小花清风藤水提物体内抗流感病毒的研究

目的:研究小花清风藤水提物体内抗流感病毒的活性。方法:采用滴鼻感染流感病毒建立BALB/c小鼠肺炎模型,以小鼠存活率、平均存活时间、肺指数、肺组织中病毒滴度及肺组织病理改变为指标,观察不同剂量小花清风藤水提物体内抗流感病毒的活性。结果:与模型组比较,小花清风藤水提物中剂量(i.g,6.5 g/kg)能提高小鼠生存率30%,延长小鼠存活时间,降低小鼠肺指数和肺组织中病毒滴度,明显改善感染小鼠肺组织炎症病变。结论:小花清风藤水提物在体内具有一定的抗流感病毒的作用。     

流感泰得在小鼠体内抗H1N1型FM1株流感病毒活性研究

目的:研究流感泰得预防给药在小鼠体内对H1N1型FM1株流感病毒的抑制作用及对病毒感染小鼠的保护作用。方法:BALB/c小鼠随机分为正常对照组,病毒对照组,磷酸奥司他韦组,流感泰得2.5、5、10 mg/kg组,提前24h和1 h滴鼻给药2次,于第二次给药1 h后滴鼻感染小鼠造成肺炎模型,连续观察14 d,统计小鼠存活率、平均存活天数、体重变化;按以上方法于造模后第5 d天取肺脏,计算肺指数、肺脏病毒滴度。结果:与病毒对照组相比,流感泰得3个剂量组可明显提高病毒感染小鼠的存活率,延长平均存活时间,缓解病毒感染引起的体重降低,降低病毒感染小鼠的肺指数和肺脏病毒滴度。结论:流感泰得在小鼠体内预防给药具有抗H1N1型FM1株流感病毒活性。     

板蓝根颗粒对甲型流感病毒小鼠的作用

目的测定板蓝根颗粒抗流感病毒的药效作用。方法 A/California/7/2009（CA7）病毒滴鼻感染BALB/c小鼠观察14d,观察板蓝根对甲型H1N1流感病毒感染小鼠的保护作用,计算小鼠存活率、存活天数以及延长生命率。感染的小鼠第5天每组小鼠处死一半,取肺组织,观察板蓝根对甲型H1N1流感病毒感染小鼠肺组织的保护作用。结果板蓝根可明显延长甲型H1N1流感病毒感染小鼠的存活天数并提高存活率,病理结果显示板蓝根对甲型H1N1流感病毒感染的小鼠的肺组织有一定程度的保护作用,与模型组比较差异显著（P〈0.05）。结论板蓝根颗粒对甲型H1N1流感病毒感染的小鼠有较好的保护作用。     

清热消炎复方制剂抗流感病毒作用的研究

为评价清热消炎复方制剂(简称AI)的抗流感病毒活性,我们以病毒唑为对照,通过在体外观察病毒致细胞病变效应(CPE)、MTT细胞染色检查病毒抑制率和检测病毒血凝滴度;在体内观察其对染毒小鼠的死亡保护作用,对小鼠流感病毒性肺炎的抑制作用,以及对小鼠肺内病毒增殖的影响,从而判定其抗流感病毒作用。结果发现AI在160ug/mL时能完全抑制流感病毒在MDCK细胞内的增殖复制作用。体内实验中0.1g/kg,0.5g/kg,1.2g/kg3个剂量均能明显降低染毒小鼠的致死率,延长平均存活时间:降低肺炎小鼠的肺指数和血凝滴度(P<0.01)。其作用与病毒唑相当。结论认为清热消炎复方制剂是一种有效的体内、体外抗流感病毒中药复方制剂。     

清热消炎复方制剂抗流感病毒作用的研究

为评价清热消炎复方制剂(简称AI)的抗流感病毒活性,我们以病毒唑为对照,通过在体外观察病毒致细胞病变效应(CPE)、MTT细胞染色检查病毒抑制率和检测病毒血凝滴度;在体内观察其对染毒小鼠的死亡保护作用,对小鼠流感病毒性肺炎的抑制作用,以及对小鼠肺内病毒增殖的影响,从而判定其抗流感病毒作用.结果发现AI在160ug/mL时能完全抑制流感病毒在MDCK细胞内的增殖复制作用.体内实验中0.1 g/kg,0.5g/kg,1.2g/kg 3个剂量均能明显降低染毒小鼠的致死率,延长平均存活时间;降低肺炎小鼠的肺指数和血凝滴度(P<0.01).其作用与病毒唑相当.结论认为清热消炎复方制剂是一种有效的体内、体外抗流感病毒中药复方制剂.     

