The Construction Of Rotavirus Group A Reassortment Strain By Reverse Genetics

Rotavirus,one genera belongs to the family Reovoridae,known as the main cause of severe,dehydrating diarrhea in infants and young children.Rotavirus is estimated to cause about 125 million cases of diarrhea annually,and the rotavirus-related diarrhea cause 900 thousands deaths in infants under 5 years old.Since there have been no specially good effect remedy,the development and universal use of a safe and effective vaccine is considered as the first strategy for prevention.This study includes:Isolation of rotavirus group A strain CC-1 detected in Wuhan with G9 genotype and.The research contents are as follows:Chapter One:A brief review of Rotavirus of the fundamental researches on viron structure,physicochemical properties,genenome and its coding proteins,culture methods, epidemiology and vaccine development,which focuses on the epidemiologic characteristics of G9 RV and the reverse genetics research on rotavirus.Chapter Two:Isolation of GARV with G9 serotype detected in Wuhan;sequence and phlogenetic analysis of vp7 gene.A human group A rotavirus strain CC-1 with G9 serotype specificity was isolated in MA104 cells.The complete vp7 gene was RT-PCR amplified and then cloned to pGEMT-easy vector to construct plasmid pGEMvp7-G9. The nucleotide sequence of vp7 gene was determined in order to determine the phylogenetic characteristics.Chapter Three:Construction and determination of the full length vp7 cDNA transcript plasmid,cDNA encoding the full-length vp7 gene was synthesized by RT-PCR from a Rotaviurs group A strain with G1 genotype.The vp7 gene flanked by T7-RNA polymerase promoter at the 5'-terminal and HDV ribozyme followed by T7-RNA polymerase terminator at the 3'-terminal was generated by OVERLAP-PCR,and then was cloned to pGEMT-easy vector to construct the plasmid pGEMvp7-Rz.The pGEMvp7-Rz was transfected to the Vero cells that was infected the recombinant vaccina virus strain vTF7-3 previously in oder to examine if pGEMvp7-Rz produces vp7 +sRNA with correct 5'and 3' ends in the mammalian cells.Chapter Four:Construction of a reassortment strain of GARV in a reverse genetic method.Recombinant vaccina virus strain vTF7-3 was treated by psoleren-UVA method to disable its replication but retain the expression of T7-RNA polymerase.The transcript plasmid of full length vp7 with G1 genotype was transfected to the Vero cells that was infected the treated vTF7-3 previously to obtain the vp7 +sRNA with correct 5'and 3' ends.Then the infected-transfected Vero cells were infected by a rotavirus strain with G10 genotype to generate a nascent rotavirus strain with a reassortment vp7 gene.The reassortment rotavirus was selected by monocloned antibodies against G1 specificity.