African green monkey Vero cells and human 293T cells were grown in DMEM supplemented with 10% FBS, penicillin, streptomycin and glutamine at 37˚C in 10% CO₂. Aedes albopictus C6/36 mosquito cells were maintained in EMEM containing Earl’s salts and non-essential amino acids supplemented with 10% FBS, penicillin, streptomycin and glutamine at 28˚C in 5% CO₂.

Virus stocks of dengue-2 16681 [28] and TSV01 (accession number AY037116) were produced as described previously [20] by inoculating monolayers of Vero or C6/36 cells in 150 cm² flasks with 100 µl of 1 ( 10⁶ FFU/ml virus stock in 5 ml complete medium. After 2 h, 15 ml of complete medium was added to infected cells. Seven days post-infection, viral supernatants were collected, filtered through a 0.2 micron filter, adjusted to 20% FBS, aliquoted and stored at -80˚C. Viral titers in focus-forming units per milliliter (FFU/ml) were determined by focus-forming assay (FFA) titration in Vero cells.

Monoclonal and polyclonal antibodies were produced by immunizing Balb/c mice and New Zealand rabbits with recombinant dengue-2 (PR-159S1 strain) capsid protein [29] (generous gift from R. Kuhn). Murine and rabbit anti-capsid IgG were purified using protein A beads.

Viruses from filtered supernatants of infected Vero cells were purified through a 30% sucrose cushion by ultracentrifugation at 23,000 rpm in an SW28 rotor (Beckman) for 2.5 h at 4˚C. Pelleted viruses were lysed and loaded onto an SDS-gel together with recombinant dengue-2 capsid as a positive control. Western blot analysis was performed according to manufacturer’s instruction for Tris-Glycine precast minigels (Invitrogen). Blots were incubated with primary monoclonal or polyclonal anti-dengue-2 IgG (5 µg/ml) overnight, incubated with secondary horseradish peroxidase (HRP)-conjugated anti-mouse or -rabbit IgG for 2 h at room temperature (Amersham) (1/10,000 dilution) and revealed by ECL (Perkin-Elmer).

Cells (2 x 10⁴) were infected with dengue at a multiplicity of infection (MOI) of 0.01 for 2 h at 37˚C, washed twice with medium, and incubated in 2 ml complete medium at 37˚C in 10% CO₂. Supernatant aliquots were collected on days 0, 3, 5, 7 and 10 post-infection. Viruses were lysed by mixing supernatant with 0.5% NP-40 in PBS for 10 min at room temperature with vigorous shaking and frozen at -20˚C. 96-well plates coated overnight at 4˚C with rabbit anti-capsid dengue-2 IgG (20 µg/ml) were washed 10 times with H₂O, blocked with 3% BSA in PBS for 1 h at room temperature, and washed 4 times with wash buffer (PBS containing 1% BSA and 0.02% Tween). Virus supernatants were added to ELISA plate for 2 h at room temperature. Wells were washed 10 times with washing buffer, incubated for 2 h at 4˚C with biotinylated rabbit or murine anti-capsid IgG (2 µg/ml), washed 10 times, incubated for 30 min at room temperature in the dark with streptavidin-conjugated HRP (Jackson Laboratories and Immunochemicals) (1/1000 dilution) and washed 10 times with washing buffer. The o-phenylenediamine dihydrochloride substrate (Sigma-Aldrich) (100 µl) was added to wells and reaction was stopped after addition of H₂SO₄ (4 N) (Fisher Scientific) (100 µl). Plates were read at 495 nm on a microplate reader (Molecular Devices).

