| Background and Objective:Newcastle disease virus (NDV) belongs to Avulavirus genera of the Paramyxoviridae family, which are enveloped viruses containing single-stranded negative-sense RNA and first isolated and named by Doyle in 1927[1], It is highly pathogenic to birds, but in rare cases it can infect humans. NDV can cause a high death rate of acute-contact infectious diseases-Newcastle disease in bird species, and chickens, turkeys and pheasants are susceptible. But in rare cases it can infect humans, and manifested as a slight conjunctivitis or mild respiratory symptoms[2], there is no human infection case was found. In recent years, Newcastle disease virus has attracted more and more attention with some characteristics such as its strong anti-tumor effects and high security to human, and so on. NDV could infect various human tumor cells, multiply and replicate independently and selectively kill them. Moreover, NDV is safe to most normal human cells[3]. In this study, the object of research is the Newcastle disease virus WDK/JX/7793/2004 (hereinafter referred to as NDV 7793, GenBank accession number is HM125898). Previous studies by our laboratory showed that NDV 7793 has a strong selective ability of anti-hepatoma cells[4], the pathogenicity test and the f gene cloning sequencing identified it as a low virulent strain[5]. From biological treatment of the safety and efficacy considerations, this strain has more potential anti-tumor biological therapy. This experiment is intended to use the cell biology, molecular biology and bioinformatics-related methods to sequence and analysis of NDV 7793 full-genome, it is beneficial to further study the strain of NDV 7793 to provide anti-cancer mechanism of molecular biology.Materials and Methods:NDV 7793 strain which was isolated in Jiangxi Poyang Lake is involved in this research. NDV 7793 strain were inoculated in chick embryo for virus amplification,48 hours later the allantoic fluid containing viruses were harvested and tested Haemagglutination(HA) titer of virus, HA titer≥1:1024 dilute the allantoic fluid to 1×108 PFU/ml.15ml collected allantoic fluid was purified by Absorption and Release-RBC, AR-RBC, and absorb the supernatant then determine its HA titer, split charging virus stored at-80℃. African green monkey kidney cell line Vero-E6 and human liver cancer cell line HepG-2 were grown according to ATCC recommendation. NDV 7793 stain liquid was triple-plaque purified in Vero-E6 cell line after preliminary purification in the chick embryo amplification, the second amplification of the purified virus was done by passage through chick embryo, and test HA titer untill≥1:1024. TCID50 was carried out to detect the cytotoxic effect of the purified virus on HepG-2.Continue to further genetic analysis with purified virus; Based on the entire genomic sequence data of Newcastle Disease Virus present in GenBank and some references[6,7],20 pairs of specific primers were designed and synthesized to acquire overlapping viral cDNA fragments of a NDV 7793 strain by highfidelity reverse transcription polymerase chain reaction(RT-PCR). The sequences of the 3'-and 5'-terminal ends of viral RNA genome were determined by sequencing cDNA fragments generated by rapid amplification of cDNA ends (RACE), and 5 gene-specific primers work for RACE. All of PCR products were purified and sequence to get full-genome sequence, and compared to the sequences of 25 NDV strains isolated from different parts of the world released in GenBank. At last, the homology, and phylogeny of NDV 7793 stain were analyzed, the molecular biological characteristics and evolutionary relationships was determined too by methods of Bioinformatics.Results:The Haemagglutination(HA) titer of NDV 7793 stain after first time amplification in the SPF chicken embryos, second time purified by Absorption and Release-RBC and third time triple-plaque purified on Vero-E6 cell line is all 1:2048. HA protein of NDV 7793 strain is certificated to have full-featured and coagulation activity; NDV 7793 strain has normal virulence.The Plaque purification experiment on Vero-E6 with NDV 7793 strain showed the PFU is 1.25×1010/ml; TCID50 on HepG-2 is 10-4.4fold diluted virus solution 0.1ml. It is proved once again the active virulence and cytotoxic effect of NDV 7793 strain.The genome of NDV 7793 strain is 15,186 nucleotides (nt) long, which share the same length with Bl, LaSota, Clone-30 and other strains published in GenBank, and was identified as a member of Genotype I, Evolution A; The results of identity analysis of nucleotide sequences showed that the identities between 7793 and Ulster67 strains were higher(95.5%) than that of the two strains of U.S(CA)/211472/02,DE-R49/99 (83.9% and 73%).The AT content of genome was 52.63%, whereas the GC content was around 47.37% respectively. NDV 7793 strain consists of 6 genes in the order of 3'-N (489aa)-P (395aa)-M (364aa)-F (553aa)-HN (616aa)-L (2204aa)-5', the 3'and 5'end of NDV 7793 strain genome contained the Leader (55nt) and Trailer sequence (144nt)The typical amino acid sequence in the protein lytic site of F protein of NDV 7793 strain is 112G-K-Q-G-R-L117, the feacture of lower virulent NDV strain with F protein cleavage sequence.Conclusions:1. NDV WDK/JX/7793/2004 strain was one virus with the cytotoxic ability and active virulence.2. The genome of NDV 7793 strain is 15,186 nucleotides is size, a low-virulent strain which belongs to genotype I3. Compared with NDV 7793 and other strains of 24 strains published in Genbank, the homology of nucleotide sequences is between 73% to 95.5%, and amino acid is between 50.7%～90.6%.4. Phylogenetic analysis indicated that NDV 7793 strain was closely related to Ulster strains which is an international standards lentogen strain belonged togenotype I...
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