BALB/c和SCID小鼠人偏肺病毒感染特点的比较研究

目的 比较研究人偏肺病毒(hMPV)在BALB/c小鼠和SCID小鼠肺内复制动力学和肺病理特点.方法 GFP-rhMPV滴鼻感染BALB/c小鼠和SCID小鼠,于感染后3、5、7、9、14d处死小鼠并无菌获取心、肝、脾、肺、肾和脑用于病毒分离和病理检查,空斑形成法检测病毒滴度,RT-PCR和real-time PCR法检测hMPV mRNA表达.结果 GFP-rhMPV滴鼻感染BALB/c小鼠和SCID小鼠后第5天肺组织均分离到病毒.感染GFP-rhMPV的BALB/c和SCID小鼠在感染后第5天肺组织病毒滴度均达峰值,分别为(5.25±1.69)×104 PFU/g和(5.83±1.21)×105 PFU/g.SCID小鼠在感染后第14天肺组织内病毒滴度仍可达(4.25±1.04) ×101 PFU/g,此时BALB/c鼠肺内已不能分离到病毒,但可检测到GFP-rhMPV F蛋白mRNA表达.感染后第5天所有小鼠的心、肝、脾、肾和脑组织内均不能分离到病毒,也无法检测到hMPV F蛋白mRNA表达.肺组织病理改变在感染后第5天最明显,为典型的间质性肺炎改变,BALB/c小鼠组肺组织病理评分略低于SCID小鼠组,差异无统计学意义.结论 GFP-rhMPV滴鼻感染后只能在小鼠肺内复制.同BAL B/c小鼠相比,SCID小鼠感染GFP-rhMPV后肺内病毒滴度高,复制时间久,但病理损伤无明显差异.     

人偏肺病毒A与B基因型对小鼠致病性的观察

目的 观察人偏肺病毒( human metapneumovirus,hMPV)A和B亚型感染BALB/c小鼠后,小鼠在体重、病毒载量、病理及气道反应性等方面的改变,探讨两亚型病毒致病性上的差异,为hMPV的进一步研究提供基础资料.方法 hMPV感染BALB/c动物模型后,通过使用real-time RT-PCR方法检测鼠肺内病毒载量的变化,肺功能检测仪监测小鼠气道反应性的变化及组织系统评分判定病理改变情况,来观测两亚型致病性的差异.结果 hMPV两亚型感染组,在体重的动态监测、病毒载量、肺组织病理改变及气道反应性上差异均无统计学意义.结论 hMPV两基因型感染BALB/c小鼠的致病性无差异.     

BALB/c鼠和裸鼠呼吸道合胞病毒感染比较研究

目的 比较普通BALB/c鼠和裸鼠呼吸道合胞病毒(RSV)感染免疫及炎症反应特点.方法 BALB/c鼠和裸鼠感染RSV后不同时间空斑形成试验检测肺组织病毒滴度,计数支气管肺泡灌洗液(BALF)白细胞总数和分类,HE染色分析肺组织病理学改变,免疫组化检测肺组织F4/80+细胞和CD49b+细胞.ELISA检测BALF中TNF-α、IFN-r、IL-12和IL-10浓度.结果 BALB/c鼠和裸鼠感染RSV后肺组织病毒滴度在第3天达峰值,感染裸鼠带毒时间更长,在感染后各天病毒滴度明显高于BALB/c鼠(P＜0.05),肺组织病理改变也更重.感染BALB/c鼠和裸鼠BALF白细胞总数明显升高,分类以淋巴细胞为主.感染裸鼠与感染BALB/c鼠比较,肺组织检测到更多的F4/80+巨噬细胞和CD49b+NK细胞(P＜0.05),BALF中TNF-α、IL-12和IL-10水平更高(P＜0.05).结论 RSV感染裸鼠与BALB/c鼠比较,病毒复制水平更高,时间更持久,炎症反应更重.单核巨噬细胞和NK细胞是RSV感染重要的免疫细胞和炎症细胞,炎症反应强度并不一定与T细胞免疫应答平行.     

