| ObjectiveAs a worldwide epidemic disease, Dengue fever is a high incidence of the disease, and spread to a wide range, which make it become the global major public health problem. After infected with dengue virus, depending on the severity of the disease is divided into DF, DHF and DSS, the former belongs to the mild, the latter two belongs to heavier conditions, and the fatality rate is higher. In recent years, guangzhou appear constantly dengue fever epidemic, since the 21st century outbreak of four large-scale dengue fever epidemic, is particularly serious, in 2014 epidemic infection the cases is close to 40000, and five deaths, guangzhou has become a national focus on prevention and control of dengue, people are paying more and more attention to the spread of dengue virus source in Guangzhou region and the spread of dengue virus in the world. In addition, now there is still no very effective in the treatment of dengue fever. Because of the lack of western medicine treatment, vaccines and anti-viral drugs, the harm of dengue virus infection after affecting is difficult to be cured. In recent years, traditional Chinese medicine (TCM) has made some progress in the treatment of dengue fever, so people give higher hope to the TCM treatment of dengue fever. Formula of cold-pungent diaphoresis and resolving dampness is conbinated after screening curative effect of TCM in the study of early antiviral drugs by Liu Xiaohong, who is the professor of Traditional Chinese Medicine University. Yet not to investigate its effect on the treatment of dengue fever.The purpose of this research is to investigate dengue transmission source in guangzhou area in 2014, combine with published research findings in the early and the knowledge of Bayesian phylogenetic geography to find ancestors of dengue virus strain in the Guangzhou region in 2014, and simulate the strain change over time in the spread of the global situation. In addition, this topic will also to explore the formula side effect of treatment of dengue fever from the Angle of cytokines. The above researches aim to provide the reference for the prevention and control strategies and treatment of dengue fever for our country, improve the efficiency of the epidemic prevention and control, save social resources, and then protect public health.Methods1. During the Guangzhou dengue pandemic in 2014, serum samples were collected from patients with dengue fever in the First Affiliated Hospital of Guangzhou University and Traditional Chinese Medicine Hospital of Guangdong Province for IgM and IgG antibody detection in the serum. Test results for positive serum was used for follow-up study.2. The positive serum was inoculated into monolayer C6/36 Aedes albopictus passaged cells, discarded it after 1 hour adsorption.5 days of culture, the culture supernatant was collected. Viral RNA was extracted and reverse transcribed into cDNA for the identification and classification of the virus according to Yong YK and others reported in the literature of RT-PCR and real-time SYBR green RT-PCR method3. To analyse the possible sources of the 2014 dengue virus strains, in the first place,It should be done that reference gene sequence of DENV-1 Hawaii strain (KM204119 GenBank accession number) and combine with Premier Primer 5.0 software to designe and synthesize DENV-1 type dengue virus E gene primers. In the next, using the technique of PCR, gel electrophoresis, DNA gel extractio and sequencing get the sequencing results. Finally compared with virus strains in GenBank and use MEGA6 to build phylogenetic tree.4. Combined with method of Bayesian phylogenetic geography trace back the spread of region source and the migration process of dengue fever in Guangzhou in 2014,.5. The effect of treatment of dengue fever by formula of cold-pungent diaphoresis and resolving dampness will be evaluated from the perspective of cytokines.Results1. During epidemics of dengue fever in Guangzhou, the serum samples from patients between 18-75 years old whose body temperature was higher than 37.5℃ within 72 hours were collected to measure IgM and IgG with ELISA kit. Normally both of the antibodies were found to be positive in serum, which could be identified as dengue virus infection.168 cases of the positive serum were collected during the time, among which 70 cases (41.7%) were males and 98(58.3%) were females. From the angle of age of the disease, there was only 1 case (0.8%) under 10 and 5 cases (4%) between 10 to 20 years old. According to the positive serum samples, There is the high incidence of dengue fever in ages 20 to 70 years old, accounting for 19.2%,12%,16.8%,28% and 12% of the total respectively.7.2% of the total cases occurred in the elderly over 70 years old. In terms of geographical distribution, mostly of the cases confirmed were located in Baiyun District (35.6%), Liwan District(23.7%), Yuexiu District(16.9%) and Haizhuqu District(11.0%). No cases is reported inNansha District and Huadu District。