Five mice were used for each experimental condition, and 3

Five mice were used for each experimental condition, and 3.4107 GCPs were inoculated per mouse and mice were followed for 15 days. Supporting Information Figure S1 Effect of mAbs around the infectious properties of standard DENV preparations. (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal contamination in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st computer virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles. Introduction Dengue computer virus (DENV) is the leading cause of mosquito-borne viral disease in the world. It is estimated that over 50 million DENV infections occur annually, resulting Rabbit polyclonal to ADD1.ADD2 a cytoskeletal protein that promotes the assembly of the spectrin-actin network.Adducin is a heterodimeric protein that consists of related subunits. in 500,000 hospitalizations and over 20,000 deaths [1]. The four antigenically distinct serotypes (DENV 1, 2, 3 and 4) are transmitted to humans by bites of female and (adapted from [35]). c in a dose-dependent manner. Open in a separate window Physique 5 Effect of anti-E mAb 4G2 around the infectious properties of immature WNV particles and experiments revealed that all mice receiving immune serum at dilutions of 1/10 to 1/104 survived contamination, whereas 3 out of 5 animals inoculated with immature WNV opsonized with serum at a dilution of 1/105 succumbed to lethal contamination ( Fig. 6E ). Open in a separate window Physique 6 Effect of immune sera around the infectious properties of immature WNV particles.Infectivity and mice experiments were performed as described in the legend to Fig. 5. (A, B) immune sera from mice prior vaccinated with E ectodomain. (D, E) Immune serum derived from mice Benzenepentacarboxylic Acid prior infected with a sublethal dose of st WNV. (A, D) Values depicted around the x axis represent dilution factors. The error bars represent standard deviations (SD); (n.d.) denotes not detectable, (PMS) denotes polyclonal mouse serum. Student’s t-tests were Benzenepentacarboxylic Acid used to determine significance; *, C6/36 cells were maintained in minimal essential medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS), 25 mM HEPES, 7.5% sodium bicarbonate, penicillin (100 U/ml), streptomycin Benzenepentacarboxylic Acid (100 g/ml), 200 mM glutamine and 100 M nonessential amino acids at 30C, 5% CO2. Baby hamster Kidney (BHK21) and BHK21 clone 15 cells (BHK21-15) cells were cultured in DMEM (Life Technologies) made up of 10% FBS, penicillin (100 U/ml), streptomycin (100 g/ml), 10 mM HEPES, and 200 mM glutamine. Human adenocarcinoma LoVo cells were cultured in Ham’s medium (Invitrogen) supplemented with 20% FBS at 37C, 5% CO2. Mouse macrophage P388D1 cells were maintained in DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 g/ml), sodium bicarbonate (Invitrogen, 7,5% answer) and 1.0 mM sodium pyruvate (GIBCO) at 37C, 5% CO2. Computer virus growth DENV-2 strain 16681 and WNV strain NY385-99 were propagated on C6/36 cells and BHK21 cells respectively, as described before [21], [38]. Immature DENV and WNV particles were produced on LoVo cells as described previously [21]. Briefly, LoVo cells were infected at MOI 5 for DENV and MOI 4 for WNV. Computer virus inoculum was removed after 1.5 hr and fresh medium was added after washing the cells three times with PBS. At 72 hpi, the medium containing the computer virus particles was harvested, cleared from cellular debris by low-speed centrifugation, aliquoted, and stored at ?80C. The specific infectivity of the DENV and WNV preparations was determined by measuring the number of infectious models by plaque assay on BHK21-15 cells and the number of GCPs by quantitative PCR (qPCR) analysis, as described previously [21], [38]. qPCR To determine the number of GCP, we extracted viral RNA by use of a QIAamp viral RNA mini kit (QIAGEN, Venlo, The Netherlands). cDNA Benzenepentacarboxylic Acid was synthesized from viral RNA by RT-PCR. For DENV we used a published protocol [38]. For WNV, the forward primer and a TaqMan probe (Eurogentec, Seraing, Belgium) was used. DNA was amplified for 40 cycles (15 s at 95C and 60 s at 60C) on a StepOne Real-Time PCR instrument (Applied Biosystems, Carlsbad, CA) and the concentration GCPs was decided using a standard curve based on a cDNA plasmid encoding the nonstructural genes of WNV NY99 (kind gift from Dr. G.P. Pijlman, Wageningen University, The Netherlands). ELISA The binding properties of anti-E antibodies to immature computer virus particles were assessed with a three-layer ELISA. Briefly, microtiter ELISA plates (Greiner bio-one) were coated with 5108 GCP of purified computer virus preparations per well in 100 l coating buffer, overnight. After blocking with 2% milk in coating buffer for 120 min, 100 l.