First Identification and Pathogenicity Evaluation of an EV-G17 Strain Carrying a Torovirus Papain-like Cysteine Protease (PLCP) Gene in China

Figure 1. The CPEs, plaque formation, and growth curves of the EV-G/YN23/2022 strain. (A) The CPEs of EV-G/YN23/2022 in various cell lines (scale bar = 100 μm). (B) Plaque formation of EV-G/YN23/2022 in BHK-21 cells (stained with crystal violet at 72 hpi). (C) Growth kinetics of EV-G/YN23/2022 in different cell lines with an MOI of 0.01.

Figure 2. Phylogenetic analysis based on the nucleotide sequences of the VP1 gene (A) and full-length genomes (B) of EV-G strains. The tree was constructed by the maximum likelihood method using MEGA 6.06 with the best-fitting model ‘GTR+G+I’. The numbers on the branches are bootstrap values (percent) from 1000 replicates. The EV-G/YN23/2022 strain isolated in this study is marked with a red dot, and the previously published EV-G17 strains are labeled with a blue box. The sequences of other representative EV-Gs are indicated as ‘Strain/Country/Year/(GenBank accession number)’.

Figure 3. Genome recombination analyses of EV-G/YN23/2022. (A) Similarity plot analysis of the genome sequences of EVG 02/NC_CHI (G1) (brown curve), KNU-1835 (G1) (blue curve), Ishi-Ya4 (G17) (purple curve), UKG/410/73 (G1) (red curve), and EV-G/YN23/2022 as query sequences; the y-axis presents the percent identity with a window size of 200 bp and a step size of 20 bp. The vertical line indicates the regions where a recombination event may occur. (B) The recombination breakpoint analysis of EVG 02/NC CHI (G1) vs. KNU-1835 (G1) (light green curve), EVG 02/NC CHI (G1) vs. EV-G/YN23 (G17) (yellow curve), and KNU 1835 (G1) vs. EV-G/YN23 (G17) (purple curve). (C) The phylogenetic tree constructed by the neighbor-joining method in MEGA 6.06 based on the concatenated regions (positions 1–708 and 3383–8033) of the EV-G/YN23/2022 genome. (D) The phylogenetic tree constructed by the neighbor-joining method in MEGA 6.06 based on the 709–3382 region of the EV-G/YN23/2022 genome.

Figure 4. The rectal temperature, weight gain, and clinical symptoms of EV-G/YN23/2022-infected piglets and mock-inoculated controls. (A) Rectal temperatures of piglets infected with EV-G/YN23/2022 and mock-inoculated piglets; mean ± SD temperature (°C) is shown, and the fever cut-off value was set at 39.5 °C. Statistical analyses were performed using the t-test. * p < 0.05. (B) Average daily weight gain of the piglets inoculated with EV-G/YN23/2022 and mock-inoculated piglets; mean ± SD weight gain (kg) are shown. Statistical analyses were performed using the t-test. * p < 0.05. (C) Mild diarrhea observed from EV-G/YN23/2022-challenged piglets at 8 dpi. (D) Skin rashes of the EV-G/YN23/2022-challenged piglets at 7, 9, and 12 dpi.

Figure 5. Gross lesions of piglets challenged with EV-G/YN23/2022 and mock-inoculated piglets. Mild hemorrhaging in the small intestine and mesenteric lymph node (green rectangle) of the challenged animals were observed.

Figure 6. Histopathological analysis of the piglets challenged with EV-G/YN23/2022 and mock-inoculated controls. (A) Hematoxylin and eosin (H&E) staining of the small intestine showed local exfoliation and defects in the epithelial mucosae (blue arrow), and the irregularly arranged and enlarged space of the lamina propria (yellow arrow) in the infected pigs when compared to the control group (scale bar = 200 μm). (B) In situ hybridization showing the distribution of viral nucleic acids in the small intestine, mesenteric lymph node, and brain (cerebellum) tissue sections (green arrow) of the piglets challenged with EV-G/YN23/2022, and mock-inoculated piglets showed no signal (scale bar = 50 μm).

Figure 7. Viral RNA copies in samples (fecal swab, heart, liver, spleen, lung, kidney, brain, small intestine, mesenteric lymph node, and stomach) and the serum neutralization antibody titers of the piglets challenged with EV-G/YN23/2022. (A) Fecal virus shedding in EV-G/YN23/2022-challenged piglets from 0 to 21 dpi. (B) Virus loads in different organs of EV-G/YN23/2022-challenged piglets at 21 dpi. (C) nAbs against EV-G/YN23/2022 of challenged piglets; the serum neutralization titer was defined as the highest serum dilution inhibiting 50% of virus growth.

Figure 8. The expression profiles of multiple immune-related cytokine genes (IL-1β, IL-6, IL-18, TNF-α, IFN-α, IFN-β, IFN-λ3, ISG-15, and IRF-7) in the PBMCs of the EV-G/YN23/2022-inoculated piglets at 2, 4, 6, 9, 12, 15, 18, and 21 dpi. The relative expression levels of the target genes were normalized to the β-actin gene and calculated using the 2−ΔΔCt method. The y-axis represents the fold change of the target gene expression in the experimental group vs. that of the control group. Statistical analyses were performed using the t-test. * p < 0.05; *** p < 0.001.

Figure 9. The protein concentration of IL-1β, IL-6, IL-18, TNF-α, IFN-α, IFN-β, and IFN-λ3 in the serum of the piglets challenged by EV-G/YN23/2022. Statistical analyses were performed using the t-test. * p < 0.05; *** p < 0.001.