Journal: bioRxiv

Article Title: Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay

doi: 10.64898/2026.03.17.712358

Figure Lengend Snippet: (A) Faceted bar charts show Ct values measured across input copy numbers (copies per µL) for each virus—dengue virus (DENV1–4), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV). Bars summarize Ct at each concentration, with points indicating individual reactions (technical replicates); red diamonds denote the mean (error bars, where shown, indicate variability across replicates). Across DENV1–4, ZIKV, and JEV (triplicates), Ct values generally decrease as input concentration increases. ZIKV shows greater replicate-to-replicate variation at some concentrations, reflected by wider error bars compared to other targets. WNV and YFV were detected at 1,000 copies per µL; only a single concentration was tested for these viruses due to limited sample material. Together, these results indicate broad target coverage and reliable amplification across a wide input range for the main panel members. (B) Limit of detection (LOD) assessment of the designed primer-probe set. Prior to testing clinical specimens, LOD was evaluated using nucleic acids from 6 arboviruses (DENV1–4, ZIKV, and JEV) at 1, 10, 1000 copies per µL, with 12 technical replicates per dilution. The assay detected DENV3, DENV4, and JEV down to 1 copy per µL, whereas DENV1, DENV2, and ZIKV were consistently detected down to 10 copies per µL. Points represent individual reaction; a diamond indicates the mean Ct, and error bars indicate ±SD. ND indicates not detected (did not meet the ≥95% positivity criterion and therefore did not qualify as the LOD). (C) Paired comparison of Ct values between assays in clinical samples. Ct values obtained with the commercial kit (Com; circles) and the designed primer–probe set (Des; triangles) are shown for each target, with paired measurements from the same sample connected by dashed lines, for 100 positive samples and 50 positive samples (20 positive with Chikungunya virus [CHIKV] and 30 others). Box plots summarize the distributions (center line, median; box, interquartile range; whiskers, range). Relative to the commercial kit, the designed primer–probe set detected DENV1–4 significantly earlier (lower Ct; p = 0.0004 [n = 25], 0.0013 [n=25], <0.0001 [n=11] and <0.0001 [n=25] for DENV1–4, respectively) but detected ZIKV later (higher Ct; p < 0.0001 [n =14]). P values were calculated using a two-sided paired t-test on matched Com–Des Ct values for each target. Overall diagnostic performance is reported in Tables 1,2 .

Article Snippet: The assay also showed the CVs less than 5% in all viruses at both dilutions, ranging from 1.15%–3.20% for DENV4 and JEV detection at high concentration (1,000 copies/μL) ( ) In addition, analytical specificity was evaluated in terms of cross reactivity using other pathogens, including Influenza A virus strain A/PR/8/34 (ATCC; VR-95), Influenza B virus strain B/Lee/40 (ATCC; VR-1535), RSV strain A-2 (ATCC; VR-1540), CHIKV strain ROSS, Enterovirus 71 (EV-71) strain H (ATCC; VR-1432), Orientia tsutsugamushi strain KARP (accession number = SAMN51290297), Rickettsia typhi strain SI-typh0423 (accession number = SAMN51290298).

Techniques: Virus, Concentration Assay, Amplification, Comparison, Diagnostic Assay

Journal: bioRxiv

Article Title: Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay

doi: 10.64898/2026.03.17.712358

Figure Lengend Snippet: (A) Faceted bar charts show Ct values measured across input copy numbers (copies per µL) for each virus—dengue virus (DENV1–4), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV). Bars summarize Ct at each concentration, with points indicating individual reactions (technical replicates); red diamonds denote the mean (error bars, where shown, indicate variability across replicates). Across DENV1–4, ZIKV, and JEV (triplicates), Ct values generally decrease as input concentration increases. ZIKV shows greater replicate-to-replicate variation at some concentrations, reflected by wider error bars compared to other targets. WNV and YFV were detected at 1,000 copies per µL; only a single concentration was tested for these viruses due to limited sample material. Together, these results indicate broad target coverage and reliable amplification across a wide input range for the main panel members. (B) Limit of detection (LOD) assessment of the designed primer-probe set. Prior to testing clinical specimens, LOD was evaluated using nucleic acids from 6 arboviruses (DENV1–4, ZIKV, and JEV) at 1, 10, 1000 copies per µL, with 12 technical replicates per dilution. The assay detected DENV3, DENV4, and JEV down to 1 copy per µL, whereas DENV1, DENV2, and ZIKV were consistently detected down to 10 copies per µL. Points represent individual reaction; a diamond indicates the mean Ct, and error bars indicate ±SD. ND indicates not detected (did not meet the ≥95% positivity criterion and therefore did not qualify as the LOD). (C) Paired comparison of Ct values between assays in clinical samples. Ct values obtained with the commercial kit (Com; circles) and the designed primer–probe set (Des; triangles) are shown for each target, with paired measurements from the same sample connected by dashed lines, for 100 positive samples and 50 positive samples (20 positive with Chikungunya virus [CHIKV] and 30 others). Box plots summarize the distributions (center line, median; box, interquartile range; whiskers, range). Relative to the commercial kit, the designed primer–probe set detected DENV1–4 significantly earlier (lower Ct; p = 0.0004 [n = 25], 0.0013 [n=25], <0.0001 [n=11] and <0.0001 [n=25] for DENV1–4, respectively) but detected ZIKV later (higher Ct; p < 0.0001 [n =14]). P values were calculated using a two-sided paired t-test on matched Com–Des Ct values for each target. Overall diagnostic performance is reported in Tables 1,2 .

