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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

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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f5: 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).

Mentions: To investigate gerbils as a model to evaluate vaccine efficacy, the protective efficacy of purified formalin-inactivated CA16 vaccines was analyzed. Groups of 7-day-old gerbils (n = 8 for each group) were immunized with the purified inactivated CA16-193 vaccine at a dose ranging from 2.5 to 640 units (U), or a control (PBS). Vaccination was repeated when gerbils were 14 days of age, and finally at 21 days of age gerbils were challenged with CA16-194 using a 100 × LD50. All gerbils of the control group rapidly developed limb paralysis during the course of infection and finally died at five to six days post-infection. In contrast, from the group of gerbils vaccinated with the lowest dose (2.5 U), 25% (2 of 8) survived the lethal challenge (Fig. 5A). Furthermore, 75% of the gerbils vaccinated at a dose of 10 U were protected and at higher vaccine doses (≥40 U), all gerbils survived (Fig. 5A). Subsequently, viral loads were analyzed in the tissues of vaccinated and control animals four days after a lethal CA16-194 challenge. Viral loads were significantly reduced in the spleen, brainstem, spinal cord and muscle tissues of vaccinated gerbils compared with control animals (Fig. 5B). No pathological changes of the brainstem, spinal cord and muscle were detected in vaccinated gerbils four days after a lethal challenge of CA16-194. However, in the PBS control group, shrinking neurons and sieve-like changes in the brainstem, neuronophagia in the spinal cord, and degeneration of skeletal muscle fibers and inflammatory cell infiltration in muscle tissues were found (Fig. 5C). The cross-protective capacity of the CA16-193 vaccine was also evaluated. Vaccinated gerbils were challenged by another clinical strain, CA16-196. Gerbils inoculated with the CA16-193 vaccine at a dose of 160 U all survived, while gerbils in the control group all died (Fig. 5D). These results indicated that the vaccine-induced immune response was sufficient to prevent viral replication and pathological changes in the challenged gerbils, which resulted in survival of gerbils from an otherwise lethal CA16 challenge.


Coxsackievirus A16 induced neurological disorders in young gerbils which could serve as a new animal model for vaccine evaluation
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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f5: 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).
Mentions: To investigate gerbils as a model to evaluate vaccine efficacy, the protective efficacy of purified formalin-inactivated CA16 vaccines was analyzed. Groups of 7-day-old gerbils (n = 8 for each group) were immunized with the purified inactivated CA16-193 vaccine at a dose ranging from 2.5 to 640 units (U), or a control (PBS). Vaccination was repeated when gerbils were 14 days of age, and finally at 21 days of age gerbils were challenged with CA16-194 using a 100 × LD50. All gerbils of the control group rapidly developed limb paralysis during the course of infection and finally died at five to six days post-infection. In contrast, from the group of gerbils vaccinated with the lowest dose (2.5 U), 25% (2 of 8) survived the lethal challenge (Fig. 5A). Furthermore, 75% of the gerbils vaccinated at a dose of 10 U were protected and at higher vaccine doses (≥40 U), all gerbils survived (Fig. 5A). Subsequently, viral loads were analyzed in the tissues of vaccinated and control animals four days after a lethal CA16-194 challenge. Viral loads were significantly reduced in the spleen, brainstem, spinal cord and muscle tissues of vaccinated gerbils compared with control animals (Fig. 5B). No pathological changes of the brainstem, spinal cord and muscle were detected in vaccinated gerbils four days after a lethal challenge of CA16-194. However, in the PBS control group, shrinking neurons and sieve-like changes in the brainstem, neuronophagia in the spinal cord, and degeneration of skeletal muscle fibers and inflammatory cell infiltration in muscle tissues were found (Fig. 5C). The cross-protective capacity of the CA16-193 vaccine was also evaluated. Vaccinated gerbils were challenged by another clinical strain, CA16-196. Gerbils inoculated with the CA16-193 vaccine at a dose of 160 U all survived, while gerbils in the control group all died (Fig. 5D). These results indicated that the vaccine-induced immune response was sufficient to prevent viral replication and pathological changes in the challenged gerbils, which resulted in survival of gerbils from an otherwise lethal CA16 challenge.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus