Identification Of The Epitope On The S Protein Of IBV And Its Interaction With Host Cells

Infectious bronchitis(IB)is an acute,highly contagious disease caused by the infectious bronchitis virus(IBV),which mainly causes respiratory and kidney diseases in chickens.IBV is one of the important pathogens harmful to the poultry industry,which has caused huge economic losses to the global poultry industry.Vaccination plays a vital role in controlling diseases caused by chicken infectious bronchitis virus.The continuously variant antigenicity of IBV limits the application of current vaccine development strategies and serological diagnostic.S protein is a key surface protein with abundant neutralizing epitopes,which is also the key protein for virus to enter host cells.However,little is known about the function of S protein and its interactions with host cells.Therefore,the objective of this study was to provide theoretical support for the development of broad-spectrum vaccines and serological detection methods.The identification of broad spectrum neutralization epitope of S protein and the interaction between S protein and host cells were studied.The main research is as follows:1.Identification of epitope the key novel on infectious bronchitis virus S proteinIn this study,the biological software was used to analyze the antigenicity of S protein of IBV.According to the antigenicity,hydrophilicity,surface antigen index,conservation and other indexes of S protein sequence,five highly conserved peptides with high antigenicity and hydrophilicity were selected and synthesized artificially.Through the cross reaction between peptides and sera to different genotype of IBV,the results showed that all the five peptides have reactivity with the sera.The Pep1 has the best reactivity among the peptides,which could cross-react well with 4 sera of serotypes of the 5 sera of genotypes detected,but Pep1 did not cross-react with the serum of CK/CH/2010/JT1 strain(New Cluster genotype).Then,this peptide sequence of CK/CH/2010/JT1 strain was synthesized and named Pep6.Peptide sequence analysis revealed several amino acid differences between Pep1 and Pep6.The results of peptide and serum cross reaction showed that Pep6 could not only react with CK/CH/2010/JT1 serum,but also cross-react with all sera tested.In order to determine the key amino acids affecting the antigenicity change,we truncated the polypeptide and mutated a single amino acid.We found the key amino acids that determination of the epitope broad-spectrum is the site 16R amino acids.With analysis of all available database sequence in the NCBI,we found that the traditional Mass type strains are 16 K strains,and the QX,TW-1 type strains currently popular in China are 16 R mutant strains.Further analysis found that 16 R mutant strains have begun to appear since the 1990s,then become the dominant epidemic dominant strains.At present,it is distributed in all continents.In this section,a broad spectrum epitope was identified and 16R amino acid was shown to be the key to the broad-spectrum epitope.This provides a reference for the development of new broad-spectrum vaccines and the establishment of detection methods.2.Establishment and preliminary application of a pELISA for detecting IBV antibodiesIn the process of the previous epidemiological investigation of IB V,we found that the existing commercial IBV antibody detection kits could not well detect the serum antibodies of the new mutated strain.The results of our previous study indicated that we had screened a broad spectrum epitope with good cross-reaction with the sera of all current strains.In this study,we established a pELISA method for detecting IBV antibody by using this broad spectrum epitope peptide as the coated antigen.The optimal reaction conditions of pELISA were determined as follows:the optimal coating concentration was 0.63?L/mL;the optimal serum dilution was 1:200;the incubation time of serum was 60min;the optimal incubation time of HRP-conjugated antibody was 60 min;the optimum reaction time of substrate was 20 min.And the cut-off value of the pELISA to be determined as 0.185.The results of inter-batch and intra-batch repeatability test showed that the coefficient of variation was less than 10%,which had good repeatability.The sensitivity,specificity,and accuracy of pELISA were 99.14%,94.12%,and 98.80%,respectively,compared with IFA results,which were significantly higher than that of commercial kits.To evaluate the suitability of pELISA in measuring immune response induced by IBV vaccine,sera collected at different time points after inoculation of IBV H52 and 4/91 vaccine were determined by pELISA and commercial ELISA kit.The results showed that pELISA was able to detect IBV positive antibodies as early as 7 days after