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Construction of Recombinant Human Metapneumovirus Expressing Green Fluorescent Protein and Study of Its Pathogenicity

Author: LiXiaoYan
Tutor: HaSiSuRong；BuZhiGao
School: Inner Mongolia Agricultural University
Course: Basic Veterinary Science
Keywords: Reverse genetics technology Recombinant human metapneumovirus Green Fluorescent Protein
CLC: R373
Type: Master's thesis
Year: 2011
Downloads: 17
Quote: 0
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Abstract


Human metapneumovirus (Human metapneumovirus, HMPV) belongs to the paramyxovirus family metapneumovirus genus the genus metapneumovirus only single- stranded negative non-segmented RNA virus that can infect humans . Available epidemiological data , HMPV was the global epidemic of common pediatric respiratory pathogen . Clinical symptoms and lung virus, respiratory syncytial virus (RSV) infection similar to a severe cough , bronchitis and pneumonia , often accompanied by fever, muscle pain and vomiting , and lead to immune dysfunction, death . HMPV main A, B two types of Canadian isolates CAN97-83 (NL/1/00) and CAN98-75 (NL/1/99) as the representative of each type can be divided into two different sub- type. In this study, using reverse genetics techniques, the exogenous gene expression of enhanced green fluorescent the HMPV NL/1/99 (B -type ) on behalf of genomic insert , successful rescue was reorganization the virus rHMPV NL/1/99-EGFP . By fluorescence confocal , electron microscopy and growth kinetics studies confirmed the final rescue of infectious reorganization rHMPV NL/1/99-EGFP virus particles and rescue by the virus to maintain the characteristics of the wild-type HMPV . Using a modified plaque formation assay viral titer was determined according to the method of Reed-Muench comparative analysis of the results showed that the the recombinant virus rHMPV NL/1/99-EGFP with the wild-type HMPV NL/1/99 curve of growth kinetics and the highest growth drops degrees . the rHMPV NL/1/99-EGFP Vero-E6 cells continuously passaged 10 times remains stable expression of green fluorescent protein and its biological characteristics remain unchanged . On this basis , comparative study rHMPV NL/1/99-EGFP (B type) and rHMPV NL/1/00-EGFP (A -type) mouse and the pathogenicity of the respective cells , using a modified plaque forming test the viral titer was determined , and the results show that , the the infection of rHMPV NL/1/00-EGFP and virulence than rHMPV NL/1/99-EGFP . The research to fill the gaps in the field of the same research , first of all , the stable expression of foreign genes in recombinant viruses HMPV have the possibility of the application as a vaccine vector . Second , EGFP protein as a viral tracer experimental conditions undemanding and easy to detect , makes in rHMPV NL/1/99-EGFP based on the virus pathogenic mechanisms and gene function studies become more convenient . Third, the two recombinant HMPV pathogenic for the transformation of the viral genome , thereby to live attenuated vaccines developed for HMPV foundation .

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus)
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