Journal: bioRxiv

Article Title: Development of RT-RPA-based point-of-care tests for epidemic arthritogenic alphaviruses

doi: 10.1101/2024.05.14.594209

Figure Lengend Snippet: Multiple sequence alignments (MSAs) were assembled for (A) CHIKV, (B) ONNV and (C) MAYV using the indicated number of sequences following which the consensus sequence was analyzed focusing on the four NSPs, the Capsid, E2 and E1 to identify the most conserved regions for each virus. The heatmaps show the percent non-homology calculated for each region and the top four most homologous sites (with the darkest shading), highlighted in a red border, were selected for primer design. For ONNV, the analysis identified NSP4, highlighted with a discontinuous border, was identified as the fourth target for primer design, however, we chose to prioritize designing primers in E1 instead.

Article Snippet: Adult AG129 mice were inoculated with 1000 PFU of either CHIKV (181/25 strain, BEI Resources), ONNV (NR-50081, BEI Resources), or MAYV (NR-51661, BEI Resources) via subcutaneous injection on the ventral side of the left footpads.

Techniques: Sequencing, Virus

Journal: bioRxiv

Article Title: Development of RT-RPA-based point-of-care tests for epidemic arthritogenic alphaviruses

doi: 10.1101/2024.05.14.594209

Figure Lengend Snippet: All 17 primers for CHIKV, 16 primers for ONNV and 17 primes for MAYV were screened in 20-minute RPA reactions using 10 viral copies with cDNA as input, The reactions were run at three temperatures as indicted. Depicted here is a representative subset of those screened primers, A) corresponding to CHIKV, (B) to ONNV, and (C) to MAYV.

Article Snippet: Adult AG129 mice were inoculated with 1000 PFU of either CHIKV (181/25 strain, BEI Resources), ONNV (NR-50081, BEI Resources), or MAYV (NR-51661, BEI Resources) via subcutaneous injection on the ventral side of the left footpads.

Techniques:

Journal: bioRxiv

Article Title: Development of RT-RPA-based point-of-care tests for epidemic arthritogenic alphaviruses

doi: 10.1101/2024.05.14.594209

Figure Lengend Snippet: Candidate (A) CHIKV, (B) ONNV, and (C) MAYV primer pairs identified in the screen were evaluated on a serial dilution of cDNA inputs ranging from 10 viral copes to 1 viral copy. Cross reactivity of these primers was also tested using 10 copies of cDNA from the other two viruses. The reaction inputs for each well are denoted in the image. The sensitivity was determined to be 10 for CHIKV and ONNV, and between 10 and 10 for MAYV, with not cross-reactivity.

Article Snippet: Adult AG129 mice were inoculated with 1000 PFU of either CHIKV (181/25 strain, BEI Resources), ONNV (NR-50081, BEI Resources), or MAYV (NR-51661, BEI Resources) via subcutaneous injection on the ventral side of the left footpads.

Techniques: Serial Dilution

Journal: bioRxiv

Article Title: Development of RT-RPA-based point-of-care tests for epidemic arthritogenic alphaviruses

doi: 10.1101/2024.05.14.594209

Figure Lengend Snippet: A) Schematic representation of the Millenia Biosciences 2T test strips utilized in this study. The strips have two test bands “T1” and “T2,” one coated with streptavidin and the other with an anti-Digitonin antibody. The gold nanoparticles (AuNPs) are conjugated to an anti-FITC antibody. Accordingly, all our reverse primers were either tagged with a 5’Biotin tag (CHIKV) or a 5’ Digitonin tag (ONNV and MAYV), and their probes-hybridization probe for CHIKV and MAYV or nfo-probe for ONNV-were all 5’FAM tagged. Shown in the figure are depictions of the reaction product from the CHIKV assay, that uses a hybridization probe, binding at T1 and the ONNV assay, that uses a nfo-probe, binding at T2. The MAYV RPA product would appear like the CHIKV product since it also uses hybridization probes, however, it would bind at T2, since its reverse primer has a 5’digitonin tag. (B-D) show the analytical sensitivity of the rapid-test versions of the CHIKV (B), ONNV, (C) and MAYV (D) assays. The reactions used a serial dilution of cDNA as their input. Also included are reactions with 10 copies of cDNA from the other two viruses to assess cross-reactivity. RPA reaction products were diluted and applied to the strips and then immersed in the manufacturer-provided assay buffer for up to 30 minutes. The strips were photographed using a smartphone camera. All three assays were able to detect down to 10 copies of viral cDNA and were not cross-reactive.

