Fig 1.
ICR suckling mice are susceptible to HY12 enterovirus infection.
Three-day old Balb/c, Kunming, and IRC neonatal mice were subcutaneously inoculated with 2×106 TCID50 HY12 viruses. Tissue samples including liver, lung, spleen, lymph node, kidney, and brain were collected from pups at 5 dpi and processed for amplification of HY12 genomic fragments using RT-PCR. Fragment with expected size was detected clearly from tissue samples in ICR (lane 2) suckling mice. No fragments were amplified from Balb/C suckling mice (lane 1) and Kunming suckling mice (lane 3) infected with HY12. Lane 4 and lane 5 were the positive and negative control, respectively. DNA ladder was presented as M and the size of expected fragment is indicated as arrow.
Fig 2.
Minimal infective dose of HY12 to ICR suckling mice.
2 × 104, 2 × 106 and 2 × 108 TCID50 HY12 viruses were injected to mice subcutaneously to determine the minimal infective dose (MID). Tissue samples were collected 5 dpi and processed for RT-PCR to detect the virus genome fragment. Representative figure showing the PCR-amplified fragments with expected size from mice infected with 2 × 106 TCID50 (lane 2) and 2 × 108 TCID50 (lane 3), respectively. The negative and positive controls were presented in lane 4 and lane 5, respectively. M stands for the DNA ladder.
Fig 3.
Confirmation of the HY12 infection by recovering the virus from the infected mice by HY12.
Representative figure showing the cytopathic effects in Vero cells after they were infected by intestine tissue samples collected from mice infected with HY12 (A) in comparison with Vero cells infected with corresponding tissue samples from normal mice (B). Immunofluorescent assay was used to detect in HY12 infected cells using polyclonal antibody generated in rabbits against HY12 VP2 protein. Rhodamine-conjugated goat anti-rabbit IgG was used as secondary antibody. The Fluorescent signals were detected in HY12 infected cells (C) with the negative control cells (D). Viral loads in tissues for three mice infected by HY12 virus were quantitated and represented as average virus copies of log 10/ g tissue(E).
Fig 4.
Determination of HY12 infective routes.
ICR suckling mice were infected with HY12 virus by administered via intranasal, oral administration, intraperitoneal injection, subcutaneous injection and intramuscular injection, respectively. Tissue samples were collected and processed for RT-PCR to detect the virus genome fragments and virus loads in each organ was quantitated. Compared with the normal controls (lane 1–5), the fragments with expected size were amplified from tissues in mice infected with HY12 via all infection routes (lane 6–10). Lane 6: intranasal route; lane 7: oral administration; lane 8 intraperitoneal injection; lane 9: subcutaneous injection; lane 10: intramuscular injection. The virus loads in each organ were quantitated and represented as average virus copies of log 10/g tissues.
Fig 5.
Effect of HY12 infection on mice.
Mice were observed every 6 hours after they were infected by HY12 viruses. Body weights of the infected mice were weighed at interval of 5 days till endpoint (23 dpi). A representative figure showed the difference of mice infected with (lower) or without (upper) HY12 via intranasal administration (A). The average body weights in mice infected intranasally and orally were significantly lower than that in mice of the normal control (P<0.05) (B). HY12 viruses persistently present in the infected mice. Tissue samples in every mouse were collected at 3, 8, 13, and 18 dpi and viruses were still detected from infected mouse tissues 18 dpi using (C). Quantitation of virus loads in tissue from mice infected by HY12 at different days postinfection (D).
Fig 6.
Histopathological lesion revealed in mice infected with HY12.
Representative figures showing histopathological lesions in mice infected via oral administration. Tissue samples were collected and processed for H&E staining and histopathological examination. Obvious histopathological lesions and inflammatory cell infiltration were observed in the lung (B), liver (D), intestine (F) and brain (H) as indicated by arrow in comparison with corresponding tissues in uninfected control mice (A, C, E and G).
Table 1.
Histopathological lesions of mice experimentally infected with HY12 with different routes.
Fig 7.
Viral tissue tropism in mice infected with HY12.
Representative figure showing the antigen distribution in tissues of mice infected via oral administration. Tissues were collected and processed for immunohistochemistry assay to examine the tissue tropism of HY12 viruses. HY12 virus antigens were detected in the majority of the tissues examined, especially in the intestine(B), brain (D), lung (F) and Liver (H) as indicated by arrow in comparison to the corresponding tissue organs in noninfected mice (A, C, E and G).
Table 2.
HY12 virus antigen detected in tissues in mice infected with HY12 by different routes.