Identification Of A New Virulent Site In HN Protein Of Genotype ? NDV And Construction Of A Live Attenuated Vaccine Candidate

Newcastle disease(ND),caused by velogenic Newcastle disease virus(NDV),could induce serious damages in the poultry industry worldwide.So far,NDV infections have been found in more than 250 species of birds and a few of mammals.In order to adapt to so many host species,like many other single-stranded RNA viruses,NDV produces a large number of variants through genetic mutations,exhibiting in a variety of genotypes.In addition to host factors,the amino acid sequence at the cleavage site of the F protein(Fcs)has been considered the basis for the virulence of NDV.Although the Fcs of genotype ? NDV is typical of virulent viruses,some of the strains show low pathogenicity to chickens,suggesting that the virulence of genotype ? NDV may be influenced by other virulent factors.Genotype ? NDVs are antigenic and host variants of NDV,which are predominantly circulating among pigeons.The low homology between genotype ? and genotype II or ?I used to produce and immunize poultry ND vaccine in China always results in only providing partial protection in pigeons.Currently,there is a lack of a specific live attenuated vaccine for genotype ? to prevent the pigeons from NDV infection.Therefore,it is of great significance for the prevention and control of pigeon derived NDV to carry out the study on the virulence of genotype ? NDV and to develop an attenuated NDV vaccine.The contents and results of our studies are following:1.Comparative biology of two genetically closely related Newcastle disease virus strains with strongly contrasting pathogenicityIn order to obtain an attenuated NDV strain by natural mutation,a mesogenic NDV isolate(CI10)from crested ibis was continuously passaging in chicken embryos in the early stage of the laboratory.After many consecutive blind passages,it was found that the pathogenicity of the passage strain(CE16)was significantly reduced.To get a comprehensive understanding of the passage strain,we compared the biological characteristics and pathogenicity with the original isolate.Compared with CI10,the plaque size produced by CE16 reduced about 50%;The proliferative activity on cells declined to1/30 of that of the original strain;The pathogenicity index(ICPI)decreased from 1.04 to0.35,and the MDT was extended from 86 h to over 120 h.Pathogenicity tests on chickens also showed that CE16 failed to cause significant clinical symptoms,and the mortality rate decreased from 100%to 0%.To elucidate the molecular reason for this biological phenotypes and virulence changes,the complete viral genomes of the original and derived strains were sequenced.Both viruses contained the same fusion(F)protein with a cleavage site(Fcs)motif that is usually associated with velogenic viruses.According to the phylogenetic analysis of F gene,both strains belong to genotype ? NDV.Molecular analysis revealed six nucleotide(nt)differences resulting in five amino acid(aa)mutations,respectively distributed in nucleoprotein(NP)(A426T),hemagglutinin-neuraminidase(HN)(A215G and T430A),and large protein(L)(E1694V and R1767G).HN is associated with NDV membrane fusion activity,NP and L are associated with viral replication.Thus,we speculated that the phenotypic difference is caused by the mutation of amino acid at these 5sites.Further predictive analysis of protein 3D structure model showed that HN215 mutation did not cause significant structural changes;The mutation of HN430 changed the?-sheet to random coil;Mutation in L1694 dramatically altered the conformation of the random coil;Mutation in L1767 turn the?-helix to random coil.Although the 426aa in NP did not have the homologous model,it lied in a region that have been confirmed to regulate the interactions between the NP and Phosphoprotein(P).Our findings make a significant contribution to the literature because these identified amino acid substitutions can provide clues into the factors contributing to high variation in pathogenicity among NDV strains despite genetic similarity and make a foundation for more in-depth investigations of the molecular mechanism underlying virulence.2.Construction and evaluation of a reverse genetic system based on NDV genotype ? NDV strain CE16In order to study the molecular mechanism of CE16 virulence weakened and to develop a live attenuated vaccine candidate,we constructed a reverse genetic system of genotype ? NDV based on T7 promoter using the CE16 strain generated by consecutive passages in chicken embryos.Firstly,we selected the modified clone vector p BR322 as the skeleton vector,which was easier for rescue NDV.A full-length c DNA clone of the viral genome was assembled by 8 different subgenomic fragments through the restriction endonuclease between repeated sequences.In order to acquire the precise genome terminals,we introduced T7 RNA polymerase promoter at the 5'end,the Hepatitis delta virus(HDV)RNA ribozyme sequence and T7 RNA polymerase terminator at the 3'end of the genomic c DNA respectively,and named the clonal plasmid inserted with the full-length c DNA of CE16 as PBR322-CE16.Meanwhile,the coding region sequences of NP,P,and L of CE16 strain were cloned into expressive vector p CAGGS to construct helper plasmid p CAGGS-NP,p CAGGS-P,and p CAGGS-L.Finally,the constructed full-length c DNA plasmid and three helper plasmids were co-transfected into human laryngeal epidermoid carcinoma(HEP-2)cells and infected with a recombinant vaccinia virus(MVA-T7)that expressed T7 RNA polymerase.Cell supernatant and cells were collected after 4 days of culture,and then inoculated into SPF chicken embryos for further amplification.Finally,hemagglutination assay(HA)was used to determine the viral titers.Through the above methods,a recombinant strain of r CE16 was successfully recovered.The recovered strain was continuously passaged in chicken embryo for 10 generations,which showed a good genetic stability.Compared with CE16,r CE16 had no significant changes in syncytium formation and cell proliferation kinetics.ICPI and MDT changed from the original 0.35 and>120 h to0.33 