The Preliminary Study Of Animal Model Of Infected The Tree Shrew In Rotavirus WA

Rotavirus?RV?infection is the most important cause of non bacterial diarrhea in infants and other young mammals.About 60%of the children who have been hospitalized for acute diarrhoea in the world are caused by RV infection,and the number of deaths caused by rotavirus is as high as 800 thousand each year.The infection rate and hospitalization rate of children in China are 28%-65%and 30-50%,causing considerable social burden and economic loss.At present,there are no effective drugs for the treatment of rotavirus.The existing vaccines in the market have a large gap in effect in different regions,and the price is expensive and there is a hidden danger of potential infection.Therefore,the research and development of a safe,reliable,efficient and cheap vaccine has become the most important project at present.Animal models can not only study the infection process and pathogenesis of virus,but also provide a basic guarantee for vaccine development and drug screening.At present,the animal models of rotavirus infection mainly include lactobacillus,aseptic pig,monkey and rat and so on.Because of the limitations of these models in relationship,experimental period and anatomical characteristics,there is no animal model verification and clear elucidation of virus pathogenesis.In recent years,tree shrews have been paid much attention due to their small size,rapid breeding,low breeding cost and similar anatomical characteristics to people.And it has been preliminarily proved to be able to infect rotavirus,so it has become a comparative advantage in the study of rotavirus.The WA strain of G1P[8]type and the JM-103 of G9P[8]isolated from the feces of clinical patients were selected as the research object.The virus was inoculated into MA-104 cells and Vero cells,and the WA strain was selected as the dominant virus by morphological observation and detection of virus titer.The MA-104 cells were identified as the host cells.Then the virus was concentrated and the TCID50 technical conditions for measuring the virus titer were optimized.At the same time,the PCR technology of rotavirus gene detection was established to prepare for the follow-up experiment.Then the primary cells of tree shrews were obtained,and the primary and small intestinal epithelial cells of tree shrews were cultured and purified,and the cells in the growing period were reinfected by the virus.The proliferation characteristics of the virus infected cells in vitro were identified by measuring the titer of the virus in the suspension.At last,the tree shrews were infected with high titer rotavirus WA by gavage.The body weight,body temperature,stool grade and activity of tree shrews were recorded.The samples of blood,feces and saliva of tree shrews in different time periods were collected,and the body tissues of tree shrews with severe diarrhea were killed,and the expression of rotavirus was detected by PCR and WB.The results of the full text of the above method are as follows:1.The highly virulent and highly infectious WA strain and the more obvious CPE MA-104 cells after infection were used for subsequent experiments.At the same time,we optimized the method of PEG-6000 concentrated virus and obtained highly virulent and high titer rotavirus WA strain as the material for subsequent infection experiments.The virus titer was measured by TCID50.By comparing the virus titers of different conditions,the optimal conditions for the activation of the virus were the final trypsin concentration 20ug/ml and incubation time 60min.Meanwhile,the detection and validation system of rotavirus RT-PCR was established according to the proliferation and cytopathic effects of rotavirus WA strain.2.Using the combined action of 0.25%trypsin-EDTA digestion and mechanical oscillation,the primary renal cells of tree shrews were successfully isolated,and small intestinal epithelial cells were successfully obtained by collagenase and neutral protease I.The primary renal cells and small intestinal epithelial cells of the tree shrews adhered quickly and grew well.By using the two characteristics of the difference of trypsin sensitivity between the primary and fibroblasts of tree shrews and the different adherence time,the cells were purified in the process of subculture,and the primary cells of tree shrews without impurity cells were obtained.At the same time,the cell proliferation curve of primary cell of tree shrew was determined by CCK,and the growth curve of cells was detected.Rotavirus infected the primary cells of tree shrew,and observed the cytopathic effect at different times.The primary cells infected with 72h appeared abscission and roundness.After the rotavirus infected the primary cell of tree shrew,it collected the cell suspension to detect the titer of the virus in the cell suspension,and proved that the rotavirus WA strain could infect the primary cells of the tree shrew.3.The best way to infect tree shrews with rotavirus is to infuse the tree shrews.The tree shrews of different ages choose different doses and virulence viruses.We choose each tree shrew for the 2m L virus titer of 1 and 10^5.8(TCID₅₀)rotavirus cell suspension.The tree shrews after infection pass through life signs,activity conditions,body temperature,and body temperature,Body weight,diarrhea and fecal grade preliminarily concluded that tree shrews can infect rotavirus WA strain,and each index is supported by each other,showing a similar rule.By detecting rotavirus in the feces,saliva and blood of infected tree shrews,it is proved that the rotavirus is replicating and proliferating in tree shrews.The excretion of the virus.We have preliminarily established the animal model of rotavirus infection of tree shrews.The preliminary study of this model has important reference significance for the pathogenesis of rotavirus and the development of vaccine.