流感病毒感染对小鼠肠道菌群的影响及桑叶的调节作用

【摘 要】 目的 探讨流感病毒对肠道菌群的影响以及桑叶提取物在治疗小鼠流感时对肠道菌群的调节作用。方法 将36只小鼠随机分为4组,正常对照组滴鼻生理盐水,其余以流感病毒鼠肺适应株FM1滴鼻3 d,建立流感小鼠模型后,正常对照组和病毒对照组灌胃生理盐水,另2组分别以高剂量(500 mg/kg)和低剂量(100 mg/kg)灌胃桑叶提取物,连续5 d。每天检测小鼠体重,并于病毒感染前、感染后及灌胃处理后3次无菌收集小鼠粪便,培养并计数小鼠粪便中肠杆菌、肠球菌、乳酸杆菌和双歧杆菌。结果 小鼠在感染病毒后体重明显降低,桑叶提取物治疗后体重降低的趋势有所减缓。小鼠肠道菌群在感染流感后有明显的变化,但桑叶提取物治疗后肠道菌群的恢复不明显。结论 流感病毒感染能引起小鼠肠道菌群的失调,桑叶不显示调节肠道菌群的效果,表明其对流感的治疗不通过调节肠道菌群平衡来发挥作用。     

异型流感病毒感染小鼠肺细胞因子水平变化

为了制备能够抵御不同型别流感病毒感染的疫苗,揭示机体对异型流感病毒感染交叉免疫保护作用的主要机制,用流感病毒疫苗免疫小鼠后分别感染同型、异型流感病毒,另设使用免疫增强剂IL-2后感染异型流感病毒组,观察小鼠的一般状况和肺指数,并用ELISA方法测定肺匀浆中细胞因子IFN-γ、IL-2、IL-4及IL-10的含量。结果显示,异型免疫组和异型免疫加强组病毒感染后细胞因子IFN-γ含量明显高于感染前（P〈0.05）。研究表明,异型病毒感染后IFN-γ水平明显增高,此细胞因子可能在流感病毒异型间交叉保护免疫反应中起重要作用,其机制有待进一步研究确定。     

北柴胡茎叶总黄酮抗流感病毒的作用

北柴胡 (BupleurumchinenseDC .)茎叶总黄酮 (TFB) 0 .0 9g kg剂量组对乙型流感病毒 (B 京防 98 76 )感染小鼠具有明显的保护作用 (P <0 .0 5 ) ,0 .0 3g kg和 0 .0 9g kg剂量组能明显降低乙型流感病毒 (B 京防 98 76 )感染小鼠肺指数值 (P <0 .0 5 ,P <0 .0 1) ,肺指数抑制率分别为 2 0 .5 %和 2 5 .4 %。组织病理学检查结果表明 ,TFB各剂量组与模型组比较 ,肺部病变均明显减轻 ,其中以TFB高剂量组肺部总体病变程度最轻 ,疗效最好。     

