Analysis of host factors for internal ribosomal entry site-mediated translation of foot and mouth disease virus and classical swine fever virus: an in vivo evaluation in single- and double-knockout mice

Figures

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  Fig 1. (A) Targeting of Pkd1l3 in the mouse genome. Exons 1–34 are indicated. The genomic region targeted for knockout by Crispr/Cas9 editing is indicated with a red line surrounded by gRNA sequences B1, A1, A2, and B2. The positions of primers for genotyping (F1, F2, and R1) and RT-PCR (RTF1 and RTR1) are also shown. (B) Targeting of Usp31 in the mouse genome. Exons 1–16 are indicated. The genomic region targeted for knockout by Crispr/Cas9 editing is indicated with a red line surrounded by gRNA sequences B1, A1, A2, and B2. The positions of primers for genotyping (F1, R1, and R2) and RT-PCR (RTF1 and RTR1) are also shown. (C) PCR genotyping for Pkd1l3. The KO genome was detected as bands with 564 bp, and the WT genome was detected as bands with 734 bp. (D) PCR genotyping for Usp31. The KO genome was detected as bands with 800 bp, and the WT genome was detected as bands with 597 bp. (E) Detection of Pkd1l3 mRNA expression in WT and Pkd1l3−/− mice (left) and Usp31 mRNA expression in WT and Usp31−/− mice (right) by RT-PCR. Reactions without mouse genomic DNA are indicated as “no.”

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  Fig 2. (A) Establishment of the DKO mouse by crossbreeding and genotyping. All mice shown here are female. (B) Transition in body weight for WT (n = 9, 5 males, 4 females), Pkd1l3−/− (n = 7, 3 males, 4 females), Usp31−/− (n = 5, 4 males, 1 female), and DKO mice (n = 9, 6 males, 3 females) from birth. Statistical analysis was performed at each time point with one-way ANOVA and Dunnett analysis, and statistically significant points are indicated (***P < 0.001). (C) Mean body weight in 1-year-old male (left) and female (right) mice from WT, Pkd1l3−/−, Usp31−/−, and DKO mice (n = 3, for each type). Vertical bars indicate standard deviation (S.D.). P values are indicated, and a value less than 0.05 is significant.

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  Fig 3. (A) Detection of IRES activities from the bicistronic FMDV-IRES mRNA (left), CSFV-IRES mRNA (right), and EMCV-IRES mRNA (lower) in liver tissue from WT, Pkd1l3−/−, Usp31−/−, and DKO mice (n = 3, respectively). P values are indicated, and a value less than 0.05 is significant. (B) CSFV-replicon structure is indicated (left), and CSFV replication is represented by the measured FLU activity in mouse liver lysate normalized with protein concentration (μg/mL) and GAPDH (copies/mL), for WT, Pkd1l3−/−, Usp31−/−, and DKO mice (right) (n = 3, respectively). Vertical bars indicate standard deviation (S.D.). P values are indicated, and a value less than 0.05 is significant.

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  Fig 4. (A) Schematic diagram of the experiment. WT, Pkd1l3−/−, Usp31−/−, and DKO mice (n = 3, respectively; male ratio in each group ranges from 0% to 66%) were inoculated (ip) with EMCV (4 × 105 TCID50), and tissues were collected after sacrifice at 1 week post-inoculation for histopathological evaluation. (B) BW change ratio (%) is shown for WT, Pkd1l3−/−, Usp31−/−, and DKO mice inoculated with EMCV over a 1-week period. Vertical bars indicate S.D.

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  Fig 5. Histopathological images (hematoxylin and eosin staining) of mouse cerebral cortices (A), lungs (B), hearts (C), spleens (D), and livers (E) from WT, Pkd1l3−/−, Usp31−/−, and DKO mice inoculated with EMCV (bars = 20–100 µm) (n = 3, representative images are shown).

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  Fig 6. EMCV RNA amounts (copies/μg total RNA/GAPDH) (A) or viral titer (TCID50/mg total protein) (B) in brains, spleens, livers, lungs, and hearts from WT Pkd1l3−/−, Usp31−/−, and DKO mice (n = 3). A P-value of less than 0.05 is indicated as statistically significant.