Generation Of The Cold-Adapted Reassortant Influenza A Virus And Immunogenicity Research

Influenza is a contagious, acute respiratory disease caused by an influenza virus. Influenza viruses are negative sense, segmented, ribonucleic acid viruses of the family Orthomyxoviridae. Influenza viruses classified into three genera or types (A, B and C) based on serological reactions of the conserved internal proteins, principally the nucleoprotein and matrix protein.Influenza virus can infect kinds of animals such as people, horses, pigs, marine mammals and poultry. Four pandemics in 20 century and appearance of high pathogenicity avian influenza virus H5N1 caused severe social impact and grievous economic loss. It's a challenge for human to prevent the influenza pandemics.The vaccination is still principal method for prophylaxis. The ways of vaccines were developed in recent yeares. The rescued of influenza virus got more and more development in recent yeares based on the cold-adapted master strains by reverse genetics. Base on this technology, LAIV production became easier and more convenient.The research is based on the cold-adapted rescued system and with reverse genetics technology, the reassorment vaccine virus rMDV-H1 which possesses 6 internal genes from the master donor cold-adapted strain, A/Ann Arbor/6/60, and the HA and NA genes from the vaccine strain A/New Caledonia/20/99(H1N1) were rescued.The rMDV-H1 possessed the attenuation phenotypes and the desired antigenic properties of the vaccine strain were verified through some ways. The mice were immunized with the pure rMDV-H1,he neutral antibodies in sera were detected by HIA, seral IgG, IgG1, IgG2a and sIgA in lung wash, nasal wash and vagina wash were detected by ELISA.At same time,the ambisense vectors which contain HA and NA gene cDNAs of A/Wisconsin/67/2005(H3N2) were constructed and this two plasmids'function were verified. 1. Generation of rMDV-H1 which is cold-adapted vaccine strain of A/New Caledonia /20/99 HA and NA genes of A/New Caledonia/20/99(H1N1) were amplified with union primers by RT-PCR and linked with pMD-19T vector and sequenced. Corrected H1HA-T and H1NA-T were cloned between the two BsmBⅠsites of the pAD3000 vector. The constructed vectors were co-transfected with 6 plasmids which contain 6 gene of PR8 into COS-1.The transfected cells were incubated at 35℃for 48 h.The recovered virus was then amplified in embryonated chick eggs directly. After 48 h, the allantoic fluid were collected and detected for rescued virus which the HA result was positive.The pMVD-H1HA and pMVD-H1NA co-transfected with 6 inter genes of donor master strain into COS-1. The transfected cells were incubated at 33℃for 48 h. The supernatant of transfected productions was injected into the allantoic cavity of embryonated chick eggs. These embryonated chick eggs were incubated at 33℃, 48 h. The reassortment virus was screened through HA test.2. The biological characteristics detection of reassortment virus rMDV-H1 The biological characteristics were detected through some ways, including stable test, CPE, indirect immunofluorescence assay (IFA), 50% embryonated chick eggs infectious dose (EID50), 50% tissue infectious dose (TCID50).3. Immunogenicity of reassortment virus rMDV-H1The fouth rMDV-H1 was injected into allantoic cavity of 10 days embryonated chick eggs. The allantoic fluid (HA titre≥28) was collected after incubating 33℃, 48 h. The pure rMDV-H1 was got by ultracentrifugation and the virulence was detected by EID50 and TCID50. 20 Balb/c mice were immunized with pure rMDV-H1 intranasally and subcutaneous injection. The immune doses were 0, 0.05EID50, 0.1 EID50 and 0.05 EID50, respectively.The interval time was 14d between two immunization. The sera were collected 7d, 14d post second immunization, neutral antibodies were detected by HIA test. Specific seral IgG and IgG subclass, IgG1, IgG2a and IgG2b, were detected by ELISA. At the same time, the local sIgA in lung wash, nasal wash and vagina wash were detected by ELISA.