人3型副流感病毒感染小鼠模型的建立及免疫应答特点

Establishment of a mouse model of human parainfluenza virus type 3 infection and characterization of immune responses+

目的建立人3型副流感病毒(human parainfluenza virus type 3,HPIV3)感染小鼠模型,并研究其免疫应答特点。方法用HPIV3兰州分离株HPIV3LZ1728C19毒株鼻腔接种BALB/c小鼠,每天检测体温和体质量。用ELISA检测血清HPIV3特异性IgG、IgA滴度,实时定量PCR检测鼻腔灌洗液和肺组织病毒滴度,流式细胞术检测外周血、肺、脾淋巴细胞中的HPIV3特异性IFN-γCD4^(+)和IFN-γCD8^(+)T细胞,酶联免疫斑点法检测分泌HPIV3特异性IgG/IgA的B淋巴细胞,肺组织切片观察病理变化。结果感染后第3天,小鼠鼻腔和肺的病毒负载量分别到达峰值104拷贝/ml鼻洗液和107拷贝/g组织,肺部发生炎症性病理变化。感染组早期体质量增加显著滞后于对照组(感染后第3天:t=4.64,P<0.05)。感染组血清中HPIV3特异性IgG(t=2.94,P<0.05)和IgA水平(t=18.66,P<0.05)显著增高,外周血、肺、脾均出现HPIV3特异性抗体分泌淋巴细胞。病毒感染诱导小鼠肺产生记忆性IFN-γCD8^(+)T和IFN-γCD4^(+)T细胞应答,脾和外周血产生IFN-γCD4^(+)T细胞应答。结论成功建立了HPIV3感染小鼠动物模型,感染小鼠产生了显著的肺黏膜体液和细胞免疫应答。

Objective To establish an animal model of human parainfluenza virus type 3(HPIV3)infection in mice,and characterize the immune responses of the HPIV3 infected mice.Methods BALB/c mice were infected intranasally with wild strain HPIV3LZ1728C19 isolated from clinical specimens.The body mass and temperature of infected mice were measured daily for 30 d.The titers of HPIV3-specific IgG and IgA in serum were detected by ELISA,and the viral titer in nasal lavage and lung tissue were detected by quantitative real-time PCR.The HPIV3-specific IgG and IgA antibody secreting cells(ASCs)and the HPIV3-specific IFN-γCD4^(+)/CD8^(+)T cells in lymphocytes isolated from lung,spleen and peripheral blood were detected by enzyme-linked immunospot assay and flow cytometry,respectively.The pathological changes in lung tissue were observed by lung slice.Results The HPIV3 replicated and viral loads peaked(104 copies/ml nasal lavage and 107 copies/g lung tissue)at post infection day 3(PID3)in infected mice.HPIV3 infection significantly lowered the body mass gain of mice at early stage of infection(t=4.64,P<0.05 at PID3),and resulted in inflammatory pathological changes in lung tissue.The HPIV3 specific IgG(t=2.94,P<0.05)and IgA(t=18.66,P<0.05)antibody levels were significantly higher in infected mice serum than control,with virus-specific ASC responses in all 3 kinds of tissues.Virus infection induced HPIV3-specific IFN-γCD8^(+)T cell response in lung,and HPIV3-specific IFN-γCD4^(+)T cell response in spleen and peripheral blood.Conclusions A mouse model of HPIV3 infection is successfully established.Lung mucosal humoral and cellular immune responses are induced in BALB/c mice infected intranasally with HPIV3.