Fig 1.
Negative staining electron micrograph of mAbs 3G1 and 2G4.
Antibody preparations were diluted in PBS and negatively stained with 1% uranyl acetate before imaging at 59000x magnification. Scale bar depicts 20 nm. Arrows denote a typical IgM pentamer confirmation.
Table 1.
Reactivity of mAbs 3G1 and 2G4 against acetone-fixed C6/36 cells infected with a panel of flaviviruses and alphaviruses in ELISA or IFA.
Fig 2.
Reactivity of mAbs 3G1 and 2G4 to heat shocked cells.
Fixed-cell ELISA was performed on C6/36 cells infected with WNVKUNV MOI: 1 7days post-infection, mock-infected or heat shocked by incubating at 41°C for 3 hours. Monoclonal antibodies used were 3G1 and 2G4, WNVKUNV NS1-specific mAb 3.1112G (IgM) or insect anti-heat shock protein 70 (HSP70) mAb. **** denotes significant difference between test samples and the negative control (mock). Statistical significance was analysed using two-way ANOVA, followed by Dunnett test for multiple comparisons (P<0.0001).
Fig 3.
3G1 and 2G4 reactivity to flavivirus-infected vertebrate cell lines.
Immunofluorescence assay performed on vertebrate cell lines African Green monkey cells (Vero) and chick embryo fibroblasts (DF-1) mock-infected or WNVKUNV-infected MOI: 10 and fixed at 48 hours post infection. MAbs 3G1, 2G4 and 3.1112G were labelled with goat anti-mouse Alexafluor 488 (green). Nuclei are labelled with Hoechst nuclear stain (blue). Slides were imaged at 40x magnification. Scale bar denotes 10 μm.
Fig 4.
Time kinetics of mAb-reactive antigen in flavivirus infected cells.
Fixed-cell ELISA was performed on C6/36 cells infected with WNVKUNV at MOI: 0.01 or mock-infected fixed over 7 days. Antibodies used were mAbs 3G1 and 2G4, flavivirus NS1-specific mAb 4G4 or flavivirus E protein-specific mAb 4G2.
Fig 5.
Co-localisation of mAb 3G1 and 2G4 staining with flaviviral replication complexes.
Dual was staining performed on Vero cells infected with WNVKUNV MOI: 10 or mock-infected and acetone-fixed at 48 hours post-infection. Merged images showing co-localisation of A) 3G1 (green), and B) 2G4 (green) with flavivirus NS1 labelled with anti-NS1 mAb (4G4, red), flavivirus NS5 labelled with anti-NS5 mAb (5H1, red) and flavivirus NS3 labelled with polyclonal anti-NS3 rabbit sera (red). Nuclei stained with Hoechst nuclear stain (blue). Images were taken at 100x magnification. Scale bar denotes 10 μm.
Fig 6.
Effect of RNAse III treatment on mAb 3G1 and 2G4 binding.
Immunofluorescence assay performed on Vero cell monolayers infected with WNVKUNV at MOI: 10 or mock infected and untreated (-RNAse III) or treated with dsRNA-specific nuclease RNAse III (+RNAse III). Shown are cells stained with mAbs 3G1 or 2G4, anti-dsRNA mAb J2 or WNVKUNV NS1-specific mAb 3.1112G. All antibodies are labelled with goat anti-mouse Alexafluor 488 (green). Nuclei are stained with Hoechst nuclear stain (blue). Images were taken at 40x magnification. Scale bar denotes 10 μm.
Fig 7.
Reactivity of mAbs 3G1 and 2G4 to synthetic dsRNA.
A) Immunofluorescence assay performed on Vero cell monolayers transfected with Poly(I:C) or treated with transfection reagent only (mock). Cells were also subjected to pre-treatment with or without RNAse III (+RNAse,-RNAse respectively). Shown are coverslips stained with mAbs 3G1 or 2G4, anti-dsRNA mAb J2 or WNVKUNV NS1-specific mAb 3.1112G. All antibodies are labelled with goat anti-mouse Alexafluor 488 (green). Nuclei are stained with Hoechst nuclear stain (blue). Slides were imaged at 40x magnification. Scale bar denotes 10 μm.