脱氧核酶抗呼吸道合胞病毒感染不同免疫功能小鼠的研究

目的 研究脱氧核酶(DZ)抗不同免疫功能小鼠呼吸道合胞病毒(RSV)感染的作用.方法 RSV感染BALB/c鼠和裸鼠滴鼻给予DZ,空斑形成试验检测肺组织病毒滴度,RT-PCR检测病毒mRNA表达、支气管肺泡灌洗液白细胞计数,ELISA检测TNF-o、IL-12、IFN-γ和IL-10水平,肺组织病理学分析炎症情况.结果 DZ治疗组BALB/c鼠和裸鼠肺组织病毒滴度比感染对照组下降(P＜0.05),裸鼠下降更明显(P＜0.01).0.2 mg、0.4 mg和0.8 mg DZ分别降低感染BALB/c鼠30.51%、47.38%(P＜0.05)、53.97%(P＜0.01)和感染裸鼠36.59%(P＜0.05)、48.72%、59.78%(P＜0.01)病毒mRNA表达.0.4 mg DZ治疗降低感染BALB/c鼠和裸鼠支气管肺泡灌洗液中白细胞总数,改善肺组织病理学损伤(P＜0.05),降低感染裸鼠气道局部TNF-α、IL-12和IFN-γ分泌(P＜0.05).结论 DZ在不同免疫功能小鼠体内有效抑制RSV复制,减轻气道炎症,对裸鼠的保护作用更突出,是有效的抗RSV制剂.     

维生素D_3对肺炎支原体感染小鼠肺组织TREM-1及HIF-1α表达的影响

目的探讨维生素D3(VD3)对肺炎支原体感染小鼠肺组织髓系细胞触发受体-1 (TREM-1)及缺氧诱导因子-1α(HIF-1α)表达的影响。方法 45只BALB/c小鼠随机分成对照组、MP感染组与维生素D3组。模型组与维生素D3组小鼠采用滴鼻法进行MP感染。模型制作7d后,处死小鼠,取肺组织,应用免疫组织化学方法和Western Blot方法检测各组小鼠肺组织TREM-1及HIF-1α蛋白的表达,real time PCR方法检测各组小鼠肺组织TREM-1mRNA及HIF-1αmRNA的表达水平。结果 MP感染组BALB/c小鼠肺组织TREM-1及HIF-1α蛋白与m RNA的表达水平显著高于对照组,P0.01;给予维生素D3处理后,感染MP的BALB/c小鼠肺组织TREM-1及HIF-1α蛋白与m RNA的表达水平显著降低,P0.01。结论维生素D3能够降低肺炎支原体感染小鼠肺组织TREM-1及HIF-1α的表达。     

维生素D3对肺炎支原体感染小鼠肺组织NF-κB及MMP-9表达的影响

目的探讨维生素D3(VD3)对肺炎支原体感染小鼠肺组织核转录因子κB(NF-κB)及基质金属蛋白酶9(MMP-9)表达的影响。方法 45只BALB/c小鼠随机分成对照组、MP感染组与VD3组。模型组与VD3组小鼠采用滴鼻法进行MP感染。取肺组织制作病理切片,HE染色观察组织病理学变化,Western blot方法与Real time PCR方法分别检测各组小鼠肺组织NF-κB及MMP-9蛋白与mRNA的表达水平。结果 MP感染BALB/c小鼠7 d后,肺组织有大量炎性细胞浸润,给予VD3处理后,炎性明显减轻。MP感染组BALB/c小鼠肺组织NF-κB及MMP-9蛋白与m RNA的表达水平显著高于对照组(P0.01);给予VD3处理后,感染MP的BALB/c小鼠肺组织NF-κB及MMP-9蛋白与mRNA的表达水平显著降低(P0.01)。结论 VD3能够降低感染肺炎支原体感染小鼠的肺内炎性反应,其作用机制可能与降低NF-κB及MMP-9表达有关。     

