The Effect Of The Polymerase-associated Proteins On The Pathogenicity Of Genotype ? Newcastle Disease Virus

Newcastle disease,caused by Newcastle disease virus(NDV),is a highly contagious and acute septic avian disease.It caused severe damages of digestive tract and respiratory tract and high levels of morbidity and mortality,resulting in considerable economic losses in the poultry industry worldwide.Therefore,it is important to understand mechanisms of pathogenesis for prevention and controlling of NDV.To study the molecular mechanism that affects NDV virulence,a reverse genetics system for LaSota strain was constructed.Based on this,sixteen chimeric viruses were recovered by exchanging the envelope-associated(F and HN)and polymerase-associated(NP,P and L)protein genes between the prevalent genotype ? strain SG10 and the genotype ? strain LaSota.We evaluated the pathogenicities and biological activities of different chimeric viruses,which showed that the envelope-associated proteins contributed greatly to NDV virulence and the polymerase-associated proteins also had a great effect on the viral replication and pathogenicity.Among the polymerase-associated proteins,the L protein was another major contributor to NDV virulence,especially when combined with the homologous NP and P proteins,as shown by an increase in the ICPI value from 0.00 for rLaSota to 0.63 for rLaSGNPPL and a decrease in the ICPI value from 1.86 for rSG10 to 1.32 for rSGLaNPPL.In NDV minigenome systems,the activity of the polymerase-associated proteins was directly related to viral RNA synthesis.The nucleocapsid protein(NP)of NDV plays an important role during viral replication.To further explore the specific functional domains in the NP protein,the protein sequences of the NDV NP genes were analyzed using three different online programs,which showed that the C terminus of NP protein may regulate the interaction of NP protein with others.Based on the predicted results,a series of NP protein plasmids encoding progressively truncated C-terminal mutants and individual amino acid mutants were constructed.Using the NDV minigenome system,we found that amino acid E402 in the C terminus of NP protein was important for the RNA synthesis.We further rescued the recombinant chimeric virus with the mutation E402A in the NP protein by reverse genetics and the relevant experiments were also carried out.These results showed that rNDV-NPE402A developed faster and larger plaques compared with rNDV-WT,and the E402A of NP directly increased the levels of mRNA transcription at the early stage of the viral infection,while the RNA replication was inhibited at the later time,and enhanced the viral virulence.In conclusion,this study demonstrated that the polymerase-associated proteins had a great effect on the NDV pathogenicity.Among the polymerase-associated proteins,the L protein was another major contributor to NDV virulence,and the E 402 of NP protein also had an important regulatory role for viral virulence.Therefore,our study provides the theoretical foundation for a further understanding of the molecular mechanisms of NDV pathogenesis.