Article Snippet: Adult AG129 mice were inoculated with 1000 PFU of either CHIKV (181/25 strain, BEI Resources), ONNV (NR-50081, BEI Resources), or MAYV (NR-51661, BEI Resources) via subcutaneous injection on the ventral side of the left footpads.

Techniques: Hybridization, Binding Assay, Serial Dilution

Journal: bioRxiv

Article Title: Development of RT-RPA-based point-of-care tests for epidemic arthritogenic alphaviruses

doi: 10.1101/2024.05.14.594209

Figure Lengend Snippet: AG129 mice were infected with 1000 PFU of (A) CHIKV (181/25 strain, n=3), (B) ONNV (NR-50081 strain, n=2) and (C) MAYV (NR-51661 strain, n=2) via subcutaneous footpad injection. Serum was collected 1-and 3-days post infection, and organs were harvested at day 3 for CHIKV and ONNV and at day 5 for MAYV, when the mice experienced mortality. RNA was extracted, reverse-transcribed and subjected to RPA using labelled primers. The reaction products were applied to the test strips and were photographed using a smartphone camera. The extracted RNA from each sample was also subjected to RT-PCR for comparison. The results of both assays for each sample are indicated with a + (or ++) and –, as observed.

Article Snippet: Adult AG129 mice were inoculated with 1000 PFU of either CHIKV (181/25 strain, BEI Resources), ONNV (NR-50081, BEI Resources), or MAYV (NR-51661, BEI Resources) via subcutaneous injection on the ventral side of the left footpads.

Techniques: Infection, Injection, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: Journal of Medical Virology

Article Title: Seroprevalence of Chikungunya and O'nyong‐nyong Viruses in Senegal, West Africa

doi: 10.1002/jmv.70282

Figure Lengend Snippet: Baseline characteristics and chikungunya virus (CHIKV) and o'nyong‐nyong virus (ONNV) serology.

Article Snippet: For each dilution, contact was performed with 100 plaque‐forming units (PFU) of CHIKV (BEI Resources: NR‐56523) or ONNV (BEI Resources: NR‐51661) for 1 h at 37°C in a 5% CO 2 incubator.

Techniques: Virus, Infection

Journal: Journal of Medical Virology

Article Title: Seroprevalence of Chikungunya and O'nyong‐nyong Viruses in Senegal, West Africa

doi: 10.1002/jmv.70282

Figure Lengend Snippet: Antibody neutralization against chikungunya (CHIKV) and o'nyong‐nyong (ONNV) viruses in Senegal. (A) Map depicts locations in Senegal (Sindia, Thies, and Kedougou) where samples were collected from non‐febrile subjects. For each location, bar charts represent the neutralizing antibody prevalence against CHIKV (C) or ONNV (C), or equivocal (E) and negative (N) results. Prevalence values are listed above each bar. (B) CHIKV virus‐like particle IgG‐positive samples ( n = 117) were tested for neutralizing antibodies against CHIKV and ONNV. Colors depict the 50% neutralizing antibody titers (NT50), each row represents one individual, and each column is the titer to CHIKV or ONNV. (C) The percentage of NT50‐confirmed CHIKV or ONNV subjects with cross‐neutralizing antibodies to the heterologous virus.

Article Snippet: For each dilution, contact was performed with 100 plaque‐forming units (PFU) of CHIKV (BEI Resources: NR‐56523) or ONNV (BEI Resources: NR‐51661) for 1 h at 37°C in a 5% CO 2 incubator.

Techniques: Neutralization, Virus

Journal: Journal of Medical Virology

Article Title: Seroprevalence of Chikungunya and O'nyong‐nyong Viruses in Senegal, West Africa

doi: 10.1002/jmv.70282

Figure Lengend Snippet: Baseline characteristics of NT50‐confirmed chikungunya virus (CHIKV) and o'nyong‐nyong virus (ONNV) subjects.

