| Dengue fever (DF)is an acute infectious disease caused by dengue virus(DENV) infection, which is transmitted through the bite of infected mosquito .There are four serotypes of dengue virus(DENV-1,DENV-2,DENV-3,DENV-4).DF is now endemic in more than 100 countries in tropical and subtropical areas, and threatens the health of approximately 40% of the world's population. The World Health Organization estimates that there may be around 100 million cases of dengue virus infection worldwide every year, resulting in approximately 500,000 cases of DHF/DSS and 25,000 deaths each year . Dengue mainly prevalent along the coast areas in China, while Guangdong province is the most affected area.Since 1978, there are dengue cases reported almost every year,all four serotypes of dengue virus have epidemics in Guangdong .But the research of DF in Guangdong was major in DENV-1 and DENV-2, however, little is known about DENV-3 and DENV-4. In the last ten years , the dengue endemic in Guangdong was mostly caused by DENV-1 ,no other serotype of DENV infected case was found . So we must pay close attention to clinical DF case, get the early specimen on time and isolate the wild strain of dengue virus, research and analyze the character of its clinic and pathology. All these are significant to understand the epidemic situation and its character, source of pathogen and control and prevention of DF in GuangdongAim of this research:Analyze the clinical and laboratory characteristics of 3 cases of a family-clustered dengue fever in city of Guangzhou in Guangdong province August 2009 . Isolate dengue virus strain in serum from the patients, then sequence and analyze the whole genome of the virus, to find out the characters of dengue virus epidemic and the genome of the pathogen in Guangdong in 2009, so as to confirm the characteristics of molecular epidemiology of dengue fever in Guangdong .Research Methods:1. Collect clinical and laboratory data of the 3 dengue fever patients, statistical analyze the data by excel; dengue virus specific IgM and IgG antibody were detected by Collidal gold method in patients'serum collected in acute phase and convalescence phase; cytokine levels of IL-6,IL-10,IFN-γ,sTNFRI,TNFαwere detected by ELISA in patients'serum collected in acute phase and convalescence phase.2. Amplify the C-PrM gene in dengue virus by RT-PCR, electrophoresis and sequence analyze the PCR product.3. Inoculate cell C6/36 with serum from patients to isolate the strain of dengue virus.4. Through software of DNA DESIGN and DNAclub, design 11 pairs of primer which could amplify the whole genome sequence of the virus, amplify by PCR and connect it with T clone vector before sequencing, bioinformatics analysis is made after we get the sequence of the genome.Results:1. All the patients were bitten by mosquito before onset of disease, have not went out in the last two weeks, and the characteristics of clinics included fever, headache, flushing, lassitude, arthalgia, myalgia, rash, tourniquet test positive and leucopenia, Serum dengue virus specific antibody tests indicate: dengue IgM antibody are positive in acute phase , while IgG antibody are positive in convalescence phase , those consistent with diagnostic criteria of dengue fever. By detecting the levels of IL-6,IL-10,IFN-γ,sTNFR1 and TNFαcytokines expression in the 3 patients in acute phase and convalescence phase,we find the IL-10,sTNFR1 and IFN-γlevel of the DF patients in acute phase are higher than the health controls. 2. Specific fragment about 290bp appear after electrophoresis using PCR products from the serum of the three patients. They all are DENV-3 which are confirmed by sequencing the products, and their genome are of 100% similarity.3. Through cell C6/36,one dengue virus strain is Succeed isolated from acute stage's serum of one patient.The length of the whole genome of DENV-3 strain in Guangdong is 10707bp after sequenced , and DENV-3 genotype III is identified while it is analyzed by phylogenetic tree analysis.4.Compared to the oldest DENV-3 genotype III strain,there are 377 nucleotide mutation and 39 amino acids diversity happens in dengue virus type 3 strain of Guangdong.Conclusions:1 All the 3 patients are primary DENV-3 infection, and this is the first time to discover endemic of DENV-3 in GD in the last 10 years.2. Succeed in cultivating and isolating DENV-3 and sequencing the whole genome of it, identify the epidemic strain this time is DENV-3 genotype III.3. Compared to the oldest DENV-3 genotype III strain,there are 377 nucleotide mutation and 39 amino acids diversity happens in dengue virus type 3 strain of Guangdong...
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