Immunization

assay and protection assay in adult Balb/c mice All procedures involving animals were approved by the Animal Experimentation Ethics Committee of Sun Yat-sen University and carried out by a licensed individual with an ethical approval number of 2012/0081. Animals were purchased from the Center of Experimental Animal of the Sun Yat-Sen University. Four groups (PL10 coupled to KLH, PH10 coupled to KLH, PM10 coupled to KLH, and PBS), each comprising of ten adult female Balb/c mice (4–6weeks old), were intraperitoneally injected with 100 μg of immunogen emulsified in complete Freund’s adjuvant for the first immunization. Mice were then injected at week 2 and 4 with the peptides and Freund’s RO4929097 purchase incomplete adjuvant. The mice were bled on week 0, 2, 4 and 6 via tail vein according to NC3Rs Selleckchem LEE011 standard procedures, and the anti-peptide antibody titer of mice sera was determined by ELISA. Two weeks after the last immunization, mice were infected with DENV2 NGC strain (106 PFU/mouse) through peritoneal injection. Blood samples were collected at day 0.25, 1, 2, 3, 4 and 5 via tail vein according to NC3Rs standard procedures. Then, all animals

were euthanized by using Carbon dioxide (CO2) according to NC3Rs standard procedures and the experiment was terminated. Viral RNA was extracted from 140 μl serum aliquots using QIAamp Viral RNA mini kit (Qiagen). The viral RNA copy numbers were quantified

by qRT-PCR. Western blot analysis DENV infected C6/36 cells were treated with 1% triton X-100, the lysates were run on 12% SDS polyacryramide gels and transferred Ergoloid onto polyvinylidene difluoride (PVDF) membranes (Amersham). The membranes were then blocked with PBS containing 5% skimmed milk and probed with prM-specific antibodies for 2 h at room temperature. Subsequently, membranes were detected with HRP-conjugated anti-mouse IgG and developed with enhanced chemiluminescence reagents (ECL, Thermo Fisher Scientific). Indirect immunofluorescence assay (IFA) C6/36 cells were infected with DENV1-4 and JEV. Cells were then fixed with acetone at −20°C for 20 min and washed three times with PBS. Cells were incubated with a 100-fold dilution of prM-specific antibodies. After 60 min of incubation at 37°C, cells were washed three times with PBS. Cells were then reacted with a 200-fold dilution of Alexa-Fluor-488-conjugated anti–mouse IgG (Invitrogen) for 45 min at 37°C, washed five times with PBS. After washing, cells were treated with DAPI and detected using a fluorescent microscope. Real-time quantitative RT-PCR (qRT-PCR) Viral RNA copy numbers were quantified by qRT-PCR as described previously [52]. Briefly, Viral RNA was extracted from 140 μl serum aliquots using QIAamp Viral RNA mini kit (Qiagen).