Article Snippet: For each dilution, contact was performed with 100 plaque‐forming units (PFU) of CHIKV (BEI Resources: NR‐56523) or ONNV (BEI Resources: NR‐51661) for 1 h at 37°C in a 5% CO 2 incubator.

Techniques: Virus, Infection

Journal: Journal of Medical Virology

Article Title: Seroprevalence of Chikungunya and O'nyong‐nyong Viruses in Senegal, West Africa

doi: 10.1002/jmv.70282

Figure Lengend Snippet: Predictors of CHIKV and ONNV exposure in Senegal. Risk factors for (A) CHIKV and (B) ONNV exposure were determined using binary logistic regression. Adjusted odd ratios (aOR), represented by the solid circular dots, are shown along with their 95% confidence intervals. The aORs were computed by adjusting for age and gender, quantifying the association between CHIKV or ONNV neutralizing antibodies and key predictors, including age groups, gender and city. Blue dots and lines indicate variables associated with a higher risk of CHIKV or ONNV exposure. Green dots and lines indicate variables associated with a lower risk of CHIKV or ONNV exposure. Black dots and lines indicate variables with no significant association with CHIKV or ONNV exposure. The vertical reference line (OR = 1) indicates no association between the predictor and the dependent variable.

Article Snippet: For each dilution, contact was performed with 100 plaque‐forming units (PFU) of CHIKV (BEI Resources: NR‐56523) or ONNV (BEI Resources: NR‐51661) for 1 h at 37°C in a 5% CO 2 incubator.

Techniques:

Journal: Journal of Medical Virology

Article Title: Seroprevalence of Chikungunya and O'nyong‐nyong Viruses in Senegal, West Africa

doi: 10.1002/jmv.70282

Figure Lengend Snippet: Comparison of the prevalence of neutralizing antibodies to CHIKV and ONNV by age group, city, and gender. Differences between (A) age group, (B) city, and (C) gender were assessed using the χ 2 test or Fisher's exact test, as appropriate. p ‐values less than 0.05 were considered to be statistically significant, while values less than 0.1 were considered to be marginally significant.

Article Snippet: For each dilution, contact was performed with 100 plaque‐forming units (PFU) of CHIKV (BEI Resources: NR‐56523) or ONNV (BEI Resources: NR‐51661) for 1 h at 37°C in a 5% CO 2 incubator.

Techniques: Comparison

Journal: Scientific Reports

Article Title: RT-RPA as a dual tool for detection and phylogenetic analysis of epidemic arthritogenic alphaviruses

doi: 10.1038/s41598-024-81763-7

Figure Lengend Snippet: Primer screens conducted by agarose gel electrophoresis of RPA products. All 17 primers for CHIKV, 16 primers for ONNV and 17 primes for MAYV were screened in 20-minute RPA reactions using 10 5 viral copies with cDNA as input, The reactions were run at three temperatures as indicated. Depicted here is a representative subset of those screened primers, ( A ) corresponding to CHIKV, ( B ) to ONNV, and ( C ) to MAYV.

Article Snippet: Viral gRNA for CHIKV (181/25, BEI Resources Repository) and for MAYV (NR-50081, BEI Resources Repository) and extracted RNA from ONNV (NR-51661, BEI Resources Repository) propagated on Vero cells (CCL-81, ATCC) was used.

Techniques: Agarose Gel Electrophoresis

Journal: Scientific Reports

Article Title: RT-RPA as a dual tool for detection and phylogenetic analysis of epidemic arthritogenic alphaviruses

doi: 10.1038/s41598-024-81763-7

Figure Lengend Snippet: Analytical sensitivities of the designed primers on an agarose gel. Candidate ( A ) CHIKV, ( B ) ONNV, and ( C ) MAYV primer pairs identified in the screen were evaluated on a serial dilution of cDNA inputs ranging from 10 6 viral copies to 1 viral copy. Cross reactivity of these primers was also tested using 10 5 copies of cDNA from the other two viruses. The reaction inputs for each well are denoted in the image. The sensitivity was determined to be 10 2 for CHIKV and ONNV, and between 10 3 and 10 2 for MAYV, with not cross-reactivity.