铁苋菜乙醇提取物抗流感活性及其机理的初步研究

研究铁苋菜乙醇提取物对感染流感病毒小鼠肺部、流感病毒神经氨酸酶活性及其抗氧化活性的影响。采用鼻腔接种建立流感病毒感染的小鼠肺炎模型,观察各组小鼠的肺部病理变化。并以2-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid(MUNANA)为底物,检测铁苋菜乙醇提取物高、中和低剂量组对流感病毒神经氨酸酶活性的影响。利用NO法、DPPH法和β-胡萝卜素漂白法测定了铁苋菜乙醇提取物抗氧化活性。结果显示,在动物实验中,与空白组相比,模型组小鼠肺泡、细支气管等正常的组织结构遭到破坏。与模型组相比,奥司他韦组和铁苋菜乙醇提取物高、中、低剂量组均能明显改善感染的小鼠肺部病理损伤,且改善效果呈明显剂量依赖关系。在流感病毒神经氨酸酶活性抑制实验中,与模型对照组相比,铁苋菜高、中、低剂量组均能明显抑制神经氨酸酶活性(P0.01),高剂量组对其活性的抑制率可达60.48%。铁苋菜乙醇提取物有较好的清除DPPH、NO自由基和抑制β-胡萝卜素氧化活性。研究说明铁苋菜乙醇提取物可能抑制流感病毒神经氨酸酶的活性和抗氧化减轻流感所致的相关炎症损伤。     

季节性流感病毒H1N1 BALB/c鼠肺适应株的建立及其分子机制

目的建立季节性流感病毒H1N1的鼠肺适应株,并对适应的分子机理进行研究。方法以病毒滴鼻感染小鼠,通过在BALB/c小鼠肺组织中连续传代,观察小鼠存活情况及肺病理改变,来获得季节性流感病毒H1N1的鼠肺适应株。结果季节性流感H1N1 A/Brisbane/59/2007病毒野生型毒株,经过在小鼠体内进行8次传代后,毒力逐渐增强,从无致病力到致死率达到100%,对鼠肺适应株与野生型毒株进行基因比对,发现适应株HA基因发生了3个有义突变。结论野生季节性低致病力H1N1流感病毒可经在小鼠中经过多次传代而获得高致病力H1N1鼠肺适应株,HA蛋白89位Thr至Ile的突变对毒力的增强起决定性作用。     

Therapeutic effect of anti-macrophage inflammatory protein 2 antibody on influenza virus-induced pneumonia in mice

We investigated the effect of anti-macrophage inflammatory protein 2 immunoglobulin G (aMIP-2 IgG) on the progression of influenza virus-induced pneumonia in mice. When mice were infected with a mouse lung-adapted strain of influenza A/PR/8/34 virus by intranasal inoculation, neutrophil counts in the bronchoalveolar lavage fluid (BALF) increased in parallel with the kinetics of MIP-2 production, which peaked 2 days after infection. After intracutaneous injection of a dose of 10 or 100 microg of aMIP-2 IgG once a day on days 0 and 1, neutrophil counts in BALF on day 2 were reduced to 49 or 37%, respectively, of the value in the control infected mice administered anti-protein A IgG. The antibody administration also improved lung pathology without affecting virus replication. Furthermore, by prolonged administration with a higher or lower dose for up to 5 days, body weight loss became slower and finally 40% of mice in both treatment groups survived potentially lethal pneumonia. These findings suggest that MIP-2-mediated neutrophil infiltration during the early phase of infection might play an important role in lung pathology. Thus, MIP-2 was considered to be a novel target for intervention therapy in potentially lethal influenza virus pneumonia in mice.     

Inhibition of lectin-mediated innate host defences in vivo modulates disease severity during influenza virus infection

Host-mediated recognition of mannose-rich glycans on the surface of pathogens represents an ancient mechanism of innate immune defence. In this study, we demonstrate that the virus strains that differ in the degree of N-linked glycosylation on the globular head of their hemagglutinin glycoprotein also differed in their (i) sensitivity to neutralization by a mannose-specific lectin in mouse lung fluids and (ii) ability to infect (and, therefore, to be destroyed) by airway macrophages. Virus strain BJx109 (H3N2), but not PR8 (H1N1), was sensitive to neutralization by mouse lung fluids and infected airway macrophages efficiently in vitro and these antiviral activities were blocked by mannan, a complex polymer of mannose residues. Although intranasal (i.n.) infection of mice with PR8 led to severe disease and mortality, mice infected with an equivalent dose of BJx109 displayed no signs of disease. However, i.n. treatment of BJx109-infected mice with mannan led to viral pneumonia, severe disease and death characterized by excessive virus replication, pulmonary inflammation, vascular leak and lung edema. Thus, when mannose-specific innate defences were inhibited in vivo, virus strain BJx109 induced severe viral pneumonia similar to that of PR8. Together, these findings highlight the importance of N-linked glycans as a target for recognition and destruction of influenza viruses by the innate immune system. Moreover, soluble and cell-associated lectins coordinate to modulate disease severity following influenza virus infection of mice.     

