重庆地区小鼠诺如病毒的感染及其对免疫功能影响的初步研究

Preliminary study on the infection of mouse Norovirus and its effect on immune function in Chongqing

摘要:

目的：建立实时荧光定量RT-PCR检测方法，普查重庆市实验小鼠诺如病毒（murine Norovirus，MNV）的感染情况及其对BABL/c-nu小鼠免疫功能影响的初步研究。方法：①根据GenBank中收录的MNV全基因组序列，设计MNV特异性引物，检测其特异性、灵敏度、重复性和稳定性，并和两个实验动物质量检测单位对123份小鼠临床样本开展比对检测，以最终构建荧光定量RT-PCR检测方法。②应用该方法比较小鼠不同肠道排泄物（盲肠内容物、新鲜粪便和排出体外24 h粪便）中MNV的检出情况。③应用该方法普查重庆市实验小鼠感染MNV的情况。④6~8周的BABL/c-nu小鼠被随机分为3组（n=3）：正常对照组、MNV感染30 d组、MNV感染60 d组，收集各组小鼠血清及肝、肺、脾、结肠，HE染色观察各脏器的病理变化；酶联免疫吸附法（enzyme linked immunosorbent assay，ELISA）检测血清、脾和结肠肿瘤坏死因子-α（tumor necrosis factor-alpha，TNF-α）及干扰素-γ（interferon gamma，IFN-γ）的含量。结果：①建立的荧光定量RT-PCR方法特异性强，与同种属其他病毒均不发生交叉反应，方法的灵敏度、重复性和稳定性良好，与另外两个实验动物质检单位的检测结果吻合度高达98%。②新鲜粪便与盲肠内容物中MNV的检出结果完全一致；24 h的陈旧粪便也能准确反映出该笼小鼠MNV的感染情况。③重庆市实验小鼠存在较高的MNV感染率，且封闭群和近交系小鼠MNV感染率无品系特异性（68.3% vs. 70.6%， χ2=0.116，P=0.733），但基因修饰小鼠比普通小鼠更易感染MNV（78% vs. 64%， χ2=6.434，P=0.011）。④与正常对照组小鼠相比，BABL/c-nu小鼠自然感染MNV后肝、肺、脾和结肠均出现不同程度的炎症细胞浸润和明显的组织病理变化，且结肠组织的TNF-α、IFN-γ水平明显升高（F=21.682，P=0.002；F=77.223，P=0.000）。结论：本研究建立的实时荧光定量PCR方法可靠，且可通过小鼠粪便样本，日常监测MNV的感染情况等；重庆市实验小鼠有较高的MNV感染率，且BABL/c-nu小鼠感染MNV会对动物实验结果造成干扰。因此，各实验动物单位应该重视MNV的传播和防控，加强实验动物的饲养管理，降低实验动物感染MNV的风险。

Abstract:

Objective：To establish a real-time fluorescent quantitative RT-PCR method to survey murine Norovirus（MNV） infection in laboratory mice in Chongqing，and explore the effect on immune function. Methods：①A pair of specific primers were designed targeting MNV GenBank genomic sequence，the specificity，sensitivity，repeatability and stability of the assay were evaluated，and comparison test of 123 clinical samples were carried out with two laboratory animal testing institutions to establish reliable real-time RT-PCR assay for MNV detection. ②The detection of MNV in different intestinal excretions（caecum contents，fresh feces，and excreted feces for 24 hours） was compared by real-time quan－titative RT-PCR. ③Real-time quantitative RT-PCR was used to investigate the infection of MNV in laboratory mice in Chongqing. ④Six to eight week-old BABL/c-nu mice were randomly di－vided into 3 groups（n=3 for each group）：control，MNV natural infection for 30 d，MNV infection for 60 d. Serum，liver lung，spleen，and colon were collected，the pathological changes of various organs were observed by HE staining；and tumor necrosis factor alpha（TNF-α） and interferon gamma（IFN-γ） levels in serum，tissue homogenates of spleen and colon were detected by enzyme linked immunosorbent assay（ELISA）. Results：①To established real-time RT-PCR assay could specifically detect MNV and had no cross reactions with mouse other virus. The sensitivity and repeatability were also excellent and the coincidence degree of comparison test in 123 clinical samples is as high as 98%. ②The detection results of MNV in fresh feces and caecum contents were completely consistent，excrement for 24 h in vitro can also accurately reflect MNV infection of mice in the cage. ③MNV infection rate in Chongqing is 65% to 85%，and no strain differences（68.3% vs. 70.6%， χ2=0.116，P=0.733），but genetically modified mice were more susceptible to MNV than normal mice（78% vs. 64%， χ2=6.434，P=0.011）. ④Compared with Control group，BABL/c-nu mice showed that inflam－matory cell infiltration and the obvious pathological changes in liver，lung，spleen and colon after natural infection with MNV，and the levels of TNF-α，IFN-γ in colon were significantly increased（F=21.682，P=0.002；F=77.223，P=0.000，respectively）. Conclusion：The established real-time fluorescent quantitative RT-PCR method is reliable and can monitor the infection of MNV through the stool samples of mice，MNV infection rate of mice in Chongqing is high and the infection in BABL/c-nu mice has interfered with the exper－imental results. Therefore，it is necessary to enhance the animal breeding management to reduce the risk of MNV infection in mice.