Article Snippet: Reference viruses were obtained from the American Type Culture Collection (ATCC), including DENV1 strain Hawaii (ATCC VR-1856), DENV2 strain TH-36 (ATCC VR-1810), DENV3 strain H87 (ATCC VR-3380), DENV4 H241 (ATCC VR-1490), and ZIKV strain PRVABC59 (ATCC VR-1843).

Techniques: Virus, Concentration Assay, Amplification, Comparison, Diagnostic Assay

Journal: bioRxiv

Article Title: Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay

doi: 10.64898/2026.03.17.712358

Figure Lengend Snippet: (A) Faceted bar charts show Ct values measured across input copy numbers (copies per µL) for each virus—dengue virus (DENV1–4), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV). Bars summarize Ct at each concentration, with points indicating individual reactions (technical replicates); red diamonds denote the mean (error bars, where shown, indicate variability across replicates). Across DENV1–4, ZIKV, and JEV (triplicates), Ct values generally decrease as input concentration increases. ZIKV shows greater replicate-to-replicate variation at some concentrations, reflected by wider error bars compared to other targets. WNV and YFV were detected at 1,000 copies per µL; only a single concentration was tested for these viruses due to limited sample material. Together, these results indicate broad target coverage and reliable amplification across a wide input range for the main panel members. (B) Limit of detection (LOD) assessment of the designed primer-probe set. Prior to testing clinical specimens, LOD was evaluated using nucleic acids from 6 arboviruses (DENV1–4, ZIKV, and JEV) at 1, 10, 1000 copies per µL, with 12 technical replicates per dilution. The assay detected DENV3, DENV4, and JEV down to 1 copy per µL, whereas DENV1, DENV2, and ZIKV were consistently detected down to 10 copies per µL. Points represent individual reaction; a diamond indicates the mean Ct, and error bars indicate ±SD. ND indicates not detected (did not meet the ≥95% positivity criterion and therefore did not qualify as the LOD). (C) Paired comparison of Ct values between assays in clinical samples. Ct values obtained with the commercial kit (Com; circles) and the designed primer–probe set (Des; triangles) are shown for each target, with paired measurements from the same sample connected by dashed lines, for 100 positive samples and 50 positive samples (20 positive with Chikungunya virus [CHIKV] and 30 others). Box plots summarize the distributions (center line, median; box, interquartile range; whiskers, range). Relative to the commercial kit, the designed primer–probe set detected DENV1–4 significantly earlier (lower Ct; p = 0.0004 [n = 25], 0.0013 [n=25], <0.0001 [n=11] and <0.0001 [n=25] for DENV1–4, respectively) but detected ZIKV later (higher Ct; p < 0.0001 [n =14]). P values were calculated using a two-sided paired t-test on matched Com–Des Ct values for each target. Overall diagnostic performance is reported in Tables 1,2 .

Article Snippet: Reference viruses were obtained from the American Type Culture Collection (ATCC), including DENV1 strain Hawaii (ATCC VR-1856), DENV2 strain TH-36 (ATCC VR-1810), DENV3 strain H87 (ATCC VR-3380), DENV4 H241 (ATCC VR-1490), and ZIKV strain PRVABC59 (ATCC VR-1843).

Techniques: Virus, Concentration Assay, Amplification, Comparison, Diagnostic Assay

Journal: Journal of Virology

Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

doi: 10.1128/JVI.02235-13

Figure Lengend Snippet: Relative RT-qPCR data from SGE treatments of mouse embryonic fibroblasts followed by infection with multiple dengue virus serotypes and isolates a

Article Snippet: Mouse-adapted dengue virus type 1 (DENV1) Hawaii strain was obtained from the ATCC (VR1254).

Techniques: Infection, Virus