The Biological Mechanism Of Coronavirus Nsp1Protein And Antibodies Development Of Coronavirus HR2Protein

Virus is a kind of tiny microorganism with simple no-cell structure and nucleotides (DNA or RNA). It has to parasitize in living cells and reproduces in the way of replication. Virus can be classified by shape and form as, spherical virus, baculovirus, Brick shaped virus, coronavirus, filamentous virus, spherical virus with envelop and virus with globular domain.Coronavirus and Influenza virus A have pathogenicity to human and animals. Most of the recent research about virus was focused on intracellular gene therapy, especially on Congenital host innate immunity However the pathogenicity mechanisms of many viruses are currently unknown. So the development of vaccines and gene therapy faced many difficulties. In this thesis, to clarify the feasibility of new development, we processed some research on pathogenicity of coronavirus Nspl protein, polyclonal antibody of HR2domain and monoclonal antibodies of influenza A virus M2e protein.Coronaviruses are divided into three groups based on serological criteria. Some members of the group II coronaviruses encode a number of proteins that antagonize host innate immunity. Nspl proteins are a major virulence and pathogenicity factor. The Nspl proteins of SARS-CoV promote host mRNA degradation and suppress host gene expressionTo under the pathogenicity of coronavirus Nspl protein, we compared the model of Nspl protein from group I coronavirus HCoV-229E, HCoV-NL63and group Ⅱ coronavirus SARS-CoV. We found they are common in folding ways and have similar structure and function. We made analysis the interaction between Nspl protein and cellar protein by immune-blotting and Co-IP. The result indicated Nspl protein from group I coronavirus HCoV-229E and HCoV-NL63could bind with ribosomal40S subunit to suppress host cell protein synthesis. Then we found IRF-3phosphorylation was not inhibited by Nsp1proteins after the analysis about the innate immune signal transduction which was stimulated by NDV infection in cells transfected with plasmids-expressing Nspl from HCoV-229E, HCoV-NL63, SARS-CoV control plasmid. The real time PCR results indicated that Nspl proteins could inhibit IFN-β and Luciferase mRNA transcription. And we found there was greater suppression of IFN-β and luciferase proteins synthesis by Nspl proteins in host cells infected by low pathogenic coronavirus than by SARS-CoV. All these results indicate that different coronaviruses might employ the same Nspl mechanism to antagonize host innate immunity and cell proliferation. However, Nspl may not be the key determinant of viral pathogenicity, or the factor used by the SARS coronavirus to evade host innate immunity.To develop new coronavirus vaccines, we analyzed the immunogenicity of HCoV-229E and HCoV-NL63using real-time PCR and western blotting. We found th at HCoV-229E has high immunogenicity and it could be made to useful coronavirus v accines.Antiviral strategies against HCoVs have been remarkably updated in recent years, especially with the emergence of the SARS-CoV. The HR region in coronavirus Spike glycoprotein is a promising therapeutic target.To investigate the Immunogenicity and the Neutralizing activity of HR region, we find out the amino acid sequences of five known coronavirus spike glycoprotein. Multiple alignments of the five HCoV HR2regions from different serogroups indicate that this region is relatively conserved. We purified the Spike fragments covering the entire HR2domain and the immediate upstream region for the five known HCoVs. After six times injection in mice, we get the five polyclonal antibodies. By the cross neutralization profile of the five anti-HR2polyclonal antibodies, we found the antibodies neutralize their antigenic virus or pseudo particles except the HCoV-HKU antibodies. To evaluate the specificity of the five HCoVs anti-HR2polyclonal antibodies, we performed Western blotting and found four of them were cross-reactive with other S proteins of the same or different serogroups. Moreover, we compared the amino acid sequence alignment in the HR2and upstream region from TGEV HR2and found it highly conserved. The neutralization assay showed the anti-TGEV-HR2antibodies have neutralization abilities with HCoV-NL63and HCoV-229E. Our data indicated the HR2and upstream regions in coronavirus spike (S) glycoprotein is a promising therapeutic target.Our results make deeper understanding on mechanisms of virus pathogenicity and bring new insight in developing vaccine and therapeutic agents against virus infections.