Fig 1.
Inhibition of YFV-17D cap0 and -Asibi cap0 in different mosquito cells.
(A) Plaque phenotypes of YFV-17D cap1 and cap0 and (D) YFV-Asibi cap1 and cap0 after performing an infectious center assay in BHK cells. At 3 days post-infection, cells were fixed and stained by crystal violet. (B, C) Growth kinetics of YFV-17D cap1 and cap0 in C6/36 and Aag2 cells. (E, F) Growth kinetics of YFV-Asibi cap1 and cap0 in C6/36 and Aag2 cells. Cells were infected at a multiplicity of infection (MOI) of 0.01 and viral titers were measured at 0, 3, 5, 7, and 10 days post-infection by titration on BHK cells. Data represent Mean ± SD of triplicates. Dashed lines: detection limit.
Fig 2.
YFV-Asibi NS4A-5 promotes replication of the cap0 virus.
The colors represent the sequence source (green: YFV-17D; yellow: YFV-Asibi), and shared sequences are those that do not differ between Asibi and 17D (grey). The red star represents the E218A exchange in the NS5 protein. A) Growth kinetics of Asibi/17D SP cap1 and cap0 in C6/36 cells. B) Growth kinetics of 17D/Asibi SP cap1 and cap0 in C6/36 cells. C) Growth kinetics of 17D/Asibi NS1-4A* cap1 and cap0 in C6/36 cells. D) Growth kinetics of 17/Asibi NS4A**-3’UTR cap1 and cap0 in C6/36 cells. E) Growth kinetics of 17D/Asibi NS5 cap1 and cap0 in C6/36 cells. F) Growth kinetics of Asibi/17D NS5 cap1 and cap0 in C6/36 cells. G) Growth kinetics of 17D/Asibi 2K-NS5 cap1 and cap0 in C6/36 cells. H) Growth kinetics of 17D/Asibi NS4A-NS5 cap1 and cap0 in C6/36 cells. The cells were infected at an MOI of 0.01. Viral titers were measured at 0, 3, 5, 7, and 10 days post-infection by titration on BHK cells. Data represent Mean ± SD of at least triplicates. Dashed lines: detection limit.
Fig 3.
Attenuation of YFV 2’-O-MTase deficient mutants is independent of Dcr-2.
(A, B) Parental Aag2 cells or Aag2 Dcr-2 knockout cells were infected with YFV-17D cap1 and cap0 or (C, D) with YFV-Asibi cap1 and cap0 at an MOI of 0.01. Viral titers were measured at 0, 3, 5, and 7 days post-infection by titration on BHK cells. Data represent Mean ± SD of triplicates. Dashed lines: detection limit.
Fig 4.
Translation efficiency of a replication-incompetent cap0 reporter YFV genome is reduced in mosquito cells.
(A) Schematic presentation of the replication-incompetent reporter YFV-17D full-length genomes. The RNA-dependent RNA polymerase activity of YFV was knocked out via mutation (GDD > AAG). In vitro transcribed YFV genomes were either modified at their 5’ end by a cap1 structure (WT/AAG genome) or a cap0 structure (E218A/AAG mutant encoding genome). Rluc: Renilla luciferase (yellow box). (B, C) C6/36 cells were electroporated with cap1 or cap0 reporter YFV-17D genome transcripts expressing Rluc. At 4 h, 8 h, and 22 h post-electroporation, cells were either lysed to measure Rluc activity (B) or lysed for RNA isolation to determine the YFV genome copies (C). YFV-17D genome copies were normalized to the housekeeping gene RPS7, with the numbers of YFV-17D genome copies depicted relative to 106 RPS7 gene copies. The means ± SD of n = 3 independent experiments each analyzed with three biological replicates are shown. Individual values are visualized as dots. The Mann-Whitney test was used to calculate statistical significance (**** p ≤ 0.0001, ns = not significant).
Fig 5.
Oral infection of mosquitoes with YFV-Asibi cap0 results in reduced infection and dissemination rates.
A, B) Female mosquitoes were orally fed with an infectious blood meal containing 1 x 107 PFU/ml virus. Engorged mosquitoes were kept at 28°C and 80% humidity and were dissected at the indicated time points. A) Viral genome copies (left) and infection rates (right) in the mosquitoes’ carcasses (mosquitoes w/o legs and wings). The viral genome copies were measured by RT-qPCR, and the infection rates were calculated as the number of mosquitoes positive in the carcasses in relation to the total number of examined mosquitoes. B) Viral genome copies in legs plus wings (left) and dissemination rates (right) into secondary tissues. Viral genome copies were determined by RT-qPCR of legs plus wings and dissemination rates were calculated as the number of mosquitoes containing viral RNA in legs plus wings in relation to the number of infected carcasses. Each point represents a single female mosquito. The blue lines indicate the median titers. Dashed line: detection limit.
Fig 6.
Oral infection with YFV-Asibi cap0 leads to lower viral loads in the midguts largely diminishing dissemination to secondary tissues.
A, B, C) Female mosquitoes were orally fed with an infectious blood meal containing 1 x 107 PFU/ml virus. Engorged mosquitoes were kept at 28°C and 80% humidity and were dissected at the indicated time points. Viral genome copies were determined by RT-qPCR in the isolated midguts (A), the legs plus wings (B), and the remaining carcasses (carcasses w/o midgut and w/o legs plus wings) (C). Each point represents a single female mosquito. The blue lines indicate the median titers. Dashed line: detection limit, n.d.: not determined.
Fig 7.
YFV-Asibi cap0 grows slower and to reduced viral titers in secondary tissues after intrathoracic injection.
A, B) Aedes aegypti mosquitoes were intrathoracically infected with 300 PFU YFV-Asibi cap1 or cap0. At 0, 3, and 7 days post-injection, viral genome copies were determined in the carcasses (mosquitoes w/o legs and wings) (A) and in the legs plus wings (B). The blue lines indicate the median titers and stars the significance calculated by the Mann-Whitney test (**** p ≤ 0.0001; ns = not significant). Dashed line: detection limit, n.d.: not determined.