Table 1.
Genetic differences between YFV-Asibi isolates and YFV-17D substrains.
Fig 1.
HA-Asibi but not 17D causes disease in hamsters.
Hamsters were infected by intraperitoneal injection with 1 × 105 focus-forming units (FFU) of HA-Asibi (yellow, n = 15), 17D (blue, n = 3), or mock (black, n = 3). Data represents a composite of 6 independent experiments. (A) Survival analysis, (B) weight change as a percentage of weight at the time of inoculation, and (C) viremia at 3 days post inoculation (dpi). Error bars show the mean standard error of mean (SEM). In C, the dashed line shows the limit of detection; statistical significance determined via unpaired two-tailed t test with Welch’s correction for unequal variance (****: p < 0.0001).
Fig 2.
Recombinant HA-YFVs generated via CPER recapitulate disease phenotypes observed with biological isolates.
(A) Schematic of the YFV genome showing amino acid differences in the polyprotein (yellow rectangle) between Asibi and 17D-204 (gray) along with mutations acquired during hamster adaptation of Asibi (purple with E-D155A shown in red). Overlapping DNA fragments amplified from YFV cDNA-containing plasmids are shown below, assembled in a circular polymerase extension reaction (CPER) with a linker sequence containing the CMV promoter and enhancer, bovine growth hormone polyadenylation (bGHpA) signal, and the self-cleaving hepatitis delta virus ribozyme (HDVr). The resulting circular CPER product was transfected into BHK-21 cells to rescue infectious recombinant YFV. The nomenclature for recombinant hamster-adapted YFV variants is shown in the box. (B) Heatmap showing the frequency of Asibi/17D mutations across biological and recombinant HA-YFVs. Viral genomes with an Asibi backbone were mapped to the Asibi reference (AY640589) and those with a 17D backbone were mapped to the 17D-204 reference (MN708488). Columns represent independently rescued virus stocks, and rows indicate individual amino acid differences between Asibi and 17D, grouped by CPER fragment, as well as all hamster adaptation mutations. Variant frequencies are color-coded: yellow indicates >50% Asibi variant, blue indicates >50% 17D variant. Frequencies of hamster adaptation mutations are shown using a red color scale. Grey boxes represent positions with low sequencing coverage (<100 reads). (C) Virus production from BHK-21 cells transfected with CPER product, quantified by focus-forming assay (FFU/mL: focus-forming units per mL of supernatant). (D) Growth kinetics of recombinant YFVs on Vero cells inoculated with passage-0 stocks (i.e., filtered and titered BHK-21 supernatants) at MOI of 0.1, quantified by focus-forming assay (E-G) 5-7-week-old female hamsters were inoculated intraperitoneally with 1 × 105 FFU of rHA1-Asibi (orange, n = 17), rHA7-Asibi (brown, n = 15), rHA1-17D (light blue, n = 5), or rHA7-17D (dark blue, n = 3). Control groups from Fig 1 are included for comparison. Data represents a composite of 9 independent experiments. (E) Survival analysis. (F) Body weight at 6 dpi, shown as a percentage of weight at the time of inoculation. (G) Viremia measured at 3 dpi, expressed as YFV genome copies per mL of serum (log₁₀). Dashed lines represent the lower limits of detection. Error bars show mean SEM. Statistical significance determined via one-way ANOVA with multiple comparisons. In F, comparisons were made to the mock-infected group. In G, rHA1-Asibi and rHA7-Asibi were compared to HA-Asibi, while rHA1-17D and rHA7-17D were compared to 17D. (**: p < 0.01; ****: p < 0.0001; ns: not significant).
Fig 3.
Attenuation determinant(s) of 17D map to Fragment 2a containing non-structural genes.
