The Whole Genome Sequencing And Virulence Determination Of An Anti-Tumor Newcastle Disease Virus

Newcastle disease virus (NDV) belongs to the paramyxovirus genus, possessing a single-stranded, negative-sense, non-segmented RNA. In recent years, the host range of NDV has been expanding, which leads to the infection of newcastle disease (ND) in variety of poultry and wild birds. However, the infection rate of NDV in human is quite low, and the infected people mainly show flu-like symptoms. NDV has become a focus of researches on anti-tumor biological agents, due to its specificity of killing tumor cells. At present, more and more NDV strains with highly efficient anti-tummor effect have been testified. Nevertheless, there are different killing ability levels of sorts of NDV strains even on the same tumor cell, which may be closely related to sequence differences among the viral genomes. In the meantime, it becomes increasingly urgent to link the viral genome sequence with the apoptotic signaling pathway of tumor cells induced by NDV in the anti-tumor mechanism investigations. An anti-tumor NDV strain is stroed in our lab, HBNU/LSRC/F3, whose anti-tumor mechanism had been preliminarily studied. Its whole genome sequence and virulence will be determined in this experiment.In the part of virulence determination, HBNU/LSRC/F3and Mukteswar were firstly prepared; the blank control group, negative control group (sterile saline), positive control group (Mukteswar) and the experimental group were then set up; finally, the mean death time of chick-embryos (MDT), intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index (IVPI) were determined. In the part of whole genome sequencing, the viral genome RNA was firstly extracted by kits, and the fragments were then amplified with the designed16pairs of primers in the one-step and two-step RT-PCR. The amplified fragments were overlapped and covered the entire genome after sequence assembly. The corresponding sequence fragments of the two ends of the genome were amplified with the anchor primers and the specific primers by simplified rapid amplification of cDNA end (RACE). All amplified fragments were identified by electrophoresis and transported to BGI (Beijing Genomics Insititute) for sequencing after purification. The results were processed and analyzed by bioinformatics softwares.The results showed that HBNU/LSRC/F3performed a MDT of105.6h, an ICPI of0.26and an IVPI of0, respectively. It is indicated that HBNU/LSRC/F3is an attenuated strain by the pathogenic indexes which are in the range of attenuated strains’. However, it is confirmed by sequence comparation and analysis that the cleavage site of the F protein of HBNU/LSRC/F3is112RRQRR↓F117, which is the characteristic structure of virulent strains. Hence, HBNU/LSRC/F3is an attenuated NDV strain with the cleavage site of virulent strains in its F proteins. It can be classified as the special NDV strain which doesn’t follow the general rule. The virulence of HBNU/LSRC/F3must be closely associated with other factors except the cleavage site of F protein.The complete genome of HBNU/LSRC/F3is15,192nt in length. The content of sequences encoding proteins is90.5%, and the G+C mol%is46.8%. The whole genome includes3’end (55nt), NP gene (1,753nt), P gene (1,451nt), M gene (1,241nt), F gene (1,792nt), HN gene (2,002nt) and L gene (6,703nt) and5’end (114nt). The six genes encode NP protein (489aa), P protein (395aa), V protein (239aa), W protein (137aa), M protein (364aa), F protein (553aa), HN protein (571aa) and L protein (2,204aa), respectively. All drawn phylogenetic trees indicate that HBNU/LSRC/F3belongs to the genotype IX of Class II lineage and the relationship between the genotype IX and the genotype III is the closest. Compared with the typical NDV strains, HBNU/LSRC/F3has the highest sequence similarity with F48E8.The virulence of HBNU/LSRC/F3was determined by the traditional methods for determination of virulence, and the whole genome of HBNU/LSRC/F3was sequenced and analyzed by the molecular biology technologies and bioinformatics softwares, which would provide the relevant basis for the subsequent researches for fully understanding of the anti-tumor mechanism of HBNU/LSRC/F3and improving its oncolytic properties.