Investigation The Human Metapneumovirus Infection In Chongqing Children And Eukaryotic Expression Of Fusion Protein By Using A Baculovirus System

PART I: Seroprevalence of human metapneumovirus infection in chongqing childrenObjection: The human metapneumovirus was firstly identified in 2001. At present, no reports have documented the seroprevalence of hMPV in chongqing, China. Our purpose is to define the seropositivity of hMPV IgG antibodies in different age group children in Chongqing.Method: The specificity of enzyme-linked immunosorbent assay (ELISA) was firstly validated by using RSV infected cell lysates substracted sero and western blotting based on anti-hMPV animal serum. This assay, was subsequently used to determine the presence of IgG antibody to hMPV and RSV in 325 serum samples from children aged 0-6 years.Results: There was no cross-reaction between hMPV and RSV ELISA observed. Seropositivity of anti-hMPV in all children was 78.8%. Seropositivity of anti-hMPV IgG antibody in children aged 0 to 5 months was 74.5%. And that was 64% for children aged 6 to 11 months, 72.7% for children aged 12 to 23 months, 90.3% for children aged 24 to 35 months and 93.1% for children 3 to 6 years old, respectively. The seropositivity rates of hMPV and RSV were considerably similar in almost all age groups.Conclusion: HMPV appears to be a common and important respiratory pathogen in Chongqing's children. Almost all individuals have exposed to hMPV by the age of 6.PART II: Analysis of neutralizing activity to human metapneumovirus in sera from children aged 0-6 yearsObjectives: Neutralizing antibody, a important part in humoral immunity, can prevent virus to attach the cells and have important role in preventation and recovery of respiratory disease. Human metapneumovirus has been identified as one of the most important viral pathogen for acute respiratory traction infections in children. This study was to observe neutralizing activity to hMPV in sera collected from children without respiratory illnesses aged 0 to 6 years and to investigate the correlation between the level of hMPV IgG antibody and neutralizing activity in a same serum.Methods: Sera of 0 to 35 months old children were collected from patients hospitalized for surgery without documented respiratory illnesses. And sera of 3 to 6 years old children were remainder of sera for physical examination. Total serum specimens were 325。Ten serum samples were selected for neutralizing activity detection in every age group and sum was 50. The neutralizing titer was assessed by microneutralization assay which had been well documented previously.Results: Among 50 serum samples, eight in which hMPV IgG antibody was proved negative by ELISA previously were mirrored by no neutralizing activity detected. Other 42 samples, as expected, showed variable neutralizing activity. The geometric mean neutralizing titer to hMPV for all the samples was 1:129. In group of 0-5 months, the geometric mean titer was 1:160. In groups of 6-11months, 12-23 months, 24-35 months, 3-6 years, the geometric mean titers were 1:58.9, 1:114.9, 1:172.8 and 1:160, respectively. The level of hMPV antibody and neutralizing activity in a same serum was significantly correlated (r=0.668).Conclusion: Most of 0-6 years old Chinese children have neutralizing antibody in their serum. Whether neutralizing activity is high enough to prevent infection of different subtype hMPVs is unknown. PART III: Eukaryotic expression of human metapneumovirus fusion protein in insect cells by using a baculovirus system Objective: Fusion protein of human metapneumovirus is the immumodominant protein based on the corresponding protein (F) of related paramyxovirus RSV. So this study was to construct the eukaryotic recombinant plasmid containing fusion protein gene of human metapneumovirus and express in insect cells.Methods: The full-length fusion protein gene of human metapneumovirus was amplified by reverse transcriptional polymerase chain reaction (RT-PCR). After being inserted at the multiple clone site of donor plasmid-pFastBacHT, target gene was delivered to bacmid to yield a recombinant bacmid. This recombinant bacmid was subsequently transfected sf-9 cells to obtain the infectious recombinant baculovirus and then, the recombinant baculovirus was used to inoculate fresh sf-9 cells to express fusion protein. Expressed protein was purified by using a 6×His tag rasin and identified by western blotting.Result: Nucleotide sequence analysis of PCR products amplified from rBacmid-hMPVF showed 100% identity to public sequence of CAN 97-83 F gene sequence. Sf-9 cells infected by recombinant baculovirus displayed typical cytopathic effects. By using 6×His tag monoclonal antibody and western blotting, a single band with molecular weight around 70KDa was identified.Conclusion: The fusion protein of hMPV was successfully expressed by using baculovirus expression system in insect cells, which will benefit the study on sero-epidemiology and pathogenesis of hMPV infection in the future.