Characteristic Analysis Of NDV Strain TL1 Full Genome Sequence And Fusional Expression Inducing Effects Of Its HN Protein Gene

Newcastle disease (ND) is regarded worldwide as one of the most devastating diseases of poultry. Its causative agent, Newcastle disease virus (NDV), is classified as a member of the newly defined genus Avulavirus in the family of Paramyxoviridae. Because of the severe nature of the disease and the associated consequences, ND is included as an Office International des Epizooties (OIE) list A disease. In the past two decades, an intensive vaccination program against ND has been practiced in both large-scale poultry operations and village poultry farming. Disease outbreaks occur infrequently in some vaccinated flocks, however, infections of atypical Newcastle disease in the chicken flocks have been more and more frequently reported since the late 1990s. Even some chicken flocks with high antibody level can also suffer ND. Some scholar think that the emergence of new genotypes or subgenotypes could be responsible for ND outbreak in vaccinated flocks. Its genome is a single-strand negative-sense RNA and comprises six genes, from the 3'-terminus to the 5'-terminus, which encode the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), haemagglutinin- neuraminidase (HN) and Large protein (L). Matrix protein can influenza the formation of Virus-like particle (VLP), which is a mimic structure of virus particle without infectious genetic material. VLP is a highly effective type of subunit vaccine which has good immunogenicity. HN and F are major virulence factors of the virus, as they play a critical role in pathogenesis by attaching the virus to host cell and mediating viral fusion to host cell membrane. HN protein has an important synergism when F protein fuses adjacent cells and forms multinuclear cells, or syncytium. NDV TL1′s biological characteristics was studied, its full genome was sequenced and analyzed, HN protein′s synergism was also analyzed. It will establish foundation for new generation vaccine to utilize reverse genetics technique and VLP technique.One strain of Newcastle disease virus (named TL1) isolated from inner mongolia current outbreak ND chicken farms, its biological characteristics was studied. Cultured by SPF chicken embryo, the results of identification showed that the isolate was able to agglutinate chicken red blood cells, which could be inhibited and neutralized by avian Newcastle disease virus (NDV) standard antiserum, but could not be inhibited by avian influenza virus (AIV) standard antiserum (H5 and H9 subtype) and EDS-76 standard antiserum. Its MDT, ICPI and IVPI are 58 h, 1.7 and 2.4 respectively. The dehaemaglutination time is fast and the haemaglutinin heat-stability is poor. The ND pathological changes and symptoms also appeared in the challenged chickens. The results disclosed that the isolate was virulent strain of Newcastle disease virus. Six pairs of specific primers were designed and synthesized based on the entire genomic sequence data of Newcastle Disease Virus (NDV) present in GenBank.Complete nucleotide sequences of TL1 strain were acquired by reverse transcription-polymerase reaction. The entire genomic sequences of TL1 strain consisted of 15 192 nt, which is the same in length to 13 NDV ZJl, SF02, chicken/China/Guangxi7/2002,NA-1 strains, but is 6 nt longer than 22 NDV LaSota, Clone-30 and B1 strains in GenBank. The additional 6 nt motif is located in the non-coding region of nucleoprotein (NP) gene, but composition and location of the additional 6 nt motif are different to the entire genomic sequences of 22 NDV LaSota, B1 and Clone-30 strains, there are five types. The entire genomic sequences of TL1 strain is 38 nt longer than Sterna/Astr/2755/2001 isolated from Russian, is 6 nt shorter than DE-R49/99 isolated from Hungary. 12 nt longer DE-R49/99 to 22 NDV LaSota, Clone-30, B1 strains is located in the coding region of phosphoprotein (P).The leader and trailer sequences of TL1 genome are 55 nt and 114 nt long respectively. The nucleotide identities of the leader region between TLl and other 36 NDVs except Sterna/Astr/2755/2001 range in 81.8 % and 100 %. The nucleotide identities of the trailer region between TLl and the 37 NDVs range in 40.7 % and 100 %. In addition, the sequences at the 3' and 5' ends of NDV TLl are completely complementary in the first 12nt, three NDV strains NA-1,SF02,ZJ1 isolated from goose are alike, while those of the other 33 NDV strains are completely complementary only in the first 8 nt. The positions of start and end, and the length of each of TL1 share high degree of identity with majority of the other NDVs. The sequences of NP-P, P-M and M-F junctions of TL1 and the other most NDVs share high degree of identity, while the sequences of F-HN and HN-L junctions of TL1 have low degree of identity with those of the other NDVs.Comparing the coding of six genes of TL1 and the other 37 NDVs, the nucleotide sequence identities between TL1 and NA-1, Guangxi7/2002, Guangxi 9/2003, ZJ1, Guangxi11/2003 and SF02 are high,the nucleotide sequence identities between TL1 and La Sota, Clone-30, B1, 01-1108, 99-1997PR-32, 99-1435, 02-1334, 99-0655, 99-0868hi, 99-0868lo, DE-R49/99 are relatively low. The results show that evolution of NDV is global, don't only locate a certain gene.In mammalian cell expression system, four recombinant expression plasmids, pCI-M, pCI-NP, pCI-F and pCI-HN that harbour M, NP, F and HN gene of TL1 strain NDV, respectively, were constructed. These recombinant expression plasmids were separately transinfected into BHK-21 cells with liposome to express proteins. The expressed proteins were detected with antisera against NDV whole molecule by indirect immunofluorescence assay. In addition, BHK-21 cells co-transfected with four recombinant expression plasmids showed syncytium formation. F protein is a fusion protein that can induce cell fusion with the presence of HN protein, which was evidenced by the fact that pCI-F and pCI-HN co-transfected BHK-21cells can express F and HN proteins with the biological functions. Based on these results, after transfection of BHK-21 cells with pCI-M+pCI-NP+pCI-F, much bigger-size VLP around 120 nm were observed by electron microscope. When the BHK-21 cells were co-transfected with four recombinant expression plasmids, the VLP were about 200 nm in diameter, similar as NDV. The results show that HN glycoprotein plays an important roles in assembly of VLP and can lead to the differences in the size of VLP.