维生素D3对肺炎支原体感染小鼠肺组织ICAM-1及IL-4表达的影响

目的探讨维生素D3(VD3)对肺炎支原体感染(MP)小鼠肺组织ICAM-1及IL-4表达的影响。方法 45只BALB/c小鼠随机分成对照组、MP感染组与维生素D3组。模型组与维生素D3组小鼠采用滴鼻法进行MP感染。取肺组织制作病理切片,HE染色观察组织病理学变化,Western blot方法与real time PCR方法分别检测各组小鼠肺组织ICAM-1及IL-4蛋白与mRNA的表达水平。结果 MP感染BALB/c小鼠7天后,肺组织有大量炎性细胞浸润,给予维生素D3处理后,炎性明显减轻。MP感染组BALB/c小鼠肺组织ICAM-1及IL-4蛋白与mRNA的表达水平显著高于对照组,给予维生素D3处理后,感染MP的BALB/c小鼠肺组织ICAM-1及IL-4蛋白与mRNA的表达水平显著降低,P0.01。结论维生素D3能够降低感染肺炎支原体感染小鼠的肺内炎性反应,可能与降低ICAM-1及IL-4表达相关。     

肠道厌氧菌失调加重流感病毒感染小鼠肺部免疫病理损伤

目的探讨肠道厌氧菌失调与流感病毒感染之间的关系,以及对小鼠肺部免疫细胞中炎性因子的影响。方法将36只BALB/c小鼠随机分为正常对照组、病毒对照组、甲硝唑组,每组12只。甲硝唑组小鼠用18 mg/m L甲硝唑灌胃8 d,造成小鼠肠道厌氧菌菌群失调;再予每只小鼠50μL/d FM1株流感病毒连续滴鼻4 d,建立厌氧菌失调小鼠感染流感病毒的动物模型。观察各组小鼠精神状态、肺指数、肺组织病理形态变化、盲肠及肠黏膜病理形态变化,ELISA测定小鼠肺组织匀浆中白细胞介素4(IL-4)、γ干扰素(IFN-γ)、IL-10和IL-17含量的变化。结果甲硝唑能使该组小鼠肺指数明显升高,其肺组织及盲肠组织病理改变较病毒对照组小鼠而言,表现出更为严重的炎症损伤。FM1甲型流感病毒感染后,甲硝唑组小鼠肺组织匀浆中IFN-γ、IL-17水平与病毒对照组相比显著降低,IL-4、IL-10含量虽有升高,但差异无统计学意义。结论肠道厌氧菌失调可能通过调节小鼠肺部炎性因子的分泌,抑制其适应性免疫应答,影响FM1流感病毒在体内复制及清除的时间,从而进一步加重流感病毒引起的免疫病理损伤。     

嵌合人偏肺病毒(hMPV)表位的重组流感病毒免疫保护效果

目的评价重组嵌合表达人偏肺病毒(hMPV)表位的流感病毒的免疫应答及免疫保护效果。方法用8质粒系统拯救出的在NS基因中嵌合hMPV表位的重组流感病毒免疫BALB/c小鼠,检测其刺激机体产生的针对hMPV和流感病毒的抗体滴度,及脾细胞分泌细胞因子含量。免疫后小鼠用hMPV和流感病毒进行攻击,对攻击后小鼠肺组织和鼻甲骨中病毒含量采用数字PCR方法进行测定,同时对肺组织进行HE染色,评价其对肺部损伤的保护作用。结果重组流感病毒株rFLU/hMPV/B(嵌入了hMPV的B细胞表位)免疫小鼠后,产生了针对hMPV和流感病毒的特异性抗体。rFLU/hMPV/CTL+Th(嵌入了CTL表位/Th细胞表位)免疫小鼠后,产生了针对流感病毒的特异性抗体及hMPV特异的细胞毒性T淋巴细胞反应(IFN-γ分泌增多)。rFLU/hMPV/B和rFLU/hMPV/CTL+Th引起了平衡的Th1/Th2免疫应答。用hMPV和流感病毒攻击重组病毒免疫后小鼠,肺组织病理HE染色结果显示重组病毒免疫后小鼠肺病变明显减轻,肺组织和鼻甲骨中hMPV和流感病毒含量也明显减少。结论重组的2株嵌合有hMPV B细胞表位和CTL表位/Th细胞表位的重组流感病毒株,可刺激机体产生针对hMPV和/或流感病毒体液免疫应答,嵌合有hMPV CTL表位/Th细胞表位的重组流感病毒株还可刺激机体产生hMPV特异性的细胞免疫应答,对hMPV和流感病毒攻击也有一定保护作用,该重组病毒株有潜在疫苗应用价值。     