小檗碱改善流感病毒性肺炎小鼠肺血管通透性的作用及机制

目的:观察小檗碱对流感病毒感染所致病毒性肺炎小鼠肺血管通透性的影响,并探讨其作用机制。方法:BALB/c小鼠108只随机分为3组,正常组、模型组、小檗碱组,25μL 50LD50病毒液滴鼻建立流感病毒感染的小鼠肺炎模型,感染后1 h,正常组和模型组予以双蒸水灌胃,小檗碱组予药物0.005 g.kg-1d-1腹腔注射;各组均给药2次/d,连续给药5 d。感染后的2 d、4 d、6 d,处死小鼠,肺组织称重以检测肺含水量;1%伊文氏兰5 mL/kg尾静脉注射检测肺血管通透性;Bicinchoninic acid(BCA)法检测肺泡灌洗液(BALF)中蛋白含量;放免法或酶免法测定肺组织中PGE2、PLA2及LT-B4含量。结果:病毒感染后,模型组肺含水量持续升高,肺血管通透性及BALF蛋白含量在感染后第4天开始明显升高,小檗碱降低了肺含水量、肺血管通透性及BALF蛋白含量(P<0.01);模型组肺组织中PGE2、PLA2、LT-B4的含量明显升高,小檗碱不同程度地抑制了PGE2、PLA2、LT-B4的表达。结论:小檗碱通过抑制流感病毒感染后肺组织中PGE2、PLA2、LT-B4的释放,降低了肺血管通透性及肺含水量,对病毒性肺炎中肺水肿的形成,起到一定的治疗作用。     

Serial histopathological examination of the lungs of mice infected with influenza A virus PR8 strain

Avian influenza H5N1 and pandemic (H1N1) 2009 viruses are known to induce viral pneumonia and subsequent acute respiratory distress syndrome (ARDS) with diffuse alveolar damage (DAD). The mortality rate of ARDS/DAD is extremely high, at approximately 60%, and no effective treatment for ARDS/DAD has been established. We examined serial pathological changes in the lungs of mice infected with influenza virus to determine the progress from viral pneumonia to ARDS/DAD. Mice were intranasally infected with influenza A/Puerto Rico/8/34 (PR8) virus, and their lungs were examined both macro- and micro-pathologically every 2 days. We also evaluated general condition, survival rate, body weight, viral loads in lung, and surfactant proteins in serum. As a result, all infected mice died within 9 days postinfection. At 2 days postinfection, inflammation in alveolar septa, i.e., interstitial pneumonia, was observed around bronchioles. From 4 to 6 days postinfection, interstitial pneumonia with alveolar collapse expanded throughout the lungs. From 6 to 9 days postinfection, DAD with severe alveolar collapse was observed in the lungs of all of dying and dead mice. In contrast, DAD was not observed in the live infected-mice from 2 to 6 days postinfection, despite their poor general condition. In addition, histopathological analysis was performed in mice infected with a dose of PR8 virus which was 50% of the lethal dose for mice in the 20-day observation period. DAD with alveolar collapse was observed in all dead mice. However, in the surviving mice, instead of DAD, glandular metaplasia was broadly observed in their lungs. The present study indicates that DAD with severe alveolar collapse is associated with death in this mouse infection model of influenza virus. Inhibition of the development of DAD with alveolar collapse may decrease the mortality rate in severe viral pneumonia caused by influenza virus infection.     

