Coxsackievirus A16 induced neurological disorders in young gerbils which could serve as a new animal model for vaccine evaluation - PubMed (original) (raw)

Ya-Jing Li 2, Yong Xia 1, Fang Xu 1, Wei-Wei Wang 2, Zhang-Nv Yang 1, Hang-Jing Lu 1, Zhi-Ping Chen 1, Zi-Ping Miao 1, Wei-Feng Liang 3, Zhi-Yao Xu 4, Hong-Jun Dong 5, Dan-Hong Qiu 6, Zhi-Yong Zhu 1, Stijn van der Veen 7, Jie Qian 8, Bin Zhou 8, Ping-Ping Yao 1, Han-Ping Zhu 1

Affiliations

Coxsackievirus A16 induced neurological disorders in young gerbils which could serve as a new animal model for vaccine evaluation

Yi-Sheng Sun et al. Sci Rep. 2016.

Abstract

Coxsackievirus A16 (CA16) is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD) in the Asia-pacific region. Although CA16 infections are generally mild, severe neurological manifestations or even death has been reported. Studies on CA16 pathogenesis and vaccine development are severely hampered because the small animal models that are currently available show major limitations. In this study, gerbils (Meriones unguiculatus) were investigated for their suitability as an animal model to study CA16 pathogenesis and vaccine development. Our results showed that gerbils up to the age of 21 days were fully susceptible to CA16 and all died within five days post-infection. CA16 showed a tropism towards the skeletal muscle, spinal cord and brainstem of gerbils, and severe lesions, including necrosis, were observed. In addition, an inactivated CA16 whole-virus vaccine administrated to gerbils was able to provide full protection to the gerbils against lethal doses of CA16 strains. These results demonstrate that gerbils are a suitable animal model to study CA16 infection and vaccine development.

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Figures

Figure 1

Figure 1. Age-related survival and severity of disease in CA16-infected gerbils.

(A) Survival curves for groups of gerbils (n = 8–10) aged 7 to 56 days when infected with CA16-194 at a TCID50 of 105.5. Curves were compared with the 7 days age group using the log-rank test. *Significantly different from the control group (p < 0.01). (B) Mean clinical scores for groups of gerbils aged 7 to 56 days when infected with CA16-194 at a TCID50 of 105.5. One representative of two independent experiments was shown in A and B.

Figure 2

Figure 2. Dose-dependent survival and severity of disease in CA16-infected gerbils.

(A) Survival curves for groups of gerbils (n = 8–10) aged 21 days when infected with CA16-194 at a TCID50 of 0.3 to 105.5. Curves were compared with the 0.3 TCID50 group using the log-rank test. *Significantly different from the control group (p < 0.01). (B) Mean clinical scores for groups of gerbils aged 21 days when infected with CA16-194 at a TCID50 of 0.3 to 105.5. One representative of two independent experiments was shown in A and B.

Figure 3

Figure 3. Virus dissemination and replication in different tissues of CA16-infected gerbils.

21-day-old gerbils were inoculated with CA16-194 at 100 × LD50. The graph shows the virus loads in the heart, liver, spleen, lung, kidney, brain, brainstem, spinal cord, muscle and blood from CA16-infected gerbils at 1, 2, 3 and 4 days post infection. Virus loads were assessed by real-time RT-PCR and compared with standard curves obtained from 10-fold serial dilutions of CA16-194. Results represent the mean ± standard error of the virus titer (log10 TCID50) per gram of tissue or per milliliter of blood from two independent experiments using three technical replicates each. *Significantly different from the day 1 group in each tissue (p < 0.01).

Figure 4

Figure 4. Pathology of CA16-infected gerbil tissues.

Gerbils aged 21 days were challenged with CA16-194 using a TCID50 of 105.5. (A) Representative images of control and CA16-infected gerbils four days post infection. The arrow indicates hind-limb paralysis observed in the CA16-infected gerbils. (B) Representative images of haematoxylin and eosin or Nissl stained brainstem, spinal cord and muscle tissues harvested from CA16-infected and control gerbils four days post infection. The arrows indicate focal shrunken neurons in the infected brainstem, swollen neurons and neuronophagia in the infected spinal cord, and inflammatory cell infiltration, severe necrotizing myositis, degeneration and swelling of skeletal muscle fibers in the infected muscle. (C) Representative images of brainstem, spinal cord and muscle tissues harvested from CA16-infected and control gerbils four days post infection. Presence of viral antigens (arrows) was visualized by incubation with peroxidase staining DAB followed by counterstaining with haematoxylin.

Figure 5

Figure 5. Protective efficacy against lethal CA16 challenges in immunized gerbils.

Gerbils were immunized with a CA16 vaccine at the age of 7 and 14 days and challenged at the age of 21 days with CA16 virus using a 100 × LD50. (A) Survival curves of groups of gerbils (n = 8) were immunized with the CA16 vaccine using a dose of 2.5, 10, 40, 160, 640 U or PBS as a negative control, then challenged with strain CA16-194. Curves were compared with the PBS control group using the log-rank test. Representative results from two similar experiments are shown. *Significantly different from the control group (p < 0.01). (B) Viral loads in the spleen, brainstem, spinal cord and muscle tissue of gerbils four days post infection. Gerbils were immunized with the CA16 vaccine using a dose of 160 U or a PBS control, then challenged with strain CA16-194. Virus loads were assessed by real-time RT-PCR and compared with standard curves obtained from 10-fold serial dilutions of CA16-194. Results represent the mean ± standard error (n = 3 each) of the virus titer (log10 TCID50) per gram of tissue. *Significantly different from the control group (p < 0.01). (C) Histology examinations of brainstem, spinal cord (Nissl stained) and muscle tissues (haematoxylin and eosin stained) harvested from CA16 vaccine-immunized and control gerbils 4 dpi. Gerbils were immunized with the CA16 vaccine using a dose of 160 U or a PBS control, then challenged with strain CA16-194. The arrows indicated shrinking neurons and sieve-like changes in brainstem, neuronophagia in the spinal cord, degeneration of skeletal muscle fibers and inflammatory cell infiltration in muscle of PBS controls. (D) Survival curves of groups of gerbils (n = 8) were immunized with the CA16 vaccine using a dose of 160 U or PBS as a negative control, and then challenged with CA16-196 using a 100 × LD50. Curves were compared with the PBS control group using the log-rank test. *Significantly different from the control group (p < 0.01).

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