小鼠感染流感病毒后可抵抗致死性流感病毒攻击的研究

目的 BALB/c小鼠初次感染流感病毒(A/California/7/2009(H1N1)(pCA07)和A/Guangzhou/333/99(H9N2)(GZ333))后,用流感病毒鼠肺适应株A/PR/8/34(H1N1)(PR8) 10倍致死剂量攻击,观察攻毒后小鼠的反应.方法 BALB/c小鼠150只,分成3组,空白对照组PBS滴鼻,实验组分别感染甲型H1N1流感病毒pCA07和禽源H9N2病毒GZ333；感染56 d后用10倍致死剂量的鼠肺适应株流感病毒PR8攻击两个实验组和对照组小鼠,比较PR8病毒攻击后,病毒载量和抗体水平,以及对小鼠存活率的影响.结果 感染过pCA07和GZ333的小鼠在致死病毒PR8攻击后全部存活,并分别在病毒攻击6d和9d后小鼠肺组织中检测不到攻击病毒.对初次感染病毒的抗体水平在病毒PR8攻击后迅速升高,在保持初次感染病毒抗体的同时能够产生针对PR8病毒的抗体.结论 不同亚型流感病毒感染小鼠后可以提供交叉保护.     

A Live Attenuated Human Metapneumovirus Vaccine Strain Provides Complete Protection against Homologous Viral Infection and Cross-Protection against Heterologous Viral Infection in BALB/c Mice

A live attenuated vaccine candidate strain (M2) of human metapneumovirus (hMPV) was generated by removing the N-linked carbohydrate at amino acid 172 in the fusion (F) protein. Previously, replication of M2 in mouse lungs could be detected by molecular assays but not by viral titration. In the present study, the protective effects of M2 against infection by homologous or heterologous viruses were evaluated in BALB/c mice. Immunization with M2 produced a high titer of serum virus-neutralizing antibodies in BALB/c mice at 4 and 8 weeks postimmunization, with the titers against the homologous virus being higher than those against the heterologous virus. Challenges at 4 and 8 weeks postinoculation with M2 or wild-type virus led to no replication when mice were challenged with a homologous virus and extremely reduced replication when mice were challenged with a heterologous virus, as determined by the detection of viral genomic RNA copies in the lungs, as well as significantly milder pulmonary pathology. Thus, M2, with only one N-linked carbohydrate removed in the F protein, provides complete protection from homologous virus infection and substantial cross-protection from heterologous virus infection for at least 56 days after inoculation. This vaccine strain may therefore be a candidate for further preclinical study. Furthermore, this attenuating strategy (changing the glycosylation of a major viral protein) may be useful in the development of other viral vaccines.     

Increased pathogenesis and inflammation of airways from respiratory syncytial virus infection in T cell deficient nude mice

Respiratory syncytial virus (RSV) infection is ubiquitous and leads to various outcomes between immunocompetent and immunocompromised individuals. This study aimed to compare RSV infection and inflammatory responses between immunocompetent BALB/c mice and immunodeficient nude mice. RSV titers in both infected BALB/c mice and nude mice peaked on the third day post-inoculation, but the nude mice had longer lasting and higher levels of viral replication. RSV infection induced a more severe grade of pulmonary histopathology and larger numbers of leukocytes in airways of nude mice than that of BALB/c mice. RSV infection increased pulmonary macrophages and natural killer (NK) cells in both strains of mice. Furthermore, infected nude mice had larger numbers of pulmonary macrophages and NK cells than infected BALB/c mice. Whereas the RSV infected BALB/c mice secreted more tumor necrosis factor -alpha (TNF-alpha), interleukin-12 (IL-12), interferon-gamma (IFN-gamma) and IL-10 than control BALB/c mice, the infected nude mice had higher levels of TNF-alpha, IL-12 and IL-10 than the infected BALB/c mice. The inflammation induced by RSV infection did not correspond with the immune response of T cells. Macrophages and NK cells were potent immunocytes and inflammatory cells in RSV infection especially when T lymphocytes were deficient. Therefore, nude mice may be a good model for severe and persistent RSV infection in immunocompromised hosts.     

