Establishment Of Animal Model Of Coxsackie B Virus Type

Objective:To provide data reference for the virus detection,pathogenic,vaccine development and drug evaluation of coxsackie virus group B type 3,the Syrian hamster and rhesus monkey infected animal models,and quick detection method of coxsackie B3 virus were established.Method:This study was mainly classified three parts.Part one:establish of CVB3 virus detection system.(1)The obtained VP1 gene of CVB3 virus by using Coxsackie B virus universal primers and PCR amplification.(2)The recombinant plasmid of VP1 gene established by transcribed in vitro to obtain RNA standard,and further established method of fluorescence quantitative detection.Part two:The established Syrian hamster model.(1)Syrian hamster was administrated with CVB3 virus and the dose of 106.75CCID50/ml by two methods of intraperitoneal injection and nasal drip.(2)The observed and record clinical state of infected hamsters,detection and analysis physiology and biochemistry,detoxification cycle or histopathological damage.Part three:The established rhesus monkey model(1)The screen of experimental group by using virus detection method of established and combine with IgG,IgM detection method.(2)rhesus monkey aged 3-4 months was administrated with CVB3 virus and the dose of 106.75 CCID50/ml 200 μl by using the method of nasal drip.(3)The observed and record clinical state of infected rhesus monkey,detection and analysis rhesus monkey of physiology and biochemistry,detoxification cycle,histopathological damage,immune factors and antibodies.Result:Part one:the established virus detection method has highly specificity,sensitivity and repeatability,and could be used for detection of CVB3.Part two(1)there were no abnormal clinical state in the control group;The two experimental groups of Syrian hamster were showed different degrees of depression and decreased appetite from the second day of infection to the 8th day;From 3rd day,the two experimental groups of Syrian hamster were appeared white herpes in lips,cheeks,etc at different time,and the herpes subsides within 2 to 4 days..(2)The viral load was detected in the nasopharyngeal swabs and stool samples of experimental group of hamsters from 1 to 14 days.(3)The biochemical results showed that serum myocardial enzymes and liver enzymes were increased on the 7th day after infection in the serum.(4)Tissue viral load detection:the viral nucleic acid was detected in the heart,liver,spleen,lungs and other major internal organs as well as nerve tissue,digestive tract tissue and herpes organization of the two experimental groups of hamsters.(5)The pathology and immunohistochemistry result shows that the pathological changes and viral antigen expression of different degrees were observed in heart,liver,spleen and herpes.Part three(1)The three infection monkeys were developed herpes symptoms on hands,feet and lips at different points during the 3 to 12 days.(2)Within 30 days after infection,there was no significant increase body temperature and no significant change body weight.(3)The blood routine results shows that the changes of white blood cells,red blood cells,hemoglobin,lymphocytes and neutrophils suggesting hematologic features of viral infection;The biochemical results shows that the LDH,CK,CKMB,AST,and UREA were increased after infection.(4)The viral load was detected in the nasopharyngeal swabs and stool samples of experimental group of monkeys from 1 to 21 days;The blood tests for the virus shows that the viral load was detected 1-21 days after infection and typical viremia was developed at 3 and 7 days.(5)The pathology and immunohistochemistry result shows that the pathological changes and viral antigen expression of different degrees were observed in heart,liver,spleen and herpes.(6)The expression levels of IL-2,TNF-α,IL-4,IL-6 and IFN-y was changed at different time points after infection.(7)Neutralizing antibody detection was up to 1:32 at 30 days after infection.Conclusion(1)In this study,the established of rapid detection method for CVB3 can rapidly screen animals and be used for laboratory sample detection,thus improving detection efficiency;(2)The animal models of Syrian hamsters and rhesus monkey of CVB3 virus infection were successfully constructed by intraperitoneal injection and nasal drip.Which was more consistent with and close to the natural infection route of the virus.(3)The rhesus monkeys of CVB3 infected presented a typical and similar HFMD animal model,and the pathophysiological results of the animal model suggested the pathological changes of viral myocarditis and liver and brain injury.