conformation of F protein changes to promote the fusion process. The HN bonding to its receptor is the key step for fusion promotion. In this study, we constructed mutations of high conserved amino acids of the global head in NDV HN to detect the biological functions of HN protein.Objective:To locate the key amino acids by determination the biological function changes of conserved amino acids mutations in NDV HN global head, analyse the effects of the hemagglutination ability, neuraminidase activity and fusion promotion ability by the decline of these biological functions in the mutations. To study the roles of the head and stalk region of NDV HN in fusion promotion process and the relationship of hemagglutination ability, neuraminidase activity and fusion promotion ability in NDV HN protein; To study the biological function of stalk region of NDV HN protein.Methods:1. To construct the mutants of high conserved amino acids in HN head. Alignment the amino acids sequence of attachment protein in NDV, hPIV 3, MV, SeV, HeV and NiV to choose 3 groups of conserved amino acids including 9 amino acids residue, E401, G402, R403, G468, V469, Y470, Y526, T52 and T528. The mutations were constructed by site directed mutagenesis methods.2. To express the mutants in BHK-21 cells. Co-transfected the HN and F gen into BHK-21 cell by TurboFect Transfection Reagent. The vaccinia virus-T7 transient expression system was used to express the interested proteins.3. To detect the expression efficiency on the cell surface of BHK-21. The indirect immunofluorescence was used to make the qualitative analysis of the mutations. Flow cytometry analysis was used to carry out the quantitative analysis expression efficiency of mutants on the cell surface.4. To detect the biological functional of the mutations. The quantitative and qualitative changes were determined by hemadsorption to analyze the receptor binding ability. R18 labeled pigeon RBCs were used to determinate the fusion promotion rate of the mutations. Giemsa stain and flow cytometry method were used to make the qualitative and quantitative analysis of the cell fusion, respectively. Colorimetric method was used to carry out the quantitative analysis of the neuraminidase activity.5. The headless NDV HN mutation was constructed and co-transfected in BHK-21 cells with wt F to determinate the syncytium formation ability by Giemsa stain at 37℃ and 39℃.Results:1. Mutants E401A, G402A, R403A, G468A, V469A, Y470A, Y526A, T527A and T528A were constructed successfully. The mutants E401A, G402A, G468A, V469A, Y526A and T527A were expressed successfully on the cell surface and the expression efficiency was 69.3%,101.4%,94.6%,92.0%,84.1% and 102.9%, respectively, compared with the wt NDV HN by flow cytometry method. The expressions of mutants R403A, Y470A and T528A could not detected by the indirect immunofluorescence and flow cytometry method.2. The hemadsorption ability of mutants E401A and Y526A decreased to 7.3% and 7.9%, respectively, of the wide type (wt) NDV HN, the neurominidase activity (NA) was 10.1% and 0.4%, respectively, compared with the wt NDV HN, cell fusion promotion ability was almost disappeared (2.4%,3.2%) compared with the wt NDV HN. Hemadsorption ability (HAD) of mutants G402A and T527A was 71.5% and 49.2%, respectively, compared with the wt NDV HN, the NA and cell fusion promotion ability declined with different degree (72.8%,35.5% and 53.4%,54.2%, respectively) compared wt NDV HN. HAD of G468A was 93.9% compared with the wt NDV HN, but the decrease of NA (35.3% of wt NDV HN) has a greater extent than that of cell fusion promotion ability (70% of wt NDV HN). On the contrary, in mutation V469A, the HAD was 46.5% of the wt NDV HN, but the cell fusion promotion (12.4%) ability has a greater decrease extent than that of NA (68.1%).3. The results of co-immunoprecipitation assay indicated that intension of HN and F protein has a reverse relationship with the cell fusion extent. Mutants of E401A and Y526A had stronger interaction than that in G468A; Result of semi-fusion assay indicated that fusion promotion rate of NDV HN mutations did not change but the syncytiums were smaller and the number was less than that in wt NDV HN.4. The headless mutant of NDV HN co-transfected with F caused syncytium formation in BHK-21 cell at 37℃ and 39℃, but the size was smaller and the number of syncytium was less than that of the wt NDV HN.Conclusions:1. E401 and Y526 residues were the key sites of NDV HN receptor binding and the mutations had very low HAD. The disappeared of the NA and fusion promotion ability in E401 and Y526 mutants was because of the lost of the HAD.2. The decline extent of the HAD, NA of mutants G402A and T527A were comparative with fusion promotion ability. It could be concluded that G402 and T527 residues participated the fusion promotion process, but they were not the key sites.3. Mutants G468A and V469A led to different extent decrease of the HAD, NA and fusion promotion ability. It indicated that G468 and V469 play an important role in keeping balance and switch the NA and HAD;4. The F protein promotion region was in the stalk region of NDV HN. And the HN protein inhibits the F protein conformation change before the F promotion.Background:Newcastle disease virus is a member of paramyxoviridae subfamily,and is a representative member of paramyxoviridae. The break of NO can cause heavy losses for poultry industry. The epidemic of ND has been controlled by vaccination with the attenuated Kve vaccine. But NDV has a high mutation rate,so the threat of NOV can not be eliminated thoroughly.NDV is a non-segmented negative chain RNA vims which is classified into