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A mouse model of peripheral nerve injury induced by Japanese encephalitis virus [1]

['Xiaoli Wang', 'The People S Hospital Of Shizuishan', 'Shizuishan', 'Ningxia Medical University', 'Yinchuan', 'Guowei Wang', 'Huan Yang', 'General Hospital Of Ningxia Medical University', 'Shihong Fu', 'Department Of Arbovirus']

Date: 2022-12 

Mice inoculated with the virus exhibited signs of neurological disease

To determine the optimal volume of virus inoculation and the optimal times for disease assessment, we exposed mice to five different virus titers (102pfu, 103pfu, 104pfu, 105pfu, and 106pfu) and evaluated the inoculated mice for 20 days. Mice treated with PBS were used as controls (n = 5). Each titer was injected into five mice (n = 5). All mice inoculated with JEV were designated as the virus group (n = 25). We discovered that mice exhibited limb paralysis and general weakness in each virus titer group. The VPS scores for each titer are shown in S1A Fig. We found that the disease severity did not directly correlate with the viral titer. Furthermore, the mice that received 104pfu (p = 0.0345) and 105pfu (p = 0.0268) presented significant advantages when evaluated. From 1 dpi to 5 dpi, the VPS of the virus group exhibited scores of 0 to 2 points. From 6 dpi to 15 dpi, the scores were between 0 to 5 points, and after 15 dpi, the scores of the virus group were between 0 to 1 point (S1B Fig). These results indicated that some symptoms, such as limb paralysis and frailty, were more severe prior to 15 dpi. On the other hand, when the body weight changes were compared between the virus and control groups, we found that there was no significant difference between the two groups prior to 5 dpi. From 5 dpi to 15 dpi, the changes in body weight between the two groups became more pronounced, indicating that the body weight of the mice in the virus group increased more slowly than in the control group. After 15 dpi, the body weights of the mice in the virus group increased more rapidly (S2 Fig). These results suggested that the mice appeared to be recovering after 15dpi. Based on this information, the virus titer used in the experiment was 105pfu, the observation period was 15 days, and the time intervals were 4 dpi, 8 dpi, 12 dpi, and 15 dpi.

Behavioral changes in mice were easy to observe and could be used as an initial assessment to determine whether mice were ill. Based on daily observations of the general condition of the mice, we determined that the mice in the control group presented a shiny hair coat, were active, moved freely with a steady gait, and ran quickly. However, the mice in the virus-exposed group exhibited different degrees of rough hair coats, weakness, and reduced activity. Some exposed mice also exhibited a hunched back, body tremors, limping on one or both hind limbs, and a weakened forelimb grip [39].

Body weight also was a good indicator of animal health [40]. The body weights of both mouse groups were recorded daily. Viral infection can lead to decreased appetite and weight loss in mice. The body weights were expressed as means ± standard deviation. We observed that mice in both groups gained weight. However, the weights of mice in the control group increased steadily, from 20.84g ± 1.07g at the beginning of the study to 22.58g ± 1.00g at the end of the investigation. The weights of the virus-exposed mice increased more slowly, with an initial weight of 20.69g ± 1.27g and a weight of 21.66g ± 0.38g at the end of the investigation, which was approximately 1g less than in the control group. Some mice exhibited significant weight loss following virus exposure. However, before 4 dpi, no significant difference in weight was observed between the two groups. While the mean body weight in the virus group was lower than the control group from 4 dpi to 8 dpi, both groups exhibited increased body weights. The mean body weight in the control group increased significantly after 8 dpi, but only a minimal increase or even a decrease in body weight was observed in the virus-exposed mice. The increase in body weight in the virus-exposed mice improved after 12dpi (Fig 1A).

