Rescue Of Recombinant LaSota Strains Of Newcastle Disease Virus With Replacement Of V Protein And Their Effects On Interferons

Newcastle disease,caused by NDV(Newcastle disease,virus,NDV)is a highly contagious disease.NDV,Paramyxoviridae type I,is a single stranded,non-segmented,enveloped RNA virus.P gene of Newcastle disease is able to code three or more proteins by RNA editing,which makes P gene be inserted 1G(V gene)and 2G(W gene)at the 484 position(AAAAAGGG).V protein can antagonize the ?/? interferon system in two ways,which contributes to immune escaping,pathogenicity and host tropism of virus.Study of V protein is mostly based on the function of V protein,but the ability to antagonize the interferon of difference virulence of V protein in NDV infection is not clear.To elucidate the capacity of different virulence strains of V protein as the antagonist of interferon during viral infection,recombinant Newcastle disease viruses,of which the V protein had been substituted with virulent,mesogenic and lentogenic strains of NDV,were rescued.The effects of different virulent V proteins in infection of NDV were compared by measuring the virulence of recombinant virus,the growth curves of recombinant Newcastle disease viruses on different cells,the m RNA expression level of interferon signaling pathway and the level of IFN in the supernatant after infection:1.Construction of full-length c DNA of NDV with V gene substitution of virulent,mesogenic and lentogenic strains of NDV: p BRN-FL La Sota plasmid was digested by Pme I.HB,Beaudette C(BC),La Sota(LA)V protein,which were virulent,mesogenic and lentogenic strains of NDV,were inserted to p BRN-FL La Sota plasmid,full-length c DNA of L-La,L-BC,L-HB were constructed.The V protein of p BRN-FL La Sota plasmid was terminated by mutated.V protein of Virulent,mesogenic and lentogenic strain as well as a red fluorescent protein(R)were inserted to the plasmid by Kfl I and Pme I digestions.Lvs-La,Lvs-BC,Lvs-HB,Lvs-R full-length c DNA were constructed.2.Recombinant virus rescue and identification of virus characteristics: The recombinant plasmid was transfected into BSR cells by calcium phosphate transfection.After 5 days,the viruses were inoculated to 9 to 11 day-old chicken embryonated eggs.5 days later,allantoic fluid was obtained and the virus was inoculated to chicken embryonated eggs to 2 passages.MDT,ICPI and IVPI of the recombinant virus were measured.MDT and IVPI were consistent with the original recombinant virus r NDV in virulence.However,ICPI of L-BC and L-HB were 0.4,0.2,the rest were 0,which showed a virulence ascension by BC,HB V protein.3.Cell pathogenicity and antagonistic action to release of IFN type I of recombinant virus: The growth curves of recombinant virus on VERO cells and DF1 cells,CPE caused by recombinant virus at 48 h,m RNA levels of type I IFN signaling pathway measured by q PCR after infection,IFN-? protein levels in DF1 cells were measured by ELISA after infection,IFN levels were measured by VSV infection after recombinant virus infection.The results showed that the insertion and replacement of different virulence V proteins could affect the level of interferon and virulence in cells.It showed a tendency that the lentogenic V protein's ability of interferon antagonism was stronger during infection,and the virulent strain of V protein ability of destroying cells was stronger during infection.The study showed the tendency that,in infection of La Sota,it was negative correlation between virulence of V protein and the ability of interferon antagonism;while,the pathogenicity of cells was positively correlated with the virulence of the V protein.