Fig 8.
ELISA was performed on 96-well plate coated with 0.25–200 ng/well Poly(I:C).
Monoclonal antibodies used were 3G1, 2G4, positive control anti-dsRNA specific mAb J2 and negative isotype (IgM) control anti-NS1 mAb 3.1112G.
Fig 9.
Reactivity of mAbs 3G1 and 2G4 to small nucleic acid oligonucleotides in capture ELISA.
Purified mAb 2G4 was coated to 96-well plates at predetermined optimal concentration. MAb 3G1 was concentrated from hybridoma supernatant and coated at a predetermined optimal dilution. Capture of biotinylated oligonucleotides was measured by probing with streptavidin-HRP. A) Reactivity to biotinylated double-stranded RNA molecules (0.25–50 pmol/well) of varying lengths was tested in capture ELISA format. Molecule sizes tested were 21 bp, 30 bp, 40 bp, 50 bp. * denotes concentration at which difference between test samples and negative control (21 bp molecule) becomes significant. Statistical significance was analysed using two-way ANOVA, followed by Dunnett test for multiple comparisons (P<0.0001). B) Reactivity of mAbs to biotinylated nucleic acid molecules was tested in capture ELISA format using predetermined optimal concentrations (5–15 pmol/well). Molecules tested were dsDNA, ssDNA, ssRNA and RNA:DNA hybrid all 50 bp in length. **** denotes significant difference between test samples and the negative control (21 bp dsRNA). Statistical significance was analysed using one-way ANOVA, followed by Dunnett test for multiple comparisons (P<0.0001).
Fig 10.
Reactivity of mAbs 3G1 and 2G4 against cells infected with various arboviruses.
Immunofluorescence assay was performed on infected and mock-infected Vero cell monolayers. A) Merged images showing dual stained Vero cell monolayers infected with the positive-sense RNA viruses WNVKUNV (Flaviviridae; MOI:1, 48 hours post-infection) or (RRV (Togaviridae; MOI: 1, 24 hours post-infection) and stained with virus-specific mAbs (green) and 3G1 or 2G4 (red). B) Merged images showing dual stained Vero cell monolayers infected with the negative-sense RNA viruses BEFV (Rhabdoviridae; MOI:0.01, 72 hours post-infection) and AKAV (Bunyaviridae; MOI: 0.1, 24 hours post-infection) and stained with virus-specific mAbs (green) and 3G1 or 2G4 (red).
Fig 11.
A) C6/36 cell monolayers infected with CASV (Mesoniviridae, MOI:1, 72 hours post-infection) or mock infected and stained with mAbs 3G1 or 2G4 (red).
B) Vero cell monolayers infected with BTV (Reoviridae; MOI:1, 24 hours post-infection) or mock-infected and stained with mAbs 3G1 or 2G4 (red) or BTV-specific mAb (green). Nuclei stained with Hoechst nuclear stain (blue). Images were taken at 40x magnification. Scale bar denotes 10 μm.
Fig 12.
Sensitivity of dsRNA detection in comparison to viral protein of circulating arboviruses at different time points.
A) C6/36 cells were infected with WNVKUNV or B) RRV T48 in serial dilutions from 10-1 to 10-12 and fixed with acetone at 24, or 48 hours post-infection or at 5 days post-infection. Fixed cell ELISA was performed using antibodies 3G1, 2G4 and the virus E protein-specific mAbs 4G2 (flavivirus) or G8 (RRV, alphavirus). Sensitivity is presented as TCID50 values for virus detected by each antibody. *** denotes significant difference between level of virus detection by mAbs 3G1 or 2G4 and virus-specific mAb 4G2 or G8. Statistical significance was analysed using one-way ANOVA comparison of means, followed by Turkey test for multiple comparisons (P<0.0005).
Table 2.
Detection of circulating arboviruses from mosquito homogenates by 3G1 and 2G4.
Table 3.
Novel virus isolates from mosquito homogenate detected in ELISA by mAbs 3G1 and 2G4.