Isolation And Identification And A Neonatal Mouse Model Of Coxsackievirus A16

Hand-foot-mouth Disease(HFMD) is common in infants, with fever and herpes as the main feature.The hospitalized patients can be self-healed,but encephalitis, aseptic meningitis, pulmonary edema can occur in serious ones, and even death. The disease is prevalent worldwide. It often occurs sporadically in a wide range and break out in locally since it was discovered. The incidence and the mortality had been rising year by year. Our country discovered first HFMD in Shanghai in1998HFMD spread throughout the country quickly. HFMD erupted in Tianjin in1983and1986, and outbreak in Taiwan of China in1998, with78death in129106cases. In2011, There were1496048cases in China Mainland,with467cases death. According to report, there is a pandemic every2to3years.The annual morbidity in China Mainland is more than one million. HFMD was grouped into class C infectious disease by the Ministry of health in May2,2008. HFMD becomes a major public health problem that threatens our people’s health.There are more than20kinds of pathogens causing HFMD. CVA16and EV71are the main pathogens. EV71infection often company with many complications, such as viral meningitis, encephalitis, pulmonary edema, pulmonary hemorrhage, and so on, which may lead to death. Thus it is more concerned for EV71. According to the data of the previous years, in most parts of China, EV71infection rate is greater than CVA16. Therefore, China for HFMD studies mainly focused on EV71, including drug and vaccine research. On the contrary, CVA16caused HFMD symptom milder and less complicated, and we rarely payed attention to CVA16. But in recent years, there have been reports that CVA16may be correlated with fatal myocarditis and refractory shock. The incidence of CVA16increased even more than EV71, which becomed the dominant serotype in some areas of China. From the time and space point of view, the situation is that CVA16and EV71alternately or co-epidemic. Therefore, we enhance the study on isolation and identification of CVA16from the different regions and pathogenesis, which may be of great significance to the prevention and control of HFMD.Objectives To explore the characterization of Coxsackievirus group A type16(CVA16) and establish suckling mice models of epidemic strains, which may attribute to clarify CVA16virus pathogenesis and immunopathogenesis and further accelerate vaccine development, drug screening process.Methods147clinical samples were collected. CVA16isolates confirmed by RT-PCR, we determine titer and analyses the gene sequences. After plaque purification, adaptation of suckling mice, eventually we get a strain, which can make11-day-old C57suckling mice die, named TS10/08(GenBank accession JX068829). Then TS10/08infected11-day-old C57suckling mice to observe and record the changes in body weight, clinical disease score and mortality, and detect the viral load in the brain, skeletal muscle, lung, myocardial muscle by real-time quantitative RT-PCR, determine tissue pathology, inflammatory molecules changes and establish neonatal animal model.Results16strains of CVA16,20strains of EV71and5strains of both were isolated from147clinical samples. The titer of these CVA16is between10-510-9.The results showed that all isolates belong to C1subtypes after we constructed phylogenetic tree based the difference of VP1nucleotide sequences. Then we determined the complete genome sequences, and submitted them to the GenBank accession. The virus can make11-day-old C57suckling mice die by intraperitoneal injection of the virus into different days old C57suckling mice, the viral virulence is50LD50/mL. Viral loads of skeletal muscle were consistently higher than ones of the heart, lung, brain, but viral loads of heart, lung, brain is not very different. The mice have lymphocytic infiltration in skeletal muscle, heart, brain, lung and skeletal muscle is the most serious. Inflammatory molecules of MCP-1, MIP-1alpha, CSF3and MIP-lbeta have significantly increased during infection.Conclusions We isolated7strains of CVA16and analysed them; We screened and adapted a virulent strain TS10/08and established CVA16animal model, which made a foundation for assaying our laboratory follow-up experimental vaccines.