腹腔注射攻毒建立蜱媒脑炎病毒感染BALB/c小鼠模型

Establishment of a BALB/c mouse model infected with tick-borne encephalitis virus via intraperitoneal injection

目的通过腹腔注射攻毒途径建立蜱媒脑炎病毒(TBEV)感染BALB/c小鼠模型,观察小鼠的感染症状和病毒在小鼠体内的复制特征。方法将6周龄雌性BALB/c小鼠随机分为未感染病毒对照组、1×10^(3)空斑形成单位(PFU)TBEV感染组(以每只小鼠1×10^(3)PFU的攻毒剂量经腹腔注射感染小鼠)、1×10^(4)PFU TBEV感染组(以每只小鼠1×10^(4)PFU的攻毒剂量经腹腔注射感染小鼠),观察小鼠的感染症状、体重变化与生存情况。通过H-E染色观察小鼠脑、脾的病理改变,采用免疫组织化学染色检测小鼠脑、脾中TBEV蛋白的表达,采用空斑试验检测小鼠脑、脾中TBEV滴度的动态变化。结果1×10^(4)PFU TBEV感染组小鼠于感染后第6天出现弓背、后肢瘫痪等感染症状,1×10^(3)PFU TBEV感染组小鼠于感染后第7天出现上述症状。与未感染病毒对照组小鼠相比,1×10^(4)PFU TBEV感染组小鼠从第5天、1×10^(3)PFU TBEV感染组小鼠从第6天开始体重明显降低,差异有统计学意义(P<0.05)。1×10^(4)PFU TBEV感染组小鼠第7天开始死亡、第8天全部死亡,1×10^(3)PFU TBEV感染组小鼠第7天开始死亡、第9天全部死亡。H-E染色结果显示,TBEV感染后第5天、第7天小鼠大脑皮质仅出现少量神经元核固缩、深染,未观察到炎症细胞浸润;TBEV感染后第5天小鼠脾红髓轻度淤血、多见中性粒细胞,第7天中性粒细胞数量增多。免疫组织化学染色结果显示,TBEV感染后第5天小鼠脑与脾少量表达TBEV蛋白,第7天TBEV蛋白表达量增加。1×10^(3)PFU TBEV感染第7天,小鼠脑内TBEV滴度高达(1.3±0.6)×10^(5)PFU/mL,而脾中TBEV滴度为(1.3±0.6)×10^(3)PFU/mL。结论经腹腔注射攻毒成功建立了TBEV感染BALB/c小鼠模型,TBEV可在小鼠体内增殖并具致病性。

Objective To establish a BALB/c mouse model infected with tick-borne encephalitis virus(TBEV)via intraperitoneal injection,and observe the infection symptoms and replication characteristics of TBEV in mice.Methods Sixweek-old female BALB/c mice were randomly assigned(1∶1∶1)to control group,1×10^(3) plaque-forming unit(PFU)TBEV infected group(infected by intraperitoneal injection of 1×10^(3) PFU per mouse),or 1×10^(4) PFU TBEV infected group(infected by intraperitoneal injection of 1×10^(4) PFU per mouse).The infection symptoms,body weight changes,and survival rates of the mice were monitored.The pathological changes in the brain and spleen of the mice were observed by hematoxylin-eosin(H-E)staining.The expression of TBEV protein in the brain and spleen of the mice was detected by immunohistochemistry.The dynamic changes of TBEV titers in the brain and spleen of the mice were detected by plaque assay.Results The infected mice developed arched back and hind limb paralysis on day 6 in the 1×10^(4) PFU TBEV infected group and on day 7 in the 1×10^(3) PFU TBEV infected group.Compared with the control group,the body weight of mice was significantly decreased from day 5 in the 1×10^(4) PFU TBEV infected group and from day 6 in the 1×10^(3) PFU TBEV infected group(P<0.05).The mice died from day 7 post-infection,and all died on day 8 in the 1×10^(4) PFU TBEV infected group and on day 9 in the 1×10^(3) PFU TBEV infected group.The H-E staining results showed that on day 5 and 7 after TBEV infection,only a small amount of neuronal nucleus pyknosis and deep staining were observed in the brain cortex of the mice,and no infiltration of inflammatory cells was observed.On day 5,the red pulp of spleen showed mild congestion and neutrophils;on day 7,neutrophils were increased.The immunohistochemical staining results showed that TBEV protein was expressed in the brain and spleen of mice on day 5 after TBEV infection,and was increased on day 7.On day 7 post-infection with 1×10^(3) PFU TBEV,TBEV titers were(1.3±0.6)×10^(5) PFU/mL in the brain of mice and(1.3±0.6)×10^(3) PFU/mL in the spleen.Conclusion A BALB/c mouse model infected with TBEV has been successfully established by intraperitoneal injection,and TBEV can proliferate and be pathogenic in mice.