Mouse strain differences in the chemokine response to acute lung infection with a murine gammaherpesvirus

Numerous mouse strain-based differences in the immune response and in susceptibility to numerous pathogens have been described, but it is not known if these differences extend to chemokine responses to viral infection of the lungs. To define mouse strain-based differences in the host chemokine response and susceptibility to infection with murine gammaherpesvirus-68 (MHV-68), we compared the induced chemokine response to MHV-68 infection in the lungs of BALB/c and C57BL/6 mice at 1-15 days post-infection. CC and CXC chemokines were induced in both BALB/c and C57BL/6 following infection but the level of chemokine induction was significantly higher in the BALB/c mice for all chemokines measured. In addition, interferon-gamma (IFN-gamma) was also induced to a significantly higher level in the lungs of BALB/c infected mice compared to C57BL/6 mice. Interestingly, viral gene expression was lower in the lungs of C57BL/6 mice during the acute phase of replication. Titers of infectious virus were also greater in BALB/c lungs, although they did not achieve statistical significance. In contrast, latent viral load in the spleen, as measured by quantitative real-time PCR, did not significantly differ between mouse strains, suggesting that the establishment of latency is not affected by the amount of virus present during acute infection. This data suggests that robust chemokine response and expression of IFN-gamma in the lungs of infected BALB/c mice does not correlate with increased resistance to infection. In addition, the significant differences in chemokine responses observed will be important factors to consider in future studies of viral pathogenesis using mouse models.     

Establishment of BALB/C mouse models of influenza A H1N1 aerosol inhalation

Influenza A (H1N1) is a rapidly spreading acute respiratory illness that remains a worldwide burden on public health. To simulate natural infection routes, BALB/C mice were challenged with the H1N1 virus by aerosol and intranasal instillation routes. We compared the weight change and survival of the mice for 14 consecutive days after infection. The infected mice were euthanized at days 3, 5, 7, and 9 to perform necropsies, lung pathological analyses, viral titers measurement, and lung cytokines examination. The aerosol-treated mice showed clinical symptoms on day 4, obvious lung lesions on day 5, rapid weight loss on day 7, peak virus replication in the lungs on days 7 to 9, and bronchial epithelial hyperplasia on day 9. However, after intranasal instillation, the mice exhibited clinical signs on day 2, rapid weight loss and obvious lung lesions on day 3, and peak virus replication in the lungs on days 3 to 5; no bronchial epithelial hyperplasia was detected. High levels of proinflammatory cytokines and chemokines were detected in the lungs of infected mice by both two routes. Disease and lung lesion progressions were slower in the mice that inhaled H1N1-containing aerosols than in those treated by intranasal instillation, and lung lesions were homogeneous in the aerosol group and heterogeneous in the intranasal group. In this study, BALB/C mouse models of H1N1 virus aerosol inhalation were successfully established and compared with mouse models of intranasal inoculation, aerosol mouse models had an infection route and lung pathology characteristics that more closely resembled those observed in humans.     

The mouse is not permissive for equine herpesvirus 2 (EHV-2), however viral DNA persisted in lung and spleen depending on the inoculation route

Summary.  BALB/c mice were inoculated with 3 EHV-2 low passage isolates. After intranasal inoculation, viral DNA was detected by virus-specific nested PCR in the lung up to day 30 post inoculation and in nasal turbinates till day 7. In trigeminal ganglia, olfactory bulb, brain and lymph nodes viral DNA was randomly shown by PCR. After intraperitoneal inoculation viral DNA was present in lymphoid tissues. The spleen was PCR positive up to day 30 and showed a splenomegaly. Clinical signs, virus replication and viraemia, were not observed and no virus strain-specific differences were obvious. Control mice inoculated with equine herpesvirus 4 were PCR negative in all tissues. Received November 5, 2001; accepted March 13, 2002 Published online May 24, 2002     

Lethal synergism induced in mice by influenza type A virus and type Ia group B streptococci.

