Construction And Immune Evaluation Of A Recombinant Newcastle Disease Virus Expressing Ebola GP Protein Mutated At Fusion Domain

Ebola haemorrhagic fever(EHF)is a zoonoses that causes the symptoms of acute severe hemorrhagic fever by the Ebola Virus.Since EHF was first reported in 1976,the 2014 West African epidemic was most serious in the outbreak affecting many regious of Africa.It caused over 11000 deaths in 28000 cases and posed a serious challenge to human public health.It is great significance to develop safe and efficient vaccine for EHF even our country has not outbreak of this disease.Currently,EBOV has been further subdivided into six species(Zaire,Sudan,Bundibugyo,IvoryCoast,Reston,Bombali),Zaire virus(ZEBOV)and Sudan virus(SUDV)are the most fatal of human infection,The Bundibugyo virus(BDBV)and IvoryCoast virus(ICEBOV)are occasionally infected in humans,The Reston virus(RESTV)and Bombali virus(BOMV)are not pathogenic to humans.Newcastle disease virus(NDV)is a negative-strand RNA virus and as a member of Paramyxoviridae,its genome encodes six structure proteins(NP?M?P?F?L?HN)and two nonstructure proteins(W?V).NDV which presents safety,high genetic stability and can induce a variety of immune responses in animals,has been widely used in the constructing of recombinant vaccines against various human diseases.Our previous study showed that the incorporation of EBOV GP protein into NDV particles significantly alters the behavior of the vector virus.On this basis,the GP of EBOV was mutated into GP(I532R),and a recombinant NDV expressing the mutant Zaire Ebola virus glycoprotein rLa-EBOVGP(I532R)was rescued via reverse genetics.The growth kinetic curve showed that rLa-EBOVGP(I532R)had a relatively consistent growth trend and maintained high titer from its parent strain.IFA and Western blot demonstrated that GP(I532R)was expressed correctly in the cells infected by rLa-EBOVGP(I532R).Meanwhile,viral spreading assay showed that GP(I532R)lost the ability to mediate recombinant virus to infect cells and spread on them.Immunoelectron microscopy confirmed the GP(I532R)was incorporated on the surface of viral particle as GP did in rLa-ZEBOVGP.Pathogenicity and safety evaluation of rLa-EBOVGP(I532R)suggested that the recombinant NDV strain had safety and low pathogenicity from its parent strain.Mice immunization assay demostrated that both rLa-ZEBOVGP(I532R)and rLa-ZEBOVGP could induce significant Ebola GP specific neutralizing antibody via intramuscular in mice,and the neutralizing antibody level of two recombinant viruses had no statistic difference.Our results thus provided a modified safer vaccine against Ebola virus,which also lays a foundation for further study of the biological function of GP(I532R).