Epidemiology Investigation Of Dengue Fever And Pathological Mechanism Research Of Severe Dengue

Dengue fever epidemiology of China were firstly discribed.749 DENV sequences selected from China were screening out from GenBank database to do evolutionary analysis. Results showed that 735735 cases of DF were reported from 1978 to 2014. In 1978-1991, DF outbreak mainly occur in guangdong and hainan province, and the epidemic centre changed to guangdong, yunnan, zhejiang and fujian provinces since 1991, which have>90% DF cases of China.507 cases of death toll were mostly occur before 1988. The age distribution of infected people shows a fan shape, almost between 20-45 years old. The epidemic strain were mainly for type Ⅰ and type Ⅱ, and shows a cross circulating infection or co-infection style. Local loop transmission and cross-provincial spread were both existed.There were 62 amino acid mutations in 2013 epidemic strains comparing with DENV serotype Ⅲ standard strain, and it related phylogenetically to Hainan epidemic strains in 2013 and Bangladesh’s in 2002. A total of 217 base change were found in17 dengue complete genome sequences of 2015 Yunnan DENV, among which 213 were located in the nonstructural protein region.Meanwhile 36 amino acid mutation were found in 42 structural protein gene sequences comparing with DENV serotype Ⅱ standard strain. Evolution analysis shows that the epidemic strains belong to the same evolutionary branches with Sri Lanka’s in 2004 and India’s in 2001.Pathological analysis of 375 cases of DF patients shows that inflammation, blood vessels leakage and liver damage were existed in all patients with different degree. The quantity of viral particals in serum were not the only reason leading to severe dengue. The content of NS1 in serum, the synthesis and consume of complement were associated with severe dengue fever.A whole blood ADE model were established using type Ⅱ dengue virus and its prM monoclone antibody. The results show that the quantity of viral RNA copies in ADE group was 2.94 times higher than that of common infection group, with a 61.5% reduction in C3. The content of C3a, C5a in supernatant of two infection groups were higher than the control, and the content of complement C3bBb were also increased significantly, of which ADE group was 2.6 times higher than that of common infection group, but the content of C1q changed little, which proved that the excessive activation of complement alternative pathway may be one of the acting mechanism in ADE infection.Monocytes were negatively separated from fresh human blood, in order to build an ADE model as before. The content of C3a, C5a in supernatant of two infection groups were higher than the control, as well as the content of IL-10. Co-IP and western Blot were used, then CD46 was screened out as a target proteins which have interaction with dengue NS1 proteins. After co-culture with monocytes ADE model in Transwell plate, the C5b-9 on the surface of HuVEC were detected by IFA, and the eosin staining were also used, to observe cytoskeleton change of HuVEC. It’s obvious that the fluorescence intensity of ADE group was higher than that of common infection group, with more obvious pathological damage on the cell shape, which remind that the excessive activation of complement may be one of the reasons for causing the endothelial cell injury and vascular leakage.Comprehensive the above results, we put forward the "complement storm" hypothesis. Dengue NS1 protein were replicated in large numbers in cells, and were circular migrated in blood throughout the body. NS1 competitively binding to the complement regulatory factor CD46, which lead to an inhibition of its functions in complement regulation, and cause to an excessive activation of complement by alternative pathway. A large number of complement splitting fragments were produced, which leading to a series of clinical symptoms similar with systemic allergic reactions.