vaccination,while the commercially available kit was able to detect IBV positive antibodies 14 days after immunization.Further analysis showed that the positive rate of pELISA was significantly higher than that of commercial ELISA kits,suggesting that pELISA has some application value in the detection of IBV antibody and evaluation of IBV vaccine3.Preliminary study on the potential of pELISA substitution neutralization test to determine serum neutralizing antibodiesThe neutralizing antibody in serum of chickens is the key indicator reflecting the immune status of chickens and the key to evaluate the vaccine efficacy.At present,neutralization test is usually used to determine neutralization antibody of serum.However,the neutralization experiment is complicated and time-consuming,which restricts the R&D efficiency of new vaccines.Meanwhile,there is currently no simple and rapid alternative method to detect neutralizing antibodies.For this reason,we explored the possibility of using ELISA instead of neutralizing test to determine neutralizing antibody.Previous studies have shown that Pep6 has a broad-spectrum reactivity to IBV positive serum.Firstly,we prepared Pep6 polyclonal antibody serum.The cross reaction results of viral and serum showed that Pep6 polyclonal antibody serum could neutralize IBV virus with a neutralization titer of 1:8.Secondly,IBV positive sera were detected by pELISA and neutralization assay,and the results showed that there was a good positive correlation between neutralization titer and ELISA titer.Finally,sera collected at different time points after immunization/challenge of M41,H52,CK/CH/2010/JT1,CK/CH/2014/FJ14 strains were further compared and detected.The results showed that the neutralization titer and ELISA titer of serum after immunization/challenge increased gradually by the time after immunization.There was a significant positive correlation between the neutralization titer and ELISA titer in serum of each strain,and the total correlation coefficient reached 0.83.The establishment of this method provides a technical condition for the evaluation of immunological effect of primary IBV vaccine in clinic4.Exploring the interaction between IBV S1 protein and host cell membrane proteinThe IBV S protein plays a key role in the infection and replication process of virus.Normally S1 is responsible for recognizing host receptors and binding,while S2 promotes the fusion of viral envelope and cell membrane.Although IBV is the earliest known coronavirus,the understanding of the interaction between IBV S1 and host cells is still very limited and no protein receptor for IBV has been identified.In addition,the mechanism of IBV cell entry is still unclear.In this study,we constructed the eukaryotic plasmid pAgGS-Sl-IgGFc that correctly expressed the fusion Protein of S1-IgGFc,and purified the fusion Protein of S1-IgGFc using Protein G purification column.Then,membrane proteins of CEK cells were extracted by cell membrane extraction kit.The purified fusion proteins and CEK cell membrane proteins were immunoprecipitated(Co-IP).The results of SDS-PAGE silver staining showed that there were three difference bands compared with the control.Finally,four host proteins,including GRP78,ANXA2,HSPA9 and Vimentin,were identified by mass spectrometry sequencing.It is suggested that these proteins may play a certain role in virus-infected cells,which provides some materials and theoretical basis for further research on the interaction mechanism between IBV and host cell.5.GRP78 protein promotes IBV replication in CEK cellsThe GRP78 protein is well known as a member of the heat shock protein family and has been reported to play a key role in the replication of several viruses.Based on the previous study,the interaction between IBV S1 protein and GRP78 protein was further verified.The inhibition of IBV M41 replication by blocking GRP78 on the membrane surface of CEK with a polyclonal antibody against GRP78 was effective.Furthermore,siRNA interference technology was used to successfully inhibit the expression level of GRP78 gene in CEK cells by transfecting siRNA targeting GRP78 protein.Western-blot and TCID50 analysis results showed that the expression of GRP78 protein in host cells was decreased,and the infection of IBV was significantly inhibited.Finally,we transfected the Eukaryotic expression plasmid GRP78(0.25 ?g,0.5?g,1.0?g,2.0?g)into non-susceptible IBV cells CEF,and then infected the IBV M41 strain.The results showed that the invasion ability of IBV into CEF cells was gradually enhanced with the increase of the concentration of GRP78 transfection,and there was a significant positive correlation.In conclusion,GRP 78 plays an important role in IBV infection of CEK cells,and this protein can help IBV to enter host cells.