Peliminary Study On Two Viruses In Reoviridae

Reoviridae is the largest dsRNA-virus family which has the most broad host and diverse range.The genomes of these viruses comprise 10,11,or 12 dsRNA segments, each encoding one to three proteins(usually one).Reoviridae is composed of 12 genusof viruses.Of Reoviridae,rotavirus is known as the main cause of severe,dehydrating diarrhea in infants children and animals.Rotavirus is assigned to seven different groups A to G, group A rotavirus have 15 different G serotype,the predominant serotype circulated in human is G1,G2,G3,G4,now G9 rotavirus is becoming the fifth major serotype of group A in human.Inprovement of hydgiene condition cannot decrease morbidity of rotavirus gastroenteristis,development and universal use of vaccine is unique and the efficence strategy for prevention of RV disease.Other members in Reoviridae can infect many other livingthings and cause different diseases,aquareovirus can cause diseases in many kinds Of fishes and other aquatic organisims.Scylla Serrata reovirus can infect several kinds of crabs and cause sleeping disease and death,uptodate there is no effective preventional and treatmental method.In this paper rotavirus and Scylla Serrata reovirus were studied,the details were as follow:Chapter One:A brief review of rotavirus was described.Disease burden,Genome Structure and Organizationvirion,structural protein and non-structureal protein,reverse genetics and vaccine of rotavirus are reviewed,especially rotavirus reverse genetics for producing attenuated reassorted was reviewed,and our strategy for reverse genetics of rotavirus is described.Chapter Two:G9 group A rotavirus strain CC-297 was isolated and characterized. The supernatant of G9 group A rotavirus positve fecal specimen detected by PAGE and genotyped by vp7 sequence analysis was inoculated to MA104 cells,the cells exhibited typical cytopathic effect,the nucleic acid extracted from culture medium of inoculated cells showed identical electrophortic pattern with the specimen,and vp7 sequence analysis showed 100%identity with the specimen.The cultured G9 group A rotavirus strain CC-297 was purified by sucrose gradient centrifugation,the purified virus was negatively stained and observed by electronic microscope,the proteins of purified virus was analysis by SDS-PAGE.Chapter Three:Infectious rotavirus was rescued from non-infectious double layer particles of rotavirus.The double layer particles of rotavirus was prepared from rotavirus LLR infected MA104 cells by EDTA treatment and sucrose cusions centrifugation,and was transfected to CV-1 cells,the transfected cells exhibited typical cytopathic effect,the supernatant of transfected cells was rotavirus positive detected by Group A Rotavirus Colloidal Gold immunochromagraphy Diagnostic Kit,MA104 cells infected with transfected cell supernantant also exhibited typical cytopathic effect and was rotavirus positive.Infectious rotavirus was recovered from the noninfectious double layer rotavirus particles transfecting CV-1 cells.Chapter Four:Infectious rotavirus was rescued from rotavirus ssRNA transcripted from double layer particles in vitro.The double layer particles of rotavirus was prepared from rotavirus LLR infected MA104 cells by EDTA treatment and sucrose cusions centrifugation,and rotavirus ssRNA were transcripted from double layer particles in vitro, rotavirus ssRNA were transfected to CV-1 cells,the transfected cells exhibited typical cytopathic effect,the supernatant of transfected cells was rotavirus positive detected by Group A Rotavirus Colloidal Gold immunochromagraphy Diagnostic Kit,MA104 cells infected with transfected cell supernantant also exhibited typical cytopathic effect and was rotavirus positive.Infectious rotavirus was recovered from ssRNA transcript.Chapter Five:PAGE and one step RT-PCR for detecting Scylla Serrata reovirus were described.The nucleinic acid of Scylla Serrata reovirus was extracted from hepatopancreas of mud crab Scylla Serrata with sleeping disease and was analyzed by PAGE,it shows the typical reovirus characteristics.The primer pair was designed based Scylla Serrata reovirus RNA polymerase gene fragment sequence published in GenBank, The nucleinic acid of Scylla Serrata reovirus was amplified by one step RT-PCR using the primer pair,an 119bp amplicon was obtained as expected,the amplicon sequence was analyzed and show 100%identity to Scylla Serrata reovirus RNA polymerase gene in GenBank.Sensitivity of PCR was analyzed and is up to 10 copies per reaction.Chapter six:The Procambarus clarkii model for Scylla Serrata reovirus was described.The Scylla Serrata reovirus was extracted from hepatopancreas of mud crab Scylla Serrata with sleeping disease and was intramuscularly inoculated into the third pygidium of healthy Procambarus clarkii purchased from market in wuhan,The Procambarus clarkii were dissected 20 days after inoculation,hepatopancreas,gill, intestine,heart,pygidial muscle and pereiopod muscle of Procambarus clarkii were collected,and were detected for Scylla Serrata reovirus by PAGE,also these tissues were sectioned and stained with haematoxylin and rosin(HE) for histopathological analysis, and were ultrathin sectioned and stained with uranyl acetate for electron microscopy.All tissues are PAGE Scylla Serrata reovirus positive and showed identical electrophoretic pattern with Scylla Serrata reovirus in mud crab Scylla Serrata with sleeping disease. Photography of HE stained tissue sections illustrated cytoplasma inclusions were formed in all these tissues.Electron microscopy of ultrathin section showed a crystalline arrangement of typical reoviruses in cytoplasma inclusions.Procambarus clarkii can be used as an animal model for Scylla Serrata reovirus.