CHIKV 37997-mCherry infection induces antibody responses against mCherry.

<p>Eight weeks old C57BL/6J mice were infected with indicated amount of CHIKV 37997-mCherry (5 mice per group) or prime immunized with 100 μg of mCherry protein followed by boosting with 50 μg of mCherry protein 2 weeks later (8 mice). (A) Titers of anti-mCherry antibodies induced in mice at 14 and 34 days post-infection were measured by end-point ELISA. The dashed line indicates the detection limit. (B) Neutralization of CHIKV 37997-mCherry by serum from immunized mice.</p

Presentation of mCherry on surface of virus particles.

Single-particle cryoEM reconstructions of CHIKV 37997-mCherry (A, B & D) and CHIK virus like particle (VLP) (C). (A) Surface-shaded and (B) cross-section views of CHIKV 37997-mCherry density map show a T = 4 icosahedral structure. (D) The comparison between CHIKV 37997-mCherry and CHIK VLP density maps reveals mCherry proteins on virus surface that are labeled in magenta.</p

Generation of a replication-competent chimeric CHIKV 37997-mCherry.

<p><b>(A)</b> Schematic illustration of the viral genomes of CHIKV 37997 <i>vs</i> CHIKV 37997-mCherry. <b>(B)</b> Localization of mCherry-E2 in CHIKV 37997-mCherry infected cells. Vero cells were infected with CHIKV 37997-mCherry and imaged with confocal fluorescence microscope. <b>(C)</b> Viral growth curves of CHIKV 37997 <i>vs</i> CHIKV 37997-mCherry. BHK21 cells were infected with CHIKV 37997 or CHIKV 37997-mCherry at a Multiplicity of Infection (MOI) 0.1. Virus release at indicated time points post-infection was measured by qRT-PCR analysis of viral genomic RNA and plaque assay of infectious viral particles in culture supernatant. The dashed line indicates the detection limit. Statistical significance was determined by ANOVA with a Sidak’s multiple comparisons test (***p<0.0002, ****p<0.0001) (n = 3). <b>(D)</b> Representative plaques formed by CHIKV 37997 and CHIKV 37997-mCherry on Vero cell monolayer. <b>(E)</b> Purified CHIKV 37997-mCherry was separated on a SDS-PAGE gel followed by Coomassie-staining and Western blot. mCherry-E2, E1 and capsid bands are indicated.</p

Pathogenesis comparison between the vaccine strain CHIKV 181/25 and mCherry tagged CHIKV 37997-mCherry.

<p>Survival of IFNαR-/- mice was monitored for 3 weeks following inoculation of (A) 10 PFU (B) 10² PFU (C) 10³ PFU and (D) 10⁴ PFU of indicated viruses in the left rear footpad (5 mice per group).</p

Attenuation of CHIKV 37997-mCherry in immunocompromised mice.

<p>IFNαR-/- mice (5 per group) were injected with 10 PFU of indicated viruses in the left rear footpad. (A) survival of mice was monitored, and (B) Left (circles) and right (squares) rear footpad sizes (width x height) were measured for 4 weeks. Data shown are pooled results of three repeated experiments.</p

Neutralization of CHIKV by serum from infected IFNαR-/- mice.

<p>Infectivity of CHIKV vaccine strain 181/25 (A&B) or 37997-mCherry (C&D) was neutralized by serum from surviving IFNαR-/- mice <b>(A&C)</b> at 3 weeks after CHIKV 181/25 and CHIKV 37997-mCherry infection and <b>(B&D)</b> at 11 days post-challenge with CHIKV LR.</p

Attenuation of CHIKV 37997-mCherry in wild-type mice.

<p>Three weeks old C57BL/6 mice were injected with 10³ PFU of indicated viruses at left rear footpad. (<b>A</b>) Rear footpad sizes (width x height) were measured at indicated time points, (<b>B</b>) virus titers in blood were quantified by qRT-PCR at 3 days post-infection, and (<b>C</b>) virus titers in rear ankles were quantified by qRT-PCR at 28 days post-infection. The dashed lines indicate the detection limits. Statistical significance was determined by ANOVA with a Turkey’s multiple comparisons test (*P<0.0332; **P<0.0021; ***p<0.0002, ****p<0.0001) (n = 5–7).</p

CHIKV 181/25 and CHIKV 37997-mCherry protected IFNαR-/- mice from lethal pathogenesis of CHIKV LR.

IFNαR-/- mice that survived CHIKV 181/25 (4 mice survived 10 PFU, 5 mice survived 100 PFU and 3 mice survived 1,000 PFU) and CHIKV 37997-mCherry (5 mice survived 10 PFU, 3 mice survived 100 PFU and 3 mice survived 1,000 PFU) infection were challenged with 10 PFU of CHIKV LR at three weeks post-infection. Mice without previous infection were used as controls. (A) Survival of IFNαR-/- mice post-challenge was monitored. (B) At 48 hours post-challenge, viruses in blood were quantified by qRT-PCR. The dashed line indicates the detection limit.</p

Antibody responses induced by vaccine strain 181/25 and 37997-mCherry.

<p>Anti-CHIKV (left) and anti-mCherry (right) total IgG antibody titers in surviving IFNαR-/- mice (A) at 3 weeks after CHIKV 181/25 and CHIKV 37997-mCherry infection and (B) at 11 days post-challenge with CHIKV LR were measured by end-point ELISA. The dashed lines indicate the detection limits. Statistical significance was determined by ANOVA with a Sidak’s multiple comparisons test (*P<0.0332; **P<0.0021; ***p<0.0002) (n = 3–5).</p

Attenuation of CHIKV 37997-mCherry at high dose in wild-type mice.

<p>Three week old C57BL/6 mice were injected with 10⁶ PFU of indicated viruses in the left rear footpad. (<b>A</b>) Rear footpad sizes (width x height) were measured at indicated time points, (<b>B</b>) virus titers in blood were quantified by qRT-PCR at 3 days post-infection, and (<b>C</b>) virus titers in rear ankles were quantified by qRT-PCR at 28 days post-infection. The dashed lines indicate the detection limits. Statistical significance was determined by ANOVA with a Turkey’s multiple comparisons test (*P<0.0332; **P<0.0021; ***p<0.0002, ****p<0.0001) (n = 5).</p