IFSA was conducted as described previously [30, 31] with minor modifications. Cells (2 x 10⁵) were infected with dengue at an MOI of 0.1 for 2 h at 37˚C, washed twice with medium, and incubated in 2 ml complete medium at 37˚C in 10% CO₂. Three days post-infection, cells were washed, trypsinized, resuspended in PBS at 1 x 10⁶/ml and fixed with 0.2% paraformaldehyde in PBS for 30 min on ice. Cells were washed, permeabilized in PBS containing 0.2% Tween for 15 min at 37˚C, washed and resuspended in FACS buffer (PBS containing 2% FBS). For intracellular staining, cells (10⁵) were incubated for 30 min at 4˚C with 10 µg/ml of isotype controls, mouse monoclonal anti-dengue capsid 9A7 IgG, or mouse anti-dengue Env complex IgG (Chemicon). Cell permeabilization was confirmed by staining cells with mouse anti-tubulin IgG (Santa Cruz Biotechnologies). Cells were washed, incubated with secondary phycoerythrin (PE)-conjugated anti-mouse IgG (10 µg/ml) for 30 min at 4˚C, washed again, resuspended in PBS, fixed in 2% paraformaldehyde and stored at 4˚C until FACS analysis.

FFA was conducted as described previously [32, 33] with minor modifications. Briefly, Vero cells (1 x 10⁴) seeded overnight in 96-well plates were incubated with supernatants containing viruses (dilutions from 10^-1 to 10^-6) for 2 h at 37˚C, washed once and incubated for 72 h at 37˚C in 10% CO₂. Cells were washed, fixed with 4% formaldehyde in PBS for 20 min at room temperature, washed again and permeabilized with PBS containing 3% BSA, 0.3% TritonX-100 and 10% FBS for 1 h at room temperature. Cells were incubated with mouse monoclonal anti-dengue envelope or anti-capsid 9A7 IgG (1 µg/ml) in 3% BSA and 0.3% Triton X-100 for 1 h at room temperature. Cells were washed 3 times with PBS and incubated with Alexa555-conjugated anti-mouse IgG (Molecular Probes) (1/1000 dilution) for 1 h at room temperature. Cell nuclei were stained with Hoechst dye. Cells were washed 3 times, resuspended in PBS and analyzed for number of infectious foci by fluorescent microscope (Zeiss).

RT-PCR was performed to determine DENV replication by quantifying DENV-2 genomic RNA copies. Viral RNA was extracted using RNA extraction kit (QIAamp Viral RNA mini kit). RT-PCR assay was performed by adding 1 µL of extracted viral RNA to the SensiMix SYBR green reagent, which contained 7.4 μL ddH2O, 10 μL 2x SensiMix One-Step, 0.4 μL 50x SYBR Green solution, 10 U of RNAse Inhibitor, 50 pmoL of forward (DNF) and reverse (D2R) primers. All samples were assayed in triplicate. The amplifications were performed using the DNA Engine Opticon system (MJ Research/Bio-Rad, Hercules, CA) with the following thermal conditions: reverse transcription at 50°C for 30 min, initial denaturation at 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec, 59°C for 30 sec and 72°C for 30 sec. Melting curve analysis was subsequently performed at temperature from 60°C to 98°C to verify the assay specificity. For absolute quantitation of the viral RNA, a standard curve was established with a serially diluted RNA extracted from DEN-2 stock.

Forty serum samples were obtained from DEN-2 infected patients, who gave a positive result for anti-DEN-2 antibodies. Ten serum samples from blood donors were collected as negative controls.

The development of assays, which rapidly and sensitively detect dengue attachment, entry and replication, is essential in order to identify the primary host cells and receptors that support viral replication in the host. To address this issue, we generated antibodies directed against the abundant structural capsid protein of dengue and developed enzyme-linked immunosorbent (ELISA), intracellular FACS staining (IFSA), and fluorescent focus (FFA) assays to detect dengue virus and infection. Balb/c mice and New Zealand rabbits were immunized with recombinant dengue-2 (PR-159S1 strain) capsid core protein purified from E. coli [29]. We produced mouse monoclonal anti-dengue-2 capsid antibodies and rabbit polyclonal antisera. We analyzed by Western blot the reactivity of the antibodies toward dengue-2 capsid derived either from E. coli or from purified viruses. Recombinant capsid derived from the dengue-2 PR-159S1 strain was purified from E. coli as described previously [29]. Dengue-2 16681 viruses were purified from supernatant of infected Vero cells by ultracentrifugation over a 30% sucrose cushion. Pelleted viruses were lysed and analyzed by Western blot. We found that both monoclonal (9A7) (Fig. 1, lane 2) and polyclonal (Fig. 1, lane 6) anti-dengue-2 capsid antibodies efficiently recognize recombinant dengue-2 capsid (at around 10 kDa). Monoclonal (9A7) (Fig. 1, lane 4) and polyclonal (Fig. 1, lane 8) anti-dengue-2 capsid antibodies recognize a single band (around 12 kDa) in viral lysates, corresponding to capsid incorporated into dengue-2 viruses.

