Relationship Between The Virulence And The Different Regions In Newcaslte Disease Virus NP Protein

Newcastle disease (ND) is a worldwide devastating disease of poultry caused by Newcastle disease virus (NDV). NDV possesses a negative-sense single-stranded non-segmented RNA genome that encodes six structural proteins, the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and large protein (L). NP ORF cosists of 1467 nucleotides and encodes 489 Amino acids (aa). The N-terminal 401 aa from NP shared high sequence identities among all NDV isolates, while the C-terminal region is highly variable. The gene sequences located in the NP non-coding region (NCR) is also variable, and additional 6nt insertion has been verified in this region among the late genotype NDVs which showed higher pathogenicity in water fowls than the NDVs without insertion. Besides, in 2007, a recombinant virus rZJ1/BNP with NP gene from Beaudette C strain was recovered and its virulence was much lower than that of ZJ1 demonstrating that NP gene was also associated with the NDV virulence. To identify the gene sequences implicated in the virulence, the biological characteristics of the non-coding, conserved and variable regions were evaluated in this paper using ZJ1 strain as a backbone.1.Relationship between 6nts insertion in the non-coding region and virulenceBased on the NP gene sequence of Beaudette C strain, two pairs of primers for inducing additional 6nt (CTCCCA) at the position of 1646-1647nt, were designed. Subsequently, the overlapping PCR was performed with these primers, and using the restriction sites Eco81 I and Sma I, the amplified fragment was cloned into pNDV7.2. The positive plasmid was then digested with Age I and Xba I, and ligated into the same sites in the full-length cDNA clone of ZJ1 strain to construct the recombinant plasmid pZJ1/SBNP6A. After cotransfection of pZJ1/BNP6A with three help plasmids pCI-NP, pCI-P and pCI-L into BSR-T7/5 cells expressing T7 RNA polymerase, an expected phenotype NDV, rZJ1/SBNP6A was rescued successfully. Pathogenicity tests showed that the newly generated chimeric virus, rZJ1/SBNP6A displayed equal ICPI and MDT to rZJ1/SBNP whose genome is 15186nt in length, which indicated that the additional 6nt insertion in NCR of NP gene has no influence with the virulence.To evaluate the role of whole NCR in NDV virulence, two chimeric NP genes with the ORF and NCR from Beaudette C strain, respectively, were constructed by two overlapping PCR procedures. The resultant two fragments were then used to replace the corresponding fragment of the full-length cDNA pNDV/ZJ1, respectively. After successful replacement, two new full-length cDNA clones, pZJ1/SNCR and pZJ1/SORF were yielded. Through cotransfection of the plasmids into BSR cells mentioned above, two recombinant viruses, rZJ1/SNCR and rZJ1/SORF were generated. The bio-properties determined by MDT, ICPI and growth curve showed that the virulence of rZJ1/SNCR is similar to that of rZJ1, while the virulence of rZJ1/SORF is lower than rZJ1 but similar to rZJ1/SBNP. The results obtained in this study demonstrated that there is no significant effect of NP NCR on NDV virulence.2. Relationship between conserved and variable region of NP gene and virulenceAlignment of the NP sequences showed that the identities of NP sequences among different genotype NDVs were from 89.8-96.5%, and that the N-terminal 400 aa sequences shared 94.2-99% identities while the C-terminal only shared 50.0-71.1%. To clarify the role of variable and conserved regions in NDV virulence, two recombinant chimeric cDNA clones, pZJ1/SHR and pZJ1/SCR with variable and conserved region from BC, respectively, were constructed via two overlapping PCR procedures. After cotransfection of these plasmids into BSR-T7/5 cells, two recombinant viruses, rZJ1/SHR and rZJ1/SCR were successfully recovered. The pathogenicity tests showed that rZJ1/SHR exhibited similar virulence to rZJ1, while rZJ1/SCR displayed equal virulence to rZJ1/SBNP. These results demonstrated that the gene sequence associated with the NDV virulence was not located in the NP variable region but in the conserved N-terminus.