Mechanism Of Vimentin Protein Regulating The Infection Of Genotype Ⅲ Newcastle Disease Virus With Different Virulence

Newcastle disease(ND),caused by virulent strains of Newcastle disease virus(NDV),is an important infectious disease of poultry and wild birds.It brings heavy economic losses to the global poultry industry.Vaccination has been proven to be the most effective way to control the epidemic of ND till now,and ND vaccine is widely administered in poultry in several countries around the world including China.Mukteswar,the classical I ND vaccine,was once widely used as an emergency immunization vaccine.As an RNA virus,genetic mutations increasing virulence have been reported from time to time due to the lack of proofreading activity and the high immune pressure.A virulent NDV strain JS/7/05/Ch was previously isolated from infected chickens in our laboratory.Whole genome sequencing showed that JS/7/05/Ch was highly homologous to the genotype Ⅲ mesogenic vaccine strain Mukteswar only with one amino acid(aa)difference in NP and six aa differences in HN.Compared with Mukteswar,JS/7/05/Ch showed more pathogenic following intravenous inoculation,but showed no significant difference after infection through the natural route(oculonasal inoculation).The aa differences in the HN protein was identified as the critical factor leading to enhanced virulence of Mukteswar.However,its internal mechanism is still unclear.Therefore,this study systematically analyzed the effect of the mutant HN protein on the biological activity of genotype Ⅲ NDV.Meanwhile,this study screened and identified the host proteins that differentially interacted with different HN proteins,which further elucidated the interaction between virus infection and host proteins.Moreover,this study evaluated the difference of infection characteristics of genotype Ⅲ ND Vs with different virulence through intravenous inoculation.The aim of this study was to elucidate the infection mechanism of genotype Ⅲ NDVs with different virulence.1.Biological characteristics of genotype Ⅲ NDVs with different virulenceMukteswar-and JS/7/05/Ch-type HN protein showed 6 aa differences.Combined with HN gene sequences of genotype Ⅲ NDVs published on NCBI,we found that N19 and A29 from Mukteswar-type HN and T145,1266 and D494 from JS/7/05/Ch-type HN were the dominant aa.Besides,both of E495 from Mukteswar-type HN and K495 from JS/7/05/Chtype HN accounted for half of the total,respectively.These aa variations affects the threedimensional structure of HN protein.The protein modification results showed that the mutant HN could affect NDV-induced protein modification,predicting that the posttranslational function of HN protein may be different.Subsequently,the biological activity assays showed that the virulent strain JS/7/05/Ch mediated the stronger receptor-binding activity,membrane fusion activity,hemolysis activity,and F protein lysis activity,but its neuraminidase activity was weaker.Moreover,the virulent strain JS/7/05/Ch showed an obvious replication advantage both in HD11 and PBMC cells,whereas it only replicated efficiently at the early stage of infection and the replication level was significantly reduced at the late stage of infection in the CEF cells,which could be associated with the high-level cell death.Additionally,the virulent strain JS/7/05/Ch induced plaque formation with larger diameter,indicating the stronger infectivity of JS/7/05/Ch.In summary,this chapter revealed the effects of the mutant HN on biological functions of genotype Ⅲ NDV5 which could help explain the differences in the infection mechanism of genotype Ⅲ NDV from the virus perspective.2.The interaction mechanism between vimentin and HN proteins of genotype Ⅲ NDVs with different virulenceVimentin,a skeleton protein that interacted differently with genotype Ⅲ NDV HN proteins,was identified through mass spectrometry analysis.Mukteswar-type HN protein could interact with vimentin,but JS/7/05/Ch-type HN protein could not,indicating that the aa variation significantly weakened the interaction between JS/7/05/Ch-type HN and vimentin.In CEF cells,the virulent strain JS/7/05/Ch obviously downregulated the level of vimentin,while two mesogenic strains Mukteswar and JS/MukHN(HN-replacement recombinant strain)showed no significant effect on the levels of vimentin.Simultaneously,vimentin in CEF cells positively regulated the viral adsorption