The Antiviral Effect Of LE-PolyICLC Against Dengue Ⅱ Virus In Established Mice Model

Dengue is an acute febrile disease caused by the dengue viruses (DENVs). It is a common concern problem to prevent and control DENVs effectively in the world with the growing epidemic of dengue fever in recent years. Currently, there are no available vaccines and antiviral drugs against DENV infection. The Mexican authorities have granted marketing authorization to Dengvaxia(?), making it the first vaccine to be licensed in the world for the prevention of dengue. However, the vaccine has not been carried out in global promotion and application. Two testing methods and a DENV2-infected mouse model were established firstly in the present study. On this basis, the study presented the first investigation of the antiviral effects of the liposome-encapsulated PolyICLC (LE-PolyICLC) on DENV2 in the BABL/c mouse model.The thesis has been divided into four sections:Section 1) Detection serum antibody levels of the mice infected by DENV2The EIII gene of DENV2 NGC strain was extracted and amplified by PCR, and then cloned into the prokaryotic expression vector pEASY(?)-Blunt E1. The obtained recombinant plasmid was transformed into Escherichia.coli BL21 (DE3). The recombinant protein EIII of approximately 17 KD was expressed successfully, the recombinant protein in the form of inclusion bodies was refolded, concentrated and purified by column. The purified recombinant protein was used to immunize BABL/c mice to make the polyclonal antibody. The result of indirect immunonuorescence assay (IFA) indicated that the polyclonal antibody could react with DENV2 virus specifically. An indirect ELISA method was established using the purified protein which showed good antigenicity. Then DENV2 serum antibody levels of the mice infected by DENV2 were detected by the established indirect ELISA method.Section 2) Detection virus titers in the brain of the mice infected by DENV2A pair of specific primers and TaqMan probe were designed for the quantitative RT-PCR according to E gene of DENV2 NGC strain. Based on the recombinant plasmid pEASY-T1-E as standard samples, a standard curve was obtained, wherein the value of R and Eff was 0.98 and 122.53%. Thus quantification was quite accurate and reliable by the method. And the lowest detection limit of the method was approximately 100 gene copies per reaction. Therefore, the method had a high sensitivity. The coefficient of variation of intra-sample and inter-sample assay was 0.53-1.90% and 0.85-2.54% respectively. Therefore, the method had a high repeatability. Then virus titers in the brains of the mice infected by DENV2 were detected by the established method. Section 3) Establishment of DENV2-infected mouse modelsWe established DENV2 NGC-infected BABL/c mouse model with intracerebral injection successfully. The mice intracerebrally (i.c.) inoculated with 1×106PFU of NGC strain virus. The mice presented several clinical signs of infection, such as leg paralysis and skin bleeding, the significant weight loss, histopathological damages to the brains, even death. And the mice presented the most severe morbidity degrees on the days 9 post-inoculation (dpi). The virus could replicate and proliferate in brains of the mice with the peak virus titers in the brain on 9 dpi. The antibodies against DENV2 were produced by the mouse to neutralize the virus with the highest level on 21 dpi during the observation period.Section 4) Antiviral effects of LE-PolyICLC against DENV2 in a mouse modelAdministration of LE-PolylCLC, at a dose of 1 mg/kg body weight, could alleviate the loss of body weight, degree of morbidity, and pathological damage in brains. DENV2 replication in the mouse was suppressed by LE-PolyICLC administration, and the data from quantitative real-time PCR and western blotting analyses further supported the conclusion. LE-PolyICLC treated by the i.v. route was more efficacious than the other routes. LE-PolyICLC could suppress DENV infection, probably through promoting cytokine expression associated with innate immunity, such as IFN-γ. And LE-PolylCLC could also enhance antibody response. In addition, the effectiveness of LE-PolyICLC was superior to PolyICLC on the basis of the data’in this study.