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TIFF original image Download: Fig 1. Behavioral changes of mice at different time. (A) Body weight change of the mice at different time; (B) VPS scores at different time. Kruskal-Wallis H test was used (****p< 0.0001); (C) Hanging wire test at different time. Kruskal-Wallis H test was applied for statistical analysis (**p = 0.005). https://doi.org/10.1371/journal.pntd.0010961.g001

In addition to observing the general condition of the mice, motor function was evaluated. Based on the viral paralysis scale (VPS), the mice in the control group (n = 18) did not exhibit any stiffness or paralysis of their limbs and received a score of 0 (normal). Virus-exposed mice exhibited progressively slower movements, tail dragging, stiff joints [40], lameness while walking, and an inability to perform normal plantar stepping. In some cases, the virus-exposed mice were unable to support their weight. The VPS ranged from 0 to 6 points. A score of ≤ 2 points indicated no apparent limb paralysis. A score of 3 to 6 points indicated the presence of limb paralysis. An increasing score indicated that the degree of paralysis increased in severity [29]. Compared to the control group at 4 dpi, the VPS of mice in the virus group was ≤ 1 point (n = 7, p > 0.9999). Three mice were scored as 0-points (3/7), and four received a score of 1-point (4/7). A lower tail position and a slight sway in their gait when the mice walked were observed. Starting at 8 dpi (n = 15, ***p = 0.0006), the number of mice with VPS between 3 to 6 points increased. Of the 15 mice that were scored, 4/15 were 0-points, 4/15 were 1-point, 1/15 were 2-points, 2/15 were 3-points, 1/15 were 5-points), and 1/15 were 6-points. Considerable limb paralysis was observed at this stage, including joint stiffness, significant weakness, limb dragging, reduced joint activity, and inability to bear weight. The VPS was between 0 to 5 points at 12 dpi (n = 14, ****p < 0.0001) and 1 to 2 points at 15 dpi (n = 14, ***p = 0.0002). The VPS assessments of 14 mice at 12 dpi were 1/14 as 0-points, 1/14 as 1-point, 3/14 as 2-points, 4/14 as 3-points, 2/14 as 4-points, and 3/14 as 5-points. At 15 dpi the scores were 4/14 as 1-point and 10/14 as 2-points (Fig 1B).

We conducted a hanging wire test, which was supplementary to the VPS assessment, to eliminate subjective bias. The motor function of the mouse limbs was assessed by recording the time each mouse was able to suspend its body on an inverted wire mesh. This test assessed the strength of their limbs, which was essential to keep them from falling. Mice in the control group exhibited a robust ability to grasp the wire mesh and were able to move continuously on the upside-down wire mesh frame without falling. The suspension time for the control mice reached even exceeded 180s. Numerous mice in the virus-exposed group fell more than once. The mice barely moved when hanging from the wire mesh and primarily stayed in one place. The lack of movement while suspended on the wire mesh might have helped maintain their limb strength to avoid falling. At 4 dpi (p > 0.9999), only one mouse fell once in the virus group and returned to normal when the experiment was repeated. On the other hand, starting at 8 dpi (*p = 0.0109), additional mice in the virus-exposed group fell one or more times. Some mice who fell on the first trial, occasionally stayed suspended on the wire mesh for 180s in the second or third trial of the day, but most mice fell all three times. Eight of 15 mice in the virus group spent less than 180s suspended on the wire mesh. At 12 dpi, the time the virus-exposed mice were able to suspend their bodies from the wire mesh also was significantly shorter (*p = 0.0283). Six of 14 mice fell and the time they remained suspended was less than 80s. At 15 dpi (p >0.9999), the amount of time the virus-exposed mice remained suspended on the wire mesh was longer than the average time observed at 12 dpi, and fewer mice fell (4/14) (Fig 1C).

The VPS and hanging wire test results indicated that, although the virus-exposed mice exhibited considerable limb paralysis (VPS ≥ 3), many mice did not exhibit a significantly shorter time during which they were able to suspend their bodies during the hanging wire test. This may be because mice also presented symptoms such as weakness and reduced activity, which increased the VPS score. However, these factors did not significantly influence the outcome of the hanging wire test. It was consistent that symptoms exhibited by the virus-exposed mice were most prominent from 8 dpi to 12 dpi, indicating that the virus-exposed mice presented considerable limb weakness during this period. After 12 dpi, mice with a score less than 3 points did not exhibit any discernable limb weakness, and the number of mice whose scores were between 1 and 2 points increased. Furthermore, the virus-exposed mice had sufficient limb strength to allow them to remain suspended from the wire mesh. Accordingly, there was no apparent reduction in the time the mice remained suspended during the hanging wire test.

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[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0010961

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