(A) Heatmap showing the frequency of Asibi/17D mutations across chimeric rHA1-YFVs in which individual CPER fragments were swapped between Asibi and 17D backbones; see description in Fig 2B for additional details. (B) Body weight of hamsters inoculated with 1 × 10⁵ FFU of each virus at 6 dpi, shown as a percentage of of starting weight. Data from animals inoculated with the recombinant parent virus are shown with bolded outlines. (C) Viremia measured at 3 dpi, expressed as genome copies per mL of serum (log₁₀). Dashed line in C represents the lower limit of detection. Error bars show mean SEM. Statistical significance was determined by one-way ANOVA with multiple comparisons, with Asibi-backbone chimeras compared to rHA1-Asibi, and 17D-backbone chimeras compared to rHA1-17D. (**: p < 0.01; ***: p < 0.001; ****: p < 0.0001; ns: not significant.).
Fig 4.
Attenuation determinant(s) of 17D map to NS2B.
(A) Heatmap showing the frequency of Asibi/17D mutations across chimeric rHA1-YFVs in which individual genes (E, NS1, NS2A, and NS2B) were swapped between Asibi and 17D backbones; see description in Fig 2B for additional details. (B) Percent body weight of hamsters inoculated with 1 × 10⁵ FFU of each virus at 6 dpi, shown as a percentage of starting weight. Data from animals inoculated with the recombinant parent virus are shown with bolded outlines. (C) Viremia measured at 3 dpi, expressed as genome copies per mL of serum (log₁₀). Dashed line in C represents the lower limit of detection. Error bars show Data represented as mean SEM. Statistical significance was determined by one-way ANOVA with multiple comparisons, with Asibi-backbone chimeras compared to rHA1-Asibi, and 17D-backbone chimeras compared to rHA1-17D. (*: p < 0.05; **: p < 0.01; ****: p < 0.0001; ns: not significant.).
Fig 5.
The NS2B-I109L mutation attenuates rHA1-Asibi.
(A) Heatmap showing the frequency of Asibi/17D mutations across rHA1-Asibi chimeras in which single or double 17D-NS2B mutations were introduced; see description in Fig 2B for additional details. (B) Percent body weight of hamsters inoculated with 1 × 105 FFU of each virus at 6 dpi, shown as a percentage of starting weight. Data from animals inoculated with the recombinant parent virus are shown with bolded outlines. (C) Viremia measured at 3 dpi, expressed as genome copies per mL of serum (log10). Dashed line in C represents the lower limit of detection. Error bars show Data represented as mean SEM. Statistical significance was determined by one-way ANOVA with multiple comparisons, with Asibi-backbone chimeras compared to rHA1-Asibi. (*: p < 0.05; **: p < 0.01; ns: not significant.).
Fig 6.
The NS2B mutations I37L and I109L cooperate to attenuate rHA7-Asibi.
(A) Heatmap showing the frequency of Asibi/17D mutations across rHA7-Asibi chimeras in which single or double 17D-derived NS2B mutations were introduced into rHA7-Asibi; see description in Fig 2B for additional details. (B) Percent body weight of hamsters inoculated with 1 × 10⁵ FFU of each virus at 5 dpi, shown as a percentage of starting weight. Data from animals inoculated with the recombinant parent virus are shown with bolded outlines. (C) Viremia measured at 3 dpi, expressed as genome copies per mL of serum (log₁₀). Dashed line in C represents the lower limit of detection. Error bars show Data represented as mean SEM. (D) Liver viral load, (E) alanine aminotransferase (ALT), and (F) prothrombin time (PT) measured at 5 dpi. Dashed lines represent the lower and upper limits of measurement range; the gray shaded area represents the reference range for hamsters, calculated from pooled data of mock-infected animals (see Methods for details). Error bars show Data represented as mean
SEM. Statistical significance was assessed by one-way ANOVA with multiple comparisons: Asibi-backbone chimeras were compared to rHA7-Asibi in panels B-C, and to HA-Asibi in panels D-F; in B and C, the two rHA7-17D viruses were compared using unpaired t-test (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; ns: not significant).