Intranasal inoculation of CD-1 or BALB/c mice with low doses of influenza A/PR8/34 (HON1) virus followed 48 h later by intranasal inoculation of low doses of type Ia group B streptococci effected a lethal synergism. At a constant input dose of virus, a direct relationship between input dose of bacteria and percent mortality was observed; the converse was also true. An inverse relationship between input dose of group B streptococci, but not input dose of virus, and mean time to death was observed in CD-1 but not in BALB/c mice. The kinetics of influenza A/PR8/34 virus and group B streptococcal replication in singly and dually infected BALB/c mice was determined by assaying samples from the lungs, liver, spleen, and blood for viable group B streptococci and infectious influenza A/PR8/34 virus. No significant difference in virus replication in the lung was observed between singly and dually infected mice. Extrapulmonary dissemination of virus was not observed. Concurrent virus infection effected a 10,000- to 100,000-fold increase in the levels of type Ia group B streptococci in the lung. Potentiation of group B streptococcal infection of the lung was not associated with bacteremia or infection of the liver or spleen, a finding contrary to previous observations of fulminant septicemia after intranasal inoculation of mice with input doses of group B streptococci less than one-tenth of the pulmonary levels observed in the present study.     

Protection from lethal infection is determined by innate immune responses in a mouse model of Ebola virus infection

A mouse-adapted strain of Ebola Zaire virus produces a fatal infection when BALB/cj mice are infected intraperitoneally (ip) but subcutaneous (sc) infection with the same virus fails to produce illness and confers long-term protection from lethal ip rechallenge. To identify immune correlates of protection in this model, we compared viral replication and cytokine/chemokine responses to Ebola virus in mice infected ip (10 PFU/mouse), or sc (100 PFU/mouse) and sc "immune" mice rechallenged ip (10(6) PFU/mouse) at several time points postinfection (pi). Ebola viral antigens were detected in the serum, liver, spleen, and kidneys of ip-infected mice by day 2 pi, increasing up to day 6. Sc-infected mice and immune mice rechallenged ip had no detectable viral antigens until day 6 pi, when low levels of viral antigens were detected in the livers of sc-infected mice only. TNF-alpha and MCP-1 were detected earlier and at significantly higher levels in the serum and tissues of ip-infected mice than in sc-infected or immune mice challenged ip. In contrast, high levels of IFN-alpha and IFN-gamma were found in tissues within 2 days after challenge in sc-infected and immune mice but not in ip-infected mice. Mice became resistant to ip challenge within 48 h of sc infection, coinciding with the rise in tissue IFN-alpha levels. In this model of Ebola virus infection, the nonlethal sc route of infection is associated with an attenuated inflammatory response and early production of antiviral cytokines, particularly IFN-alpha, as compared with lethal ip infection.     

病毒性心肌损伤时总RNA表达及cTnI基因的扩增分析

目的研究心肌型肌钙蛋白(cTnI)-mRNA在监测病毒性心肌损伤发生发展和预后中的价值。方法于BALB/c小鼠腹腔接种1×108TCID50柯萨奇病毒B3(CVB3)液诱发心肌损伤发生,分别在CVB3感染后第3、6、9、12、15、18和21天采集外周血后处死小鼠,留取鼠心脏作病理组织学检查,并以逆转录-聚合酶链反应(RT-PCR)分析心肌及外周血中cTnI基因的表达状态。结果CVB3感染后,鼠心肌组织及循环血中cTnI-mRNA均表达增加,且与心肌细胞肿胀、炎性细胞浸润、核固缩及碎裂、变性、坏死、钙化等组织学改变相关。cTnI-mRNA基因扩增,心肌组织全数阳性;外周血阳性率在对照组及感染后第3、6、9、12、15、18和21天分别为0、0、0、16.7%、40.0%、71.4%、83.3%和87.5%。结论病毒性心肌损伤时cTnI-mRNA上调表达并释放入血,循环血cTnI-mRNA为监测心肌损伤发生发展及预后的灵敏基因标志物。