The difference in size between recombinant and viral dengue-2 capsid proteins may result from post-translational modifications such as protein cleavage or phosphorylation. No reactivity was observed in ultracentrifuged supernatant from uninfected Vero cells (mock) (Fig. 1, lanes 3 and 7). Moreover, no reactivity was observed toward other dengue serotypes such as dengue-1, -3 and -4 (data not shown). This demonstrates that our monoclonal and polyclonal antibodies recognize dengue-2 capsid specifically.

Using our monoclonal and polyclonal anti-dengue-2 capsid antibodies, we developed a rapid, reproducible and sensitive ELISA. Briefly, 96-well plates were coated with 20 µg/ml of protein A-purified rabbit anti-capsid dengue-2 IgG, washed and blocked with 3% BSA. Increasing concentrations of recombinant dengue-2 capsid protein diluted in PBS containing 0.5% NP-40 were added to ELISA plates (triplicates). Captured dengue-2 capsid was detected with biotinylated mouse monoclonal anti-dengue-2 capsid 2 µg/ml IgG and streptavidin-conjugated HRP. The threshold of detection of recombinant dengue-2 capsid by the ELISA was 100 to 300 pg/ml (Fig. 2).

We then asked if our ELISA can detect dengue replication over time by measuring levels of capsid released into the supernatant of infected cells. Vero or 293T cells were infected with either 16681 or TSVO1 dengue-2 viruses at an MOI of 0.01. Supernatants from infected cells were collected at different time points and amounts of virus quantified by ELISA. Viral replication can be detected as early as 3 days post-infection and reaches a plateau between day 7 and 10. Note that if we increase the MOI, capsid release into supernatant of infected cells can already be detected 48 h post-infection (data not shown). We found that 16681 replicates more robustly than TSV01 in both Vero and 293T cells (Fig. 2). Specifically, 16681 reaches a growth peak of 30-40 ng/ml of capsid in supernatant of infected cells, whereas TSV01 reaches a growth peak of 5-15 ng/ml (Fig. 3). We obtained similar growths in human Huh-7 and mosquito C6/36 cells (data not shown). This demonstrates that our capsid ELISA permits rapid and sensitive detection of dengue replication in various cell lines.

We then asked whether our ELISA is as accurate as other previously established assays, which measure dengue infection and replication. Specifically, we developed an intracellular FACS staining assay (IFSA) as well as a fluorescent focus assay (FFA) using anti-capsid antibodies. We then compared ELISA, IFSA and FFA for their capacities to monitor dengue infection in various cell lines. First, we developed an IFSA using anti-capsid antibodies. Vero cells (2 x 10⁵) were infected with 16681 dengue-2 virus at an MOI of 0.1 for 2 h at 37˚C, washed twice and incubated in complete medium for three days. Cells were then washed, trypsinized, fixed and permeabilized. Permeabilized infected cells were incubated with the monoclonal anti-dengue capsid 9A7 antibody followed by a secondary phycoerythrin (PE)-conjugated anti-mouse antibody. Cells were then washed, fixed in paraformaldehyde and analyzed by FACS. Efficiency of permeabilization was verified by anti-tubulin staining. Ninety-five to 99% of the cells were positive for tubulin staining after permeabilization (Fig. 4, bottom panels), whereas less than 0.01% of the cells were positive for tubulin staining without permeabilization (data not shown). Importantly, we found that 75 to 80% of infected cells were positive after anti-dengue capsid 9A7 antibody staining (Fig. 4, top right panel) compared to 0.03 to 0.07% of non-infected cells (Fig. 4, top left panel). We obtained similar results using mouse anti-dengue envelope complex antibodies (Chemicon) (data not shown). Our data indicate that anti-dengue-2 capsid antibodies can be used to measure dengue replication by ELISA (Fig. 3) as well as by IFSA (Fig. 4).