and replication of three NDV strains.JS/7/05/Ch infection induced vimentin rearrangement earlier in CEF cells,but all three NDV strains could induce vimentin rearrangement around the nucleus without significant difference.In addition,the rearrangement region of vimentin in CEF cells did not completely coincide with the viral replication,indicating that the rearranged vimentin in CEF cells was not the viral replication center of genotype Ⅲ NDV.In HD 11 cells,JS/7/05/Ch downregulated the levels of vimentin more significantly compared with that in CEF cells,while Mukteswar and JS/MukHN only took a weak effect on the expression of vimentin.Vimentin in HD11 cells significantly affected the adsorption and replication of JS/7/05/Ch but exhibited no obvious effect on Mukteswar and JS/MukHN infection.JS/7/05/Ch infection rapidly downregulated the levels of vimentin and formed obvious "cage" structure in HD 11 cells,and the rearranged vimentin became the center of viral replication.By contrast,Mukteswar and JS/MukHN could not induce vimentin rearrangement.Besides,vimentin rearrangement and virus replication were mutually dependent both in CEF and HD 11 cells.That is,disruption of the vimentin rearrangement would impair virus replication,whereas inactivation of the virus would hinder vimentin rearrangement.Furthermore,we deterrmined vimentin-related biological functions.Briefly,the virulent strain JS/7/05/Ch induced the stronger cell damage,apoptosis and autophagy,and the.effects of apoptosis and autophagy on JS/7/05/Ch infection was more significant.Simultaneously,vimentin knock-down enhanced NDV-induced apoptosis and autophagy.Notably,JS/7/05/Ch enhanced the cell death,apoptosis and autophagy by weakening the activation of NF-κB.To conclude,this chapter identified a host protein vimentin that differentially interacted with genotype Ⅲ NDV HN proteins and analyzed the differential regulation between vimentin and NDV infection.This study provides important clues for further understanding the pathogenic mechanism of genotype Ⅲ NDVs with different virulence.3.In vivo infection characteristics of genotype Ⅲ NDVs with different virulenceTo elucidate the underlying mechanism of the mutant HN protein affecting the pathogenicity of genotype III NDV in chickens,SPF chickens were infected with genotype III NDVs carrying differential HN proteins by intravenous route.Thereby,we analyzed the differences in infection characteristics of genotype III NDV in vivo.Compared with Mukteswar and JS/MukHN,the virulent strain JS/7/05/Ch caused more severe clinical symptoms and mortality,induced earlier and higher rates of virus shedding.Notably,JS/7/05/Ch efficiently replicated in the peripheral blood and then spread to multiple organs throughout the body,subsequently causing serious infection.Moreover,JS/7/05/Ch exhibited stronger tissue tropism,which mainly caused inflammatory cell infiltration in the lung and spleen and T lymphocyte depletion in the thymus.Simultaneously,JS/7/05/Ch showed a stronger affinity for macrophages in vivo organs,which was characterized by obvious colocalization of the virus with macrophages in the lung,spleen,and thymus.To verify the effect of the mutant HN protein on the regulation of vimentin in vivo by NDV,we detected the vimentin level and its related functional proteins in different tissues of infected chickens.The results showed that JS/7/05/Ch significantly downregulated the vimentin level in the bursa,spleen,thymus and glandular stomach,and enhanced the activation of autophagic proteins in the bursa,thymus and glandular stomach,as well as apoptotic proteins in the spleen,thymus and glandular stomach.These results were consistent with in vitro results.Furthermore,transcriptome sequencing revealed significant differences in the regulation of host genes by genotype III NDVs with distinct virulence.The differentially expressed genes were mainly enriched in blood and cytoskeleton functions,which further supported the differences in virulence of NDV after intravenous inoculation and the differential interaction with cytoskeletal proteins.In conclusion,this chapter comprehensively investigated the differences in pathogenicity of genotype III NDV in chickens and revealed that the mutant HN protein affected in vivo infection of NDV in multiple ways.This study further elucidates the mechanism of virulence and pathogenicity differences of genotype III NDV.