Effects Of The Lysine Of F Protein Virulent Cleavage Site And Specific Sites Of HR2 In Newcastle Disease Virus On Their Virulence

Newcastle disease virus?NDV?infection causes severe disease in avian species.The fusion protein?F?cleavage site?Fcs?of Newcastle disease virus?NDV?is a major virulence factor.The Fcs of mesogenic and velogenic strain contains multiple basic amino acids with a common motif of“¹¹²R/K-R-Q/R/K-R/K-R?F¹¹⁷”that can induce cell membrane fusion to form syncytia,which results in cytopathic effect?CPE?and disease.The Fcs lentogenic strain has only 1 or 2 basic amino acids with a common motif of“¹¹²G/E-K/R-Q-G/E-R?L¹¹⁷”that can not induce membrane fusion and CPE.It is not pathogenic.Previous studies in our laboratory showed the different basic amino acids of virulent Fcs?VFcs?in its motif played a key role in the ability to cause cell membrane fusion and CPE in vitro assay.Besides,the specific amino acid position of the heptad repeat region 2?HR2?of the F protein has a certain effect on the ability to induce cell membrane fusion in vitro.However,the effects of these important amino acid sites on viral characteristics and virulence are unknown.The aim of this study is to use the reverse genetics technology with the lentogenic strain La Sota as the backbone for rescuing the recombinant viruses with these specific mutation sites of the VFcs and HR2 regions that will explore the effects of these specific sites on the biological characteristics and pathogenesis of these recombinant viruses.This study provides a theoretical basis for in-depth understanding of the F protein functions in NDV.The results are as follows:1.Construction of recombinant plasmids and virus rescueUsing the La Sota vaccine strain as a backbone,several VFcs and HR2 region-specific mutation recombinant plasmids were constructed by mutating its Fcs“¹¹²GRQGR?L¹¹⁷”,and nine recombinant viruses were successfully rescued using reverse genetics technology,including five VFcs mutants(3K:“¹¹²RRKRR?F¹¹⁷”;23K:“¹¹²RRKKR?F¹¹⁷”;25K:“¹¹²KRRKR?F¹¹⁷”;235K:“¹¹²KRKKR?F¹¹⁷”;P₁'L:“¹¹²RRQRR?L¹¹⁷”)and four HR2 region mutants?N479D;R486S;S497G;N479D+R486S?.2.Biological characteristics of recombinant virusesThe VFcs mutated recombinant viruses could normally grow in BHK-21 cells and caused obvious cell membrane fusion,and formed significant plaques,indicating that the VFcs mutations enhanced membrane fusion and CPE ability.Compared with the La Sota,there are no significant changes in the biological characteristics of recombinant viruses with mutations of specific sites in the HR2 region.MDT and ICPI experiments showed that the virulence of recombinant virus with the VFcs mutations was significantly enhanced.The virulence of r-23K,r-25K,r-235K could reach to mesogenic and velogenic.The virulence of r-3K,r-P₁'L was between lentogenic and mesogenic,It shows that when the number of basic amino acid lysine is 2 or 3,the NDV virulence is obviously enhanced.The virulence of recombinant viruses with HR2 specific site mutations was similar as lentogenic virus.3.Pathogenesis of the recombinant mutated virusesAfter the chicks were infected with the VFcs mutated recombinant viruses,the sick birds showed varying degrees of appetite,depression,diarrhea and lying on the floor.Some chickens also showed neurological symptoms of angulation and paralysis.The symptoms were most obvious in the r-25K group.Compared with the La Sota,the mortality of chicks in the r-3K,r-23K,r-25K,and r-235K groups were increased.The mortality of r-25K was as high as 90%,indicating that the pathogenicity of the recombinant mutated viruses in chicks was enhanced.The virus titers of thirteen tissues and organs in the infected with recombinant VFcs viruses were determined.The results showed that the viral titers of r-23K and r-235K were lower than r-3K and r-25K,of which r-25K was the highest viral titer.4.Effect of the recombinant mutated viruses on the permeability of the chicken brain The dying chicks in the infected with recombinant VFcs viruses were intravenously injected with fluorescein sodium?Na F?to determine the permeability of brains.The results showed that the cerebellum of r-235K and r-3K groups contained less amount of Na F.The r-25K group had the highest Na F content,while La Sota and r-23K groups had no Na F detection.The data suggested that the r-3K,r-25K,and r-235K viruses could increase the permeability of chicken brain,among which the r-25K virus has the strongest ability.In summary,this study used the La Sota vaccine strain as a backbone to construct the site mutations of the VFcs and HR2.The recombinant mutated viruses were successfully rescued by reverse genetics technology.The biological characteristics and pathogenicity of the recombinant viruses showed that the VFcs mutants enhanced the ability of membrane fusion and CPE,and increased the pathogenicity in chicks.The r-25K caused most obvious pathogenicity.Comprehensive analysis shows that when the number of lysine is 2 or 3,the NDV virulence,cell membrane fusion ability and pathogenicity to chicks are significantly enhanced.The r-3K,r-25K and r-235K viruses could increase the permeability of chicken brains,of which r-25K has the strongest ability.This study provided a more theoretical basis for further understanding the virulence and pathogenesis of the VFcs and HR2 in NDV.