In order to compare the accuracies of ELISA and IFSA, we tested them for their abilities to measure the potency of anti-dengue agents. Specifically, we compared side-by-side ELISA and IFSA capacities to detect inhibition of dengue-2 infection by neutralizing antibodies. Vero cells were exposed to dengue-2 16681 virus at an MOI of 0.1 in the presence or absence of the neutralizing anti-dengue-2 envelope 3H5 antibody (hybridoma supernatant diluted at 1/100, 1/1000 and 1/10,000) (kindly provided by R. Kinney). Three days post-infection, infection was monitored either by IFSA, by measuring amounts of intracellular capsid, or by ELISA, by measuring amounts of capsid released in the supernatant. By IFSA, we found that the neutralizing 3H5 antibody efficiently reduces dengue-2 infection. The percentage of capsid positive Vero cells drastically decreases (60.5%, 27.8% and 4.25% of positive cells) in the presence of increasing concentration of 3H5 antibody (Fig. 4). Specifically, we found that dengue-2 infectivity goes from 100 down to 76, 16 and 5.6% in the presence of 3H5 hybridoma diluted at 1/10000, 1/1000 and 1/100, respectively. Importantly, we observed a very similar pattern of inhibition of dengue-2 replication by 3H5 when we quantified amounts of capsid released in the supernatant of infected cells by ELISA (Table 1, right column). Dengue-2 infectivity monitored by ELISA is gradually reduced from 100 to 75, 36 and 5.4% when 3H5 is added at a dilution of 1/10000, 1/1000 and 1/100, respectively. These data indicate that ELISA and IFSA assays using anti-capsid antibodies represent accurate tools to measure dengue-2 replication as well as to evaluate the potency of anti-dengue agent candidates.

We then developed an FFA using anti-capsid antibodies to compare its potential to measure dengue infection with that of ELISA and IFSA described above. FFA was conducted as described previously [32-33] with minor modifications. Briefly, supernatants from Vero and 293T cells infected with 16681 dengue-2 virus were harvested on days 0, 3, 5, 7 and 10 and immediately used to infect naïve Vero cells. Infected Vero cells were washed, fixed with formaldehyde and permeabilized with TritonX-100. Cells were then incubated with monoclonal anti-dengue-2 capsid or anti-envelope antibodies. Cells were washed and incubated with Alexa555-conjugated anti-mouse antibodies. Infectious foci were visualized using a fluorescent microscope.

We found that the subcellular localization of capsid and envelope in infected Vero cells greatly differs (Fig. 5A). The anti-capsid antibody mainly stains the nucleus and nucleolus (Fig. 5A, bottom panels), whereas the anti-envelope complex antibody stains the cytoplasm (Fig. 5A, top panel). This is in accordance with previous studies, which showed that capsid localizes in the nuclear compartment [34, 35]. Importantly, dengue-2 replication monitored by FFA using the anti-capsid antibody perfectly correlates with that using the anti-envelope antibody (Fig. 5B). More importantly, dengue-2 replication monitored by FFA using anti-capsid antibodies perfectly correlates with that using our capsid ELISA and FFA (Fig. 5C). RT-PCR analysis of dengue-2 replication match well the capsid ELISA data, although there is a small window period between when viral RNA is detectable by the sensitive RT-PCR and when capsid is detected by ELISA (Fig. 5D). Altogether these data indicate that antibodies directed against the structural capsid protein of dengue-2 represent ideal tools to measure dengue-2 replication in various detection assays.

We then asked whether this capsid ELISA can be used as a diagnostic tool to identify infected patients. Specifically, we collected 10 donor sera and 50 dengue-2 patient sera and tested them for capsid content by ELISA. Importantly, we constantly found that patient sera gave capsid values superior to those of donor sera (Table 2). This demonstrates that the capsid ELISA assay could be of a great utility for screening Dengue 2 infected patients. It also suggests that similar assays can be developed when similar antibodies will be generated for the detection of the 3 other serotypes.