and>120 h;There was also no significant difference in virulence.Therefore,the above results indicated that our study successfully constructed a reverse genetic system of genotype ? NDV based on T7 promoter,providing a technical means for the subsequent research on the molecular mechanism of NDV virulence and develop attenuated live vaccine.3.Identification of new virulence site in HN proteins by reverse genetic techniqueThe HN protein of NDV is an important virulence factor which is confirmed to affect virulence and pathogenicity.To investigate the role of aa215 and aa430 mutations in the HN protein of NDV CE16 strain on the biological characteristics and pathogenicity,wild type(wt)and single point mutant HN proteins of wt CE16,wt CI10,G215A and A430T were constructed.Cell surface expression efficiency,hemagglutinin(HAd)hemadsorption,Neuraminidase(NA)activity,membrane fusion activity promotion and F protein cleavage ability were evaluated to test these HNs functions.The results showed that the expression efficiency on the cell surface of the 4 HN proteins were almost consistent,indicating that the amino acid mutation did not affect the expression of HN.Other biological studies found that,compared with wt CE16,A430T and wt CI10 decreased the HAd by 27.5%and 29.9%,increased NA activity by 78.3%and 95.5%,improved fusion index by 56.6%and 66.3%,and promoted F protein cleavage index by 40.9%and 51.4%,respectively;G215A increased HAd activity by 19.6%,improved NA activity by 17.9%,promoted fusion index by 12.9%,and promoted cleavage index by 5%,all of which showed no significant differences.All the above results proved that HN430 is an important functional site affecting the biological activity of HN protein from the protein level.In order to further study the effect of HN430 amino acid mutation on the virus,A430T was introduced into PBR322-CE16 by point mutation to construct PBR322-CE16-HNA430T.r CE16-HNA430T was successfully obtained by virus rescue,and confirmed to has a good genetic stability after being continuously passaged for 10 generations in chicken embryos.Compared with r CE16,r CE16-HNA430T produced larger syncytium by about 40%,increased HAd ability by about50%,improved NA activity by about 130%,and showed higher replication ability by about8 times.The pathogenicity test showed that the pathogenicity index(ICPI)increased from0.33 to 1.38,and the MDT was from>120 h reduced to 86 h;Pathogenicity tests on chickens also showed that r CE16-HNA430T could cause severer clinical symptoms and a mortality rate of 100%,indicating that the pathogenicity of r CE16-HNA430T was significantly enhanced.Therefore,the HN430 amino acid site identified in this study is a newly identified virulence site different from the previously reported with important roles in cell fusion,virus proliferation and pathogenicity.These results lay a foundation for the study of functional sites of HN proteins.4.Construction of the genotype ? NDV live attenuated vaccine by reverse genetic techniqueAt present,the ND vaccine commonly used on pigeons is the traditional vaccine for chickens,the genetic distance of which with the prevalent gene ? NDV in pigeons is about20%.Although NDV has only one serotype,the mismatch between genotypes and antigens prevents traditional vaccines from fully controlling the infection and spread of endemic strains.Therefore,in order to study the live attenuated vaccine specifically targeting genotype ? NDV for pigeons,we used the genotype ? NDV strain CE16 attenuated strain as the research object and replaced the Fcs of PBR322-CE16 with ¹¹²RRQKR/F¹¹⁷of the typical virulent strain of La Sota with¹¹²GRQGR/L¹¹⁷of the traditional vaccine strain through infectious cloning technology.r CE16-La(Fcs)was obtained through virus rescue.F gene sequencing revealed that the strain contained a lentogenic strain Fcs sequence and no syncytium formation was observed in infected cells.It was found that r CE16-La(Fcs)had good reproductive activity in chicken embryo,with an HA titer of 2⁹,and the virus titer in cells higher than 10⁸EID₅₀/m L.The pathogenicity results showed that ICPI was further reduced from 0.33 to 0.00 and MDT was still>120 h,suggesting that r CE16-La(Fcs)was a typical lentogenic strain.Then,the r CE16-La(Fcs)was continuously passaging in chicken embryos for 15 generations and in the pigeon brain for 5 generations.Respectively we selected 5,10 and 15 passages of chicken embryos and 1,3,and 5 generations of pigeon brain to check the virulence and Fcs sequence.Results showed that the strain in chicken embryos did not significantly change the pathogenicity index(ICPI:0-0.05),cause any symptoms in the pigeons,and the Fcs sequence were not found revert mutation in every generation of virus,proved that the strain has good genetic stability.In order to evaluate the protection efficiency of r CE16-La(Fcs),pigeons were incubated one and two inoculations respectively with r CE16-La(Fcs)and La Sota,and then the production of serum antibodies post immunization,the protection ability and virion shedding post challenged with velogenic genotype ? were all detected.The results showed that after 28 days of immunization,the HI titer responding to genotype ? strain was 2¹¹,while that of La Sota was 2^8.5.No clinical symptoms or death occurred after challenged with the velogenic strain,indicating that r CE16-La(Fcs)and La Sota had the same immune protection efficiency.In addition,the detection of virus in oral and cloacal swabs post challenge with velogenic strain showed that r CE16-La(Fcs)significantly reduced the positive rate of swabs and shortened the time of virion shedding.In summary,r CE16-La(Fcs)showed good proliferative activity in chicken embryos,and its virulence and genetic stability conformed to the OIE standards of vaccine.In addition,compared with La Sota,when the serum antibodies produced by r CE16-La(Fcs)responded to the homologous strains can induce stronger neutralizing antibody activity,prevent the virion shedding out.Above all,r CE16-La(Fcs)can be used for pigeons to effectively prevent and control genotype ? NDV to spread to wild birds,and even poultry.