Reporter Coxsackievirus A5 Expressing iLOV Fluorescent Protein or Luciferase Used for Rapid Neutralizing Assay in Cells and Living Imaging in Mice

Figures

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  Figure 1. Construction and rescue of recombinant mouse-adapted CV-A5-611. (A) Genome organization and construction strategy of the infectious cDNA clone of rCV-A5-611 are shown. Fragment A represents linearized pBR322 vector. Two PCR fragments B (T7 promotor + nucleotides 1–3325) and C (nucleotides 3306–7402 + polyA25) represented by black lines were amplified from the first strand cDNA by PCR with specific primer pairs. (B) The amplified fragments A, B and linearized plasmid were detected by agarose gel electrophoresis. (C) Detection of the in vitro transcribed recombinant virus RNA from an insert-positive plasmid after the denature at 65 °C, 5 min. (D) Viral RNAs were extracted from RD cells infected with rescued rCV-A5-611 and parental CV-A5-611 and PCR fragments amplified using specific primers of virus genes were detected in gel electrophoresis. Mock represents RT-PCR using RNA extracted from uninfected cells. (E) Virus plaque assay of rescued rCV-A5-611 and parental virus CV-A5-611 in RD cells on day 3 post infection.

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  Figure 2. Schematic representation for construction and rescue of the CV-A5 reporter viruses. (A) Construction strategy of the CV-A5-611-DsRed, CV-A5-611-iLOV and CV-A5-611-Nluc reporter viruses. Fragment A represents linear pBR322 vector. Fragments D (T7 promotor + nucleotides 1–3325) and E (nucleotides 3326–7402+ polyA25) indicated by lines were amplified from the first strand cDNA by specific primer pairs. Fragment F was amplified by PCR that represents DsRed, iLOV or Nluc gene with homological arms, respectively, for linking to fragments D and E. (B) DsRed or iLOV or Nluc gene was inserted between cleavage site of VP1/2A of 2A protease. The TSITTTGKFGQQ represents amino acid residues up- and downstream of 2A protease cleavage site TG that was introduced into pBR322-CV-A5-611 for expression of the exogenous reporter genes. (C) The r611-iLOV passages P1 and P10 were detected. (D) Bioluminescence induced by r611-Nluc P1 and P10 was quantified after adding nano-Glo substrate solution. The background bioluminescence was defined by the cut-off value induced in the uninfected cell.

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  Figure 3. Genetic stability of tagged CV-A5 recombinants. (A) RNAs were extracted from cells transfected with viral RNA (P0) and infected with passage P1 of r611-DsRed, infected with passages 1/10 of r611-iLOV and r611-Nluc and parental r611. PCR fragments were amplified from reversely transcribed RNAs using specific primers to cover the exogenous gene and partial CV-A5 sequences and analyzed on agarose gel. (B) Viral structural proteins of the rescued r611-iLOV P1, P10, r611-Nluc P1, P10 and r611 recombinants in both infected cells and cultured supernatants were analyzed by Western blotting using anti-CV-A5 antiserum. The supernatants were detected directly while the cell lysates were diluted 10 fold before loading. VP0, VP1, VP2 and VP3 are indicated with arrows. Molecular weight markers are indicated with lines (in base pair and kilodalton). The loading control was included using an anti-β-Actin antibody. Parental virus r611-infected and mock-infected RD cells were used as controls.

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  Figure 4. Growth ability and virulence of r611-iLOV and r611-Nluc. (A) Comparison of the growth kinetics of recombinants was performed on RD cells infected with r611-Nluc, r611-iLOV and r611 at the same M.O.I. (MOI = 10) *, **, ***, and **** indicate p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 (using one-way ANOVA), respectively. (B) Plaque formation of r611, r611-iLOV and r611-Nluc was observed on RD cells on day 3 post infection. (C) The 14-day-old suckling Kunming mice were infected with r611, r611-iLOV and r611-Nluc and their survival rates were calculated 14 days post infection. (D,E) Mean clinical scores and body weight changes of the recombinants were recorded during the 14-day observation period post infection.

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  Figure 5. Microneutralization assay based on r611-iLOV and r611-Nluc for rapid analysis of neutralizing antibody levels in serum samples. (A) Schematic representation of a method designed for performing antibody neutralization assay in a format of 96-well plates (left panel). For example, serum neutralizing titers were determined to be 384 by 4 times repeated experiments. Green and white wells represent fluorescence, caused by infection of r611-iLOV, with CPE and fluorescence-free without CPE, respectively. All viruses were back-titrated to make sure that viruses of 30-320 CCID50 were used (right panel). Mock represents uninfected normal cell controls. (B) The rabbit serum titers were detected by the r611-iLOV, r611-Nluc and r611 using iLOV fluorescence, luciferase and conventional neutralizing assays, respectively, for comparison. (C) The mouse serum titers were determined by the r611-iLOV, r611-Nluc and r611. Each symbol represented a mouse. The titers of rabbit and mouse antisera based on r611 (assayed by conventional method) were calculated on day 7, while those based on r611-iLOV and r611-Nluc on day 2 post-neutralizing. One-way ANOVA was used for statistical significance analysis.…

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  Figure 6. In vivo imaging of CV-A5-r611-iLOV and CV-A5-r611-Nluc infected Kunming mice. (A) Fluorescence measurement of Kunming mouse infected with r611-iLOV at days 1, 3 and 6 post infection. Mock represents the uninfected mouse inoculated with equal volume of PBS. Color scale, Min = 9.50 × 107, Max = 1.00 × 109. (B) Bioluminescence measurement of Kunming mouse infected with r611-Nluc at days 1, 3 and 6 post infection using furimazine substrate. Color scale, Min = 3.00 × 104, Max = 3.00 × 105. Mock represents the uninfected mouse inoculated with equal volume of PBS and substrate. Radiance, p/sec/cm2/sr. D, dorsal; V, ventral.