Table 1.
Primer and probe sequences for rapid Iso-CHIKV-Dx assay.
Fig 1.
Phylogenetic tree of 141 CHIKV E1 gene sequences.
CHIKV E1 sequences are named by their corresponding GenBank accession number/strain/country of origin, with text being coloured according to their continent of isolation. The midpoint-rooted maximum likelihood phylogenetic trees were constructed using IQTREE2, which automatically incorporates the most appropriate nucleotide substitution model (TN + F + G4). The scale bar indicates the number of nucleotide substitutions per site. Bootstrap values above 80% are displayed next to the node of each clade and are coloured according to the figure legend.
Table 2.
Virus strains used in this study.
Fig 2.
Analytical sensitivity of Iso-CHIKV-Dx using synthetic RNA transcripts.
Assays were conducted against 10-fold dilutions of synthetic CHIKV RNA. The presence of two lines (control and test) on scanned images of the lateral flow strips is indicative of a positive result (far left). RNA concentration/µL and No template control (NTC) consisted of nuclease free H2O in place of RNA (left). Numeric analysis of normalised pixel density of bands contrasted with the white space (middle). Number of positive samples detected over number of samples tested along with calculated percentage accuracy of positive results for each dilution series (right).
Fig 3.
Analytical specificity of Iso-CHIKV-Dx using alphaviruses and co-circulating flaviviruses.
Specificity assays used synthetic RNA from CHIKV (PTC) and TRIzol extracted RNA from O’nyong nyong virus (ONNV), Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), Barmah Forest virus (BFV), Mayaro virus (MAYV), Zika virus (ZIKV), Japanese encephalitis virus (JEV) and all 4 Dengue virus (DENV) serotypes. CHIKV 104 copies/µL used as positive control; whilst No template control (NTC) consisted of nuclease free H2O in place of RNA (left). Numeric analysis of normalised pixel density of bands contrasted with the white space (middle). Number of positive samples detected over number of samples tested along with calculated percentage accuracy of positive results for each dilution series (right).
Fig 4.
Inactivation of CHIKV (Mauritius strain) culture using TNA-Cifer Reagent E.
Inactivation of 2.5 x 107 FFU/mL CHIKV (Mauritius strain) culture by TNA-Cifer Reagent E (TCE). Samples were mixed in at 1:1 and 2:1 ratio (sample to TCE) and incubated between 0 and 10 minutes at room temperature. *The limit of detection (LOD) for CHIKV (Mauritius strain) via immuno plaque assay.
Fig 5.
Rapid Iso-CHIKV-Dx of mock urine samples spiked with CHIKV (Mauritius).
A) Workflow, equipment needed and time frame of Iso-CHIKV-Dx. B) Sample processing conditions including sample to reagent ratio and processed sample dilution ratio. C) Sample description and quantities (NTC, non-template control; PTC, positive template control, synthetic CHIKV RNA transcripts 106 copies/µl); NVC, no virus control, urine. Scanned lateral flow strips showing test and control bands observable by naked eye. Normalised pixel densities (black values) from the displayed lateral flow strips. D) Comparative Ct values and copies/reaction quantified via TaqMan qRT-PCR. E) Workflow, equipment and time frame involved in conventional CHIKV RT-PCR diagnostic. (Images used to create this figure was obtained from Bio render-https://www.biorender.com).