Article Snippet: Viral gRNA for CHIKV (181/25, BEI Resources Repository) and for MAYV (NR-50081, BEI Resources Repository) and extracted RNA from ONNV (NR-51661, BEI Resources Repository) propagated on Vero cells (CCL-81, ATCC) was used.

Techniques: Agarose Gel Electrophoresis, Serial Dilution

Journal: Scientific Reports

Article Title: RT-RPA as a dual tool for detection and phylogenetic analysis of epidemic arthritogenic alphaviruses

doi: 10.1038/s41598-024-81763-7

Figure Lengend Snippet: Detecting RPA reactions on LFA strips. ( A ) Schematic representation of the Millenia Biosciences 2T test strips utilized in this study. The strips have two test bands “T1” and “T2,” one coated with streptavidin and the other with an anti-Digitonin antibody. The gold nanoparticles (AuNPs) are conjugated to an anti-FITC antibody. Accordingly, all our reverse primers were either tagged with a 5’Biotin tag (CHIKV) or a 5’ Digitonin tag (ONNV and MAYV), and their probes- hybridization probe for CHIKV and MAYV or nfo-probe for ONNV- were all 5’FAM tagged. Shown in the figure are depictions of the reaction product from the CHIKV assay, that uses a hybridization probe, binding at T1 and the ONNV assay, that uses a nfo-probe, binding at T2. The MAYV RPA product would appear like the CHIKV product since it also uses hybridization probes, however, it would bind at T2, since its reverse primer has a 5’digitonin tag. (B-D) show the analytical sensitivity of the rapid-test versions of the CHIKV ( B ), ONNV, ( C ) and MAYV ( D ) assays. The reactions used a serial dilution of cDNA as their input. Also included are reactions with 10 5 copies of cDNA from the other two viruses to assess cross-reactivity. RPA reaction products were diluted and applied to the strips and then immersed in the manufacturer-provided assay buffer for up to 30 min or until the strips were dry. The strips were photographed using a smartphone camera. All three assays were able to detect between 100 and 10 copies of viral cDNA, with “eq” indicating an equivocal test result and were not cross-reactive.

Article Snippet: Viral gRNA for CHIKV (181/25, BEI Resources Repository) and for MAYV (NR-50081, BEI Resources Repository) and extracted RNA from ONNV (NR-51661, BEI Resources Repository) propagated on Vero cells (CCL-81, ATCC) was used.

Techniques: Hybridization, Binding Assay, Serial Dilution

Journal: Scientific Reports

Article Title: RT-RPA as a dual tool for detection and phylogenetic analysis of epidemic arthritogenic alphaviruses

doi: 10.1038/s41598-024-81763-7

Figure Lengend Snippet: Detecting alphaviruses in serum and tissues from mouse infection models using RT-RPA rapid tests. IFNRα/β/γ -/- mice were infected with 1000 PFU of ( A ) CHIKV (181/25 strain, n = 3), ( B ) ONNV (NR-50081 strain, n = 2) and ( C ) MAYV (NR-51661 strain, n = 2) via subcutaneous footpad injection. Serum was collected 1 day post infection (for all three) and 3-days post infection (for CHIKV and ONNV), and organs were harvested at day 3, when the mice experienced mortality. RNA was extracted, reverse-transcribed and subjected to RPA using labelled primers. The reaction products were applied to the test strips and were photographed using a smartphone camera. The extracted RNA from each sample was also subjected to RT-PCR for comparison. The results of both assays for each sample are indicated with “+” for positive, a “-” for negative, or “eq” for equivocal, as observed.

Article Snippet: Viral gRNA for CHIKV (181/25, BEI Resources Repository) and for MAYV (NR-50081, BEI Resources Repository) and extracted RNA from ONNV (NR-51661, BEI Resources Repository) propagated on Vero cells (CCL-81, ATCC) was used.

Techniques: Infection, Injection, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Comparison