A Neonatal Murine Model for Caprine Enterovirus Infection and the Viral Tissue Tropism

Figures

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  Figure 1. ICR suckling mice are susceptible to CEV-JL14 infection. Three-day-old ICR, BALB/c, and Kunming suckling mice were injected subcutaneously with 50 μL virus suspension containing 2 × 108 TCID50 of CEV-JL14. The suckling mice injected subcutaneously with the same volume DMEM were used as control groups. RNAs were extracted from mixed tissue samples of mice infected by CEV-JL14 and used to amplify CEV-JL14 virus gene fragments. Fragments with expected sizes were only detected in ICR (Lane 1), while no fragments were detected in BALB/c (Lane 2) and Kunming (Lane 3) suckling mice. Lane 4–Lane 6 were the results from non-infected mice of ICR, BALB/c, and Kunming strains. Lane 7 and Lane 8 were positive and negative PCR controls, respectively. M stands for the DNA ladder.

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  Figure 2. Determination of CEV-JL14 infective routes. Three-day-old ICR suckling mice in experimental groups were injected with 2 × 108 TCID50 of CEV-JL14 virus by intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and intranasal administration. CEV-JL14 was detected in suckling mice inoculated with CEV-JL14 via all infective routes (Lane 1–5). No fragments were detected in control groups (Lane 6–10). Lane 1: intraperitoneal injection; Lane 2: intramuscular injection; Lane 3: subcutaneous injection; Lane 4: oral administration; Lane 5: intranasal administration. Lane 11 and Lane 12 were positive and negative control, respectively. M stands for the DNA ladder.

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  Figure 3. The minimal infective dose for CEV-JL14 to ICR suckling mice. Three-day-old ICR suckling mice were injected with 2 × 104, 2 × 106, and 2 × 108 TCID50 CEV-JL14 by subcutaneous injection, respectively. Mixtures of tissue samples were collected at 5 dpi for RT-PCR to amplify CEV-JL14 genomic fragments. No fragment was observed in mice infected by 2 × 104 TCID50 viruses (Lane 1). Fragments from ICR suckling mice inoculated with 2 × 106 (Lane 2) and 2 × 108 TCID50 (Lane 3) CEV-JL14 were amplified with expected size. No fragments were detected in the control groups (Lane 4). Lane 5 and Lane 6 were positive and negative control, respectively. M stands for the DNA ladder.

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  Figure 4. Recovery of the virus from infected mice. Tissue samples from infected mice were collected, homogenized, and used to inoculate the Vero cells. Tissue samples from non-infected mice were used as control. Typical cytopathic effects (CPE) were usually observed in the 36–48 h post inoculation (A). No CPEs were noticed in Vero cells inoculated with normal mice tissue samples (B). IPMA revealed the strong signals in Vero cells infected with CEV-JL14 (C). No signals were observed in the control groups (D). Three-day-old ICR suckling mice were injected with 2 × 106 TCID50 CEV-JL14 by subcutaneous injection. Tissues including heart, liver, spleen, lung, kidney, intestine, brain, and muscle were harvested after 5 dpi for RT-qPCR. Viral loads in the corresponding tissues from three infected mice (5 dpi) by CEV-JL14 virus were quantitated and represented as average virus copies of log 10/g tissue (E).

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  Figure 5. Persistence of CEV-JL14 infection revealed in ICR suckling mice. Three-day-old ICR suckling mice were injected with 2 × 106 TCID50 CEV-JL14 by subcutaneous injection. Suckling mice in the control groups were injected with DMEM. Tissues samples were collected at 5, 11, 14, 16, 18, and 21 dpi, respectively. RT-PCR were performed to amplify CEV-JL14 genomic fragments. Fragments with expected size were present at 5 (Lane 1), 11 (Lane 2), 14 (Lane 3), and 16 dpi (Lane 4) in infected mice. No fragments were observed at 18 (Lane 5) and 21 dpi (Lane 6) in infected mice. Similarly, no fragments were seen at 5 (Lane 9), 11 (Lane 10), 14 (Lane 11), 16 (Lane 12), 18 (Lane 13), and 21 dpi (Lane 14) in control mice. Lane 7 and Lane 8 were positive and negative control, respectively. M stands for the DNA ladder.

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  Figure 6. Histopathological lesions revealed in mice infected with CEV-JL14. Three-day-old ICR suckling mice were injected with 2 × 106TCID50 CEV-JL14 via different infective routes. The mice were euthanized 5 dpi. Tissue samples were collected and processed for histopathological examination using H&E staining. The representative results are shown. Obvious histopathological lesions and inflammatory cell infiltration were observed in the heart (B), liver (D), spleen (F), lung (H), kidney (J), intestine (L), brain (N), and muscle (P), as indicated by arrow in comparison with corresponding tissues in control mice (A,C,E,G,I,K,M,O). Tissues including the heart (B), liver (D), and kidney (J) were obtained from mice inoculated via intramuscular injection. Spleen (F) and muscle (P) were from mice inoculated via intraperitoneal injection. Lung (H) was harvested from mice inoculated via intranasal administration. Intestine (L) was collected from mice inoculated via subcutaneous injection. Brain (N) was from mice inoculated via oral administration.

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  Figure 7. Representative results showing the viral tissue tropism in mice infected with CEV-JL14. Three-day-old ICR suckling mice were injected with 2 × 106 TCID50 CEV-JL14 via different infective routes. Tissue samples were collected from the infected mice (5 dpi) and processed for immunohistochemistry assay to examine the tissue tropism of CEV-JL14. CEV-JL14 antigens were observed in the majority of the tissues examined, especially in the heart (B), liver (D), spleen (F), lung (H), kidney (J), intestine (L), brain (N), and muscle (P) as indicated by arrow in comparison with corresponding tissues in control mice (A,C,E,G,I,K,M,O). Heart (B) was obtained from mice inoculated via intramuscular injection. Liver (D) and brain (N) were from mice inoculated via intraperitoneal injection. Tissues including spleen (F), kidney (J), and intestine (L) were from mice inoculated via subcutaneous injection. Lung (H) and muscle (P) were from mice inoculated via intranasal administration.