2. The result of identification and typing of dengue viruses showed that 17 strains of dengue viruses in total could display about 350bp electrophoretic bands with Tm value of 82.5℃.3. The data provided by phylogenetic tree constructed with MEGA6 showed that dengue virus type 1 in Guangzhou in 2004 includes two genotypes (I、V), of which six strains of the virus(KT037100、KT037101、KT037103、KT037105、 KT037106、KT037109) of the genotype I were similar to Guangdong virus strain in 2006、2009、2013 and 2015, Singaporean virus strain in 2013, Thailand virus strain in 2007、2008、2009;Meanwhile, it was revealed that the other two strains(KT037100 and KT037108) had high homology with the Guangzhou virus 2004、2005、2007、2009, Malaysian virus strain in 2005、2008、2012、2013、 2014 and Singaporean virus strain in 2005、2006、2008、2009、2014, Indonesia virus strain in 2007、2008、2010、2011、2012、2013, and Taiwan virus strain in 2014; The 9 virus strains(KT037099、 KT037102、 KT037104、KT037110、KT037111、 KT037112、KT037113、KT037114、KT037115) of the genotype V showed a high sequence similarity to Guangdong virus strain in 2009、2010、2011、 2013, Singaporean virus strain in 2008、2009、2011、2012、2014, and Bhutanese and Malaysian virus strain in 2013, india virus strain in 2005、2008、2009、 2010、011.4.Genotype I MCC tree shows the ancestor nodes located in Thailand, a posteriori probability of 0.8679. Geographic spread of space-time diagram and the significant main road map shows that the main region of the virus spread is south east Asia. Group dynamics results show that the highest species diversity is in 2007, then slow down. Genotype V MCC tree shows the ancestor nodes located in India, a posteriori probability of 0.7429. Geographic spread of space-time diagram and the significant main road map shows that the main region of the virus spread is Asia and the americas. Population diversity rise rapidly in 1970-1975 and remains high.5. Compared with normal control group, the serum levels of IL-1, IL-6, IL-10, IL-17, TNF-a and IFN-y were significantly increased (P<0.05 or P<0.01) besides an ascending trend of IL-1 β (P>0.05) before treatment and the levels of cytokines mentioned after treatment didn’t change apparently (P>0.05) except IL-10 and IL-17, both of which were increased markedly (P<0.05). Moreover, IL-1, IL-6, IL-10, IL-17, TNF-a and IFN-y levels after treatment were obviously decreased (P<0.01) also besides a decreasing trend of IL-1β (P>0.05), as compared with those before treatment.Conclusion1. In 168 cases of patients with gender, female with excess male. The lowest incidence of the disease, but adults’is higher. The numbers of Patients in the old city such as Baiyun, Liwan, Yuexiu, Haizhu and other old district is in the majority, the number of cases is low in the other district, for example cases are not counted in Nansha and huadu district. This may be affected by the hospital’s geographical area.2. To investigate dengue virus amplification of 168 patients with dengue fever clinical samples for identification and classification, The results of Identification and classification, a total of 17 virus strains were isolated, 17 strains of dengue virus belong to Type 1 dengue virus3. Combined with large sample size to construct genetic evolutionary tree and analyze genotype I evolutionary tree and the spread of genotype V source, it can be seen that the spread of genotype I source is divided into two, and six genotype I strains of the virus may spread to guanzghou in 2009 from Thailand, and formation location spread, and Formed pandemics in 2013、 2014, and spread to 2015. Other two strains of the virus spread source may be Malaysia, and it may spread to Guangzhou in 2004、2005、2007、2009、2014, Taiwan’ s strains may be passed by Malaysia in 2014. Genotype V 9 strains of the virus may be passed from Singapore, and it may spread to Guangzhou in 2009、2010、 2011、2013、2014.4. Genotype I dengue virus source is Thailand, mainly popular in southeast Asian countries and the surrounding. Genotype V dengue virus is the source of India, is popular in Asia and the Americas.5. The infection of dengue virus can prompt the release of cytokines, Formula of cold-pungent diaphoresis and resolving dampness can effectively regulate the level of cell factors, such as IL-1/IL-10, IL-6, IL-10, IL-17, TNF-a, IFN-y and so on, it has good therapeutic effect on dengue fever. |
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