denv4 h241  (ATCC)

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: 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: Microbiology Spectrum

Article Title: A laboratory-adapted and a clinical isolate of dengue virus serotype 4 differently impact Aedes aegypti life-history traits relevant to vectorial capacity

doi: 10.1128/spectrum.00001-25

Figure Lengend Snippet: DENV4 TVP/360 and LRV 13/422 infection dynamics in Ae. aegypti mosquitoes. ( A ) Experimental design. Mosquitoes were fed with human blood containing different concentrations of two strains of DENV4-TVP/360 and DENV4-LRV 13/422. ( B )Viral load was measured by plaque assay immediately following feeding (0), 7, 14, and 21 days post-infection (DPI). Each point represents one female mosquito. The number of samples tested is indicated above each group. Red dots represent the virus concentration in the blood offered to mosquitoes in each replicate experiment. Bars in each column represent medians. This figure represents the sum of three to four independent experiments for each group. N.d.: not determined. ( C ) Input is defined as the infectious particle concentration presented in the blood meal offered to mosquitoes (expressed as PFU/μL of blood—red text in the left Y-axis). Intensity is defined as the number of infectious particles per mosquito measured at 14 days post-infection (PFU/mosquito in the left Y-axis). Prevalence is defined as the percentage of infected mosquitoes at 14 days post-infection (% infection in the right Y-axis). Statistical analysis: intensity—Kruskal-Wallis test. Prevalence—Fischer’s exact test.

Article Snippet: Vero cells (ATCC, CCL-81) were used to quantify DENV4 TVP/360 and Vero E6 cells (ATCC, CRL-1586) were used to quantify DENV4 LRV 13/422 based on plaque optimization for each viral strain.

Techniques: Infection, Plaque Assay, Virus, Concentration Assay

Journal: Microbiology Spectrum

Article Title: A laboratory-adapted and a clinical isolate of dengue virus serotype 4 differently impact Aedes aegypti life-history traits relevant to vectorial capacity

doi: 10.1128/spectrum.00001-25

Figure Lengend Snippet: Survival curves of Ae. aegypti infected with DENV4 strain TVP/360 and DENV4 strain LRV 13/422. ( A ) Experimental scheme. Three to four days following adult emergence, females were fed with blood supplemented with two concentrations of DENV4 strains, as indicated in panel C (input—infectious doses—PFU/μL of blood). ( B through E ) Survival curves were performed at least twice in batches of 20 fully engorged females per cage. Survival was scored six times per week until all the mosquitoes died. Note that the survival of DENV4 (TVP/360) was already tested in much higher numbers in our previous work with similar results . Horizontal bars in panels B and D represent medians. Statistical analysis in —Kruskal-Wallis test.

Article Snippet: Vero cells (ATCC, CCL-81) were used to quantify DENV4 TVP/360 and Vero E6 cells (ATCC, CRL-1586) were used to quantify DENV4 LRV 13/422 based on plaque optimization for each viral strain.

Techniques: Infection

Journal: Microbiology Spectrum

Article Title: A laboratory-adapted and a clinical isolate of dengue virus serotype 4 differently impact Aedes aegypti life-history traits relevant to vectorial capacity

doi: 10.1128/spectrum.00001-25

Figure Lengend Snippet: Fecundity and fertility of Ae. aegypti mosquitoes infected with DENV4 strains TVP/360 and LRV 13/422. Female mosquitoes were fed with human blood containing two concentrations of DENV4 strains, TVP or LRV, as indicated in (input—infectious doses—PFU/μL of blood). ( A ) Each point represents fecundity (eggs/female) measured through the individual oviposition of each mosquito. ( B ) Fertility represents the percentage of viable eggs laid by each mosquito individually measured 7 days post-egg laying. Mosquitoes infected with DENV4 were compared with their respective controls: blood and mock (RBCs containing C6/36 cell supernatant instead of serum, as detailed in the Materials and Methods). The number of samples tested is indicated at the bottom of each column. Experiments were conducted at least three times and presented mean plus standard error. Statistical significance was determined using the Kruskal-Wallis test, followed by Dunn’s multiple comparison test.

Article Snippet: Vero cells (ATCC, CCL-81) were used to quantify DENV4 TVP/360 and Vero E6 cells (ATCC, CRL-1586) were used to quantify DENV4 LRV 13/422 based on plaque optimization for each viral strain.

Techniques: Infection, Comparison

Journal: Microbiology Spectrum

Article Title: A laboratory-adapted and a clinical isolate of dengue virus serotype 4 differently impact Aedes aegypti life-history traits relevant to vectorial capacity

doi: 10.1128/spectrum.00001-25

Figure Lengend Snippet: Comparative analysis of the induced flight activity of Ae. aegypti mosquitoes infected with DENV4 strains TVP/360 and LRV 13/422. ( A ) Experimental design of the INFLATE assay. See details in the Materials and Methods. Female mosquitoes were fed on blood containing the highest doses available for each strain—see (input). ( B through E ) The induced flight activity of each group was measured at 1, 7, 14, and 21 DPI. Mosquitoes challenged with DENV4 were compared with the mock/uninfected group. Each dot represents the inflate index (five mosquitoes per cage tested 10 times in a row). The experiments were conducted at least three times, and statistical significance was determined by the Tukey multiple comparison test and ANOVA.

Article Snippet: Vero cells (ATCC, CCL-81) were used to quantify DENV4 TVP/360 and Vero E6 cells (ATCC, CRL-1586) were used to quantify DENV4 LRV 13/422 based on plaque optimization for each viral strain.

Techniques: Activity Assay, Infection, Comparison