Construction Of An Infectious Clone Of Influenza B Virus And Study On Its Immunogenicity

Influenza B virus belongs to the Orthomyxoviridae family. The genome of IBV has a negative-sense,single-stranded v RNA, which contains two lineages of B/Yamagata/16/88-like and B/Victoria/2/87-like strains. Influenza B virus is similar to Influenza A virus in form, but can only infect human hosts. As a member of seasonal influenza, influenza B virus circulated with regional outbreaks and epidemics generally.Epidemiological surveillance showed that the predominant circulating lineage of influenza B virus was distinct annually. This phenomenon posed a challenge for the immunoprophylaxis of flu. With the establishment of a reverse genetics system of influenza virus, we can operate gene reassortment and gene recombination. This provided a new significant direction of using reverse genetics in the development of efficient influenza vaccine rapidly and the research of influenza vaccine.Experimental objective:Our study intends to establish a reverse genetics system of influenza B virus B/Yamagata / 16/88. We evaluate its pathogenicity,immunogenicity and immunoprotection to the rescued influenza B virus. Based on this reverse genetics system, we can study the attenuated vaccine of influenza B virus by genetic engineering nextly.Experimental contents and methods:1.The establishment and identification of a reverse genetics system of influenza B virus B/Yamagata/16/88.(1)The reverse primer designed and genome synthesis. We used the internal eight gene segments’ sequences reported in Genbank.(2)We constructed 8 plasmids used for transfection by molecular biology methods. The eight genes of influenza B virus linked with PBD vector. The identification of PCR and sequencing were positive.(3)The eight plasmids were cotransfected into 293 T cells and incubated in embryonated eggs. As the mother virus of influenza B virus,the rescued virus was named B-S9.(4)We studied the biological characteristics of B-S9 and the cell growth wave on MDCK.2.The pathogenicity experiment of the rescued virus B-S9.(1)The viral titer of B-S9 was detected by 50% embryonated eggs infectious dose(EID50). BALB/c mice were infected with 106 EID50/50μl、105EID50/50μl dose of the B-S9 virus intranasally.(2)In 106EID50 group, the mice were euthanized on 3 dpi after inculation and harvested the lungs, intestines, brains, spleens, kidneys. We harvested the lungs on 6 days.The lungs of mice in 105EID50 was harvested at 3 and 6 dpi. The virus titers of organs were determined by titration in chicken eggs.(3)Survival and body weight were recorded for 14 days.3.The immunogenicity experiment of the rescued virus B-S9.(1)The BALB/c mice were divided into two groups. One were intranasally inculated with 105EID50/50μl B-S9, the other was intramuscularly inoculated 256 hemagglutination unit inactivated B-S9.(2)The mice were immuned 3 weeks. We harvested serum of two groups and detected neutralization antibody and hemagglutination inhibition antibody titers by the B-S9 virus.(3)The HI titers in mice serum were cross-tested by NYMC BX-51 B vaccine candidate strain in 2013-2014.4.Protective efficacy of the rescued virus B-S9.(1)BALB/c mice were intranasally and intramuscularly immuned with the rescued virus B-S9. Then mice were challenged with viruses three weeks after immunization.(2)The mice were intranasally inoculated with 106EID50/50μl dose of B-S9 and 106EID50/50μl dose of NYMC BX-51 B vaccine candidate strain in 2013-2014. We observed mice deaths and weighted weight of mice.(3)We harvested the lungs on 3 dpi and 6 dpi after inoculation.The experiment results:1.We rescued the B/Yamagata/16/88 strain in vivo, the rescued virus named B-S9. The gene segments were consistent with that reported in Genbank by PCR. The sequences of B-S9 were correct through comparison. Influenza viral particle were observed by electronic microscope. The result of virus infectivity in MDCK was 107.5 TCID50/ml and infectivity in eggs was 108.75 EID50/ml. Also we measured the growth wave of B-S9 on MDCK.2.The mice infected with 106EID50 and 105EID50 of B-S9 lost weight rapidly. The weight of mock mice were normal. 100% mice inoculated with different dose survived. We determined B-S9 MLD50 was over 106.5EID50. The B-S9 virus replicated to high titers in lungs and turbinates and couldn’t replicate in other organ systems. We detected higher titers in 3 dpi than 6 dpi.3.The sera from immunized mice was collected and the serum antibody responses were measured by NT and HI assays. Intranasally immunization had a higher leval than muscle immunization. We also determined HI titers in the serum with NYMC BX-51 B vaccine strain in 2013-2014. The result was a lower titer.4. When immunized mice was challenged by B-S9, the mock mice lost weight rapidly, the intranasal mice had a normal weight and muscle group lost a little weight. The virus titers in the lungs were largerly in mock mice, but none in instranasally. When immunized mice was challenged by NYMC BX-51 B vaccine strain in 2013-2014. The uninfected mice lost weight kindly and other groups had a normal weight. The virus replicated to high titers on 3 dpi than of 6 dpi in muscle immunization. These results suggest B-S9 could provide protective efficacy.The experiment conclusions:1.We established a reverse genetics system of influenza B virus B/Yamagata/16/88 and identified primarily.2.We evaluated the mice pathogenicity of the rescued influenza B virus B-S9. We established a BALB/c infection model for influenza B virus.3.After 3 weeks, the sera in the immunized mice could test NT and HI titers. The mice intranasally inoculated had a higher titer than intramuscularly inoculated.4.The protective efficacy were better for the mice in intranasal immunization group.The rescued virus B-S9 could provide protection against the challenge of mother virus B-S9 and NYMC BX-51 B vaccine strain in 2013-2014.The innovation of this study:1.We establish a reverse genetics system of influenza B virus B/Yamagata/16/88 and lay a foundation for further research.2.We establish a BALB/c mice infection model for influenza B virus B/Yamagata/16/88 and evaluate the pathogenicity, immunogenicity and immune protective efficacy.