Rubulavims, and Peeters suggested that NDV should be a single genus called Avulavirus basing on the immunology, serology and gene characteristic analysis.NDV can be divided into three classes, velogenic, mesogenic and lentogenic. The phylogenetic analysis of high variety region(47-435nt) showed that NDV includes 9genotypes, I-IX. The genotype VI, VII and VIII appear since 1960 s, IX is also appears after 1960 s, but it has been considered as a classic branch of genotype III.NDV has an extensive host range in nature. It can infect all kinds of the birds almost. The chicken is susceptible to NDV. Waterfowls have a high resistance of NDV,and the infection only causes mild symptoms or only causes suppressive infection.The wild birds are considered as the storage pool of NDV. It has been reported several ND breaks in water birds recent years. In mainland of China, it has been reported that ND brreks out in domestic goose and duck. If it was the adaptive variation causing the virulence increasing in the NDV evolution process or not needed to be verified.Phylogenetic analysis is a useful tool to study the NDV evolutionary history and plays an important role in NDV evolution analysis and classification. In this study, we analyzed the genetic characteristic trying to find NDV evolution and original evidence by phylogeny and discuss the reasons and mechanism of NDV sub-genotype appearance.Reverse genetics is also called Kscue of virus which is a new technique by conistmction the infectious foil length cDNA to study the pathogenic mechanism ofviras. Several NDV strains have been rescued with the advanced reverse genetics technology. The platform of NDV reverse genetics system provides better conditions1:0 study the evolutionary history and pathogenic mechanism. Moist of the reported Kscued NDV strains were lentogenic strains, and just few velogenic strains were rescued successfully study,we sequenced a velogenic strain NDV BJ,and constructed the full length cDNA and the helper plasmids. The purpose of the research is a rescue the velogenic NDV BJ strain to construct the NDV reverse genetic platform.Objectives:To gain the purified NDV BJ strain and detect the virulence. To amplify genome of NDV BJ strain by PCR and sequence the foil length genome of NDV BJ. To analyze the evolutionary history and probable origin of NDV BJ strain. To construct the foil length cDNA and helper plasmids system of NDV BJ strain for the rescue of NDV BJ strain.Methods:1.NDV BJ strain was purified by limiting dilution method and determinate the mean death time by allantoic cavity inoculation in 9 days SPF embryo eggs.Intracerebral inoculatio n was used a detect virulent of NDV BJ strai n by i ntracerebral pathogenicityindex.2.12 pairs of primers of NDV BJ strain were designed 1:0 amplify the gene fragments of NDV BJ genome. The PCR products were inserted into the T-vector.PCR productions were sequenced a get the fiill length genome of NDV BJ strain.Phylogenetic analysis of F gene and L gene of NDV BJ strain was made a find clues of the evolutionary evidence and potentially origin of NDV BJ strain.3.Available restriction sites were chose in the NDV BJ genome base on the sequence to construct the fill! length cDNA. The full length genome of NDV BJ strain was inserted into pVAX-1 vector.4.Primers of NP, P and L genes of NDV BJ strain were designed and the genes were amplified by PCR. NP and L genes were inserted into T-vector,sequenced and cloned into pVAX-1 vector to construct helper plasmids named pVAX-R pVAX-NP and pVAX-L.5. Primers of leader and trailer in genome 3’and 5, end of NDV BJ strain were designed. Primers of EGFP gene was designed and amplified by PCR from pEGFP-N3 vector; Recombined ae leader region,trailer regien and EGFP gene by homologous recombinant PCR,ae product was name过 TGL. TGL DNA firagment was inserted into pVAX-1 vector a construct pVAX-TGL vector a COnfirm the function of helper plasmids.Results:1.The purified NDV BJ strain was isolated from plaque by limting dilutiem method,ae foil length genome was amplified by PCR and aquenced. The sequence was uploaded a GeneBank and the accession number is HQ317394.2.The virulence of NDV BJ strain was determinated by MDT and ICPI. MDT was 52.4h. Tissue adhesion, hemorrhage and histolysis were found in dead embryo eggs. ICPI of NDV BJ strain was 1.92, the 1 day chicken showed serious clinical symptoms including blinking quickly, shaking head,astasia, wings ptosis and paralysis and chicken died in 3 Oh. NDV BJ strain was an NDV isolation with very high virulence.3.Phylogenetic analysis of high variety region of F gene(47-435nt)demo n strated NDV BJ strain being a member of genotype IX of NDV. NDV BJ strain belonged to genotype IX with a closed phylogenetic relationship to I-IV genotypes from the phylogenetic tree. NDV BJ strain was a sister branch of genotype ni.aprobably plays an important role in evolutionary process of NDV.4.Helper plasmids and mini-genome of NDV BJ were co-transfected into BHK-21 cells. The results demonstrated that NP, P and L genes could expression and formed the RNPs with biological fimetion.Conclusions;1 NDV BJ strain was a member of IX genotype with high virulence. The infection of NDV BJ strain caused death of embryo eggs and 1 day chicks within a short time.2.NDV BJ strain was a potential recombination strain of III/IV and VI-VIII genotypes. It was a transkion genotype of NDV between the isolation before and after1960 s.3.Co-transfection the helper plasmids and TGL can produce green fluorescence in BHK-21 cells.It demonstrated that the helper plasmids formed RNPs wiabiological function. |
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