The Functional Analysis And Application Of Vital Virulent Proteins Of Genotype ? Newcastle Disease Virus

The virulence of Newcastle disease virus(NDV)is regulated by several viral proteins together,including fusion(F)glycoprotein,hemagglutinin-neuraminidase(HN)glycoprotein,large polymerase(L)protein and nucleocapsid(NP)protein.First,single amino acid(AA)mutation was performed in the 457-461 AA motif of NDV F protein and 5 mutated F proteins were constructed.After transfection,all mutants of F protein could be expressed on the cell surface,furthermore,the F mutants(excluding N460)can significantly enhance the fusion activity with homotypic HN protein,which is manifested as the increased number and the expanded area of syncytia.In addition,the results of syncytium formation assay indicated that both T458 D and G459 D mutation conferred fusion activities without HN protein,which is in cell type-independent manner.Mutated viruses,rNDV-V457 D,rNDV-T458 D,rNDV-G459 D,rNDV-N460 D,and rNDV-L461 D were rescued by reverse genetic system successfully.The characteristic of rescued viruses showed that the efficiencies of viral replication of rescued viruses were decreased in DF-1 cells compared to that of rNDV,and the highest viral titers of rescued viruses were lower than that of rNDV.Importantly,the viral titers of rNDV-T458 D and rNDV-L461 D were significantly lower than that of rNDV at 24 h post infection(hpi),the viral titers of rNDV-T458 D and rNDV-G459 D were significantly lower than that of rNDV at 40 hpi and 48 hpi.The results also showed that both rNDV-T458 D and rNDV-G459 D have attenuated virulence compared to wild-type NDV.In addition,all 5 mutants reduced the viral titer in infected organs but rNDV showed a high virus titer in distributed organs,rNDV-T458 D,rNDV-G459 D and rNDV-L461 D were mainly found in the immune organs(spleen and bursa of fabricius);and rNDV-V457 D was mainly found in the intestine.The animal study showed that the pathogenicity of two mutated NDVs,rNDV-T458 D and rNDV-G459 D,were attenuated in chickens.Therefore,our data demonstrated that two newly-identified mutations in the F protein trigger a HN-independent fusion and attenuate the pathogenicity in chickens.Compared to rNDV,the NDV F protein mutants increased the ability to induce syncytia;however,they did not confer increased replication or virulence in vivo.L protein is the largest protein in the NDV,which is a multifunctional enzyme catalyzing viral RNA synthesis and processing(mRNA capping,cap methylation,and polyadenylation),and it is associated with virulence of NDV.The amino acid in conserved K-D-K-E motif of L protein was change individually and corresponding mutant viruses were successfully recovered.The results showed that NDV L mutations decreased the growth capacity in DF-1 cells and attenuated the virulence in vivo.The four mutated viruses were continuously passaged to 15 generations,sequencing proved that the mutation sites still exist in mutated viruses,which are genetically stable.SPF chickens were inoculated with those attenuated mutants and challenged with same genotype NDV-74 strain at day 21 post-vaccination,the result indicated that all immuned SPF chickens can be protected after challenge,among those mutants,there is no viral shedding in rNDV-E1954 Q group.Based on rNDV-E1954 Q construct,the recombinant virus rNDV-F+E1954Q was successfully recovered and the virulence was significantly lower than that of wild-type NDV.SPF chickens,which were immuned with rNDV-F+E1954Q,could be completely protected after challenge with virulent NDV-74,and viral shedding of rNDV-F+E1954Q group was significantly reduced compared to LaSota group.In addition,the mutated NDV bearing 18 AAs deletion of NP protein and the changed cleavage site of F protein was constructed.The rescued virus rNDV-NP?+F was avirulent in vivo,and high viral titer in day 9 embryonated eggs.They are genetically stable during 15 consecutive passages in chickens.Serological analysis showed that rNDV-NP?+F induced higher titers of neutralizing antibody than commercial LaSota vaccine,those neutralizing antibodies have the activity of hemagglutination inhibition.SPF chickens,which were inoculated with rNDV-NP?+F,were protected completely from challenging with same genotype NDV-74 strain at day 21 post-vaccination,and the viral loads in the infected organs of rNDV-NP?+F group were lower than that of LaSota group.The rNDV-NP?+F immunized chickens have the ablity of increased effectiveness against circulating virulent genotype VII strains,and of reducing virus shedding.These results demonstrated that genotype VII NDV vaccine candidate strains generated by reverse genetics can provide better protection than commercial vaccines.These results will provide a promising system for development of novel NDV vaccines.