Respiratory disease and wasting in athymic mice infected with pneumonia virus of mice

Although pneumonia virus of mice (PVM) is ubiquitous among rodent colonies in the United States, it has not been reported to cause clinically apparent disease in euthymic mice. However, PVM has been reported to cause respiratory disease and death in experimentally infected euthymic and athymic mice. A group of nu/nu mice, housed in quarantine in a Trexler-type isolator, had weight loss and dyspnea. Gross necropsy findings included cachexia and diffuse pulmonary edema or lobar consolidation. Histologically there was diffuse interstitial pneumonia. Electron microscopy revealed filamentous virions budding from plasma membranes, and immunohistochemical staining of lung tissue was positive for PVM antigen. PVM was isolated from affected lung tissue in BHK 21 cells and mouse antibody production tests resulted in seroconversion to PVM. Experimental inoculation of athymic mice with lung homogenate from spontaneously infected mice resulted in clinically apparent respiratory disease and histologic lung changes similar to those in naturally infected mice. Inoculation of athymic mice with infected BHK 21 cell culture fluid resulted in pneumonia which was qualitatively similar to, but less severe than, that observed in mice with spontaneous disease. These findings indicate that naturally occurring PVM infection in athymic mice may cause respiratory disease and wasting.     

Defective interfering influenza virus inhibits immunopathological effects of infectious virus in the mouse.

Mice inoculated intranasally with a lethal dose of standard influenza virus die with an immune-mediated pneumonia but are protected by coinoculation with defective interfering (DI) virus. Here we show that recruitment of immune cells into the infected lung is halved by treatment with DI virus although the CD4+/CD8+ cell ratio is not affected. Responsiveness of lung T and B cells to lectins is inhibited by standard virus, but coinoculation of mice with DI virus causes a 13-fold increase in T-cell proliferation and up to a 100-fold increase in immunoglobulin production. This effect appears to be due to lymphocyte-specific DI virus-mediated interference, since there is no inhibition of virus multiplication in the lungs. The net result is a shift from a lethal to a beneficial immune response.     

Molecular and cellular basis of the retrovirus resistance in I/LnJ mice

Previously, we showed that IFN-gamma elicited by mouse mammary tumor virus (MMTV) infection in I/LnJ mice stimulated production of virus-neutralizing Abs, mostly of the IgG2a isotype. These Abs coated virions secreted by infected I/LnJ cells, and thus completely prevented virus transmission to offspring. However, the mechanism of virus neutralization by isotype-specific Abs remained unknown. Ab coating is capable of blocking virus infection by interfering with receptor-virus binding, by virus opsonization, by complement activation, and via FcgammaR-mediated effector mechanisms. The aim of the studies described in this work was to uncover the cellular basis of anti-virus Ab production, to evaluate the importance of the IgG2a subclass of IgGs in virus neutralization, and to investigate which of the blocking mechanisms plays a role in virus neutralization. We showed that I/LnJ-derived bone marrow cells, specifically IFN-gamma-producing CD4+ T cells, were key cells conferring resistance to MMTV infection in susceptible mice upon transfer. We also established that a unique bias in the subclass selection toward the IgG2a isotype in infected I/LnJ mice was not due to their potent neutralizing ability, as anti-virus Abs of other isotypes were also able to neutralize the virus, but were a product of virally induced IFN-gamma. Finally, we demonstrated that F(ab')2 of anti-MMTV IgGs neutralized the virus as efficiently as total IgGs, suggesting that Ab-mediated interference with viral entry is the sole factor inhibiting virus replication in I/LnJ mice. We propose and discuss possible mechanisms by which infected I/LnJ mice eradicate retrovirus.     

Laninamivir Octanoate and Artificial Surfactant Combination Therapy Significantly Increases Survival of Mice Infected with Lethal Influenza H1N1 Virus

Background

Patients with influenza virus infection can develop severe pneumonia and acute respiratory distress syndrome (ARDS) which have a high mortality. Influenza virus infection is treated worldwide mainly by neuraminidase inhibitors (NAIs). However, monotherapy with NAIs is insufficient for severe pneumonia secondary to influenza virus infection. We previously demonstrated that mice infected with a lethal dose of influenza virus develop diffuse alveolar damage (DAD) with alveolar collapse similar to that seen in ARDS in humans. Additionally, pulmonary surfactant proteins were gradually increased in mouse serum, suggesting a decrease in pulmonary surfactant in the lung. Therefore, the present study examined whether combination therapy of NAI with exogenous artificial surfactant affects mortality of influenza virus-infected mice.

Methodology/Principal Findings

BALB/c mice were inoculated with several viral doses of influenza A/Puerto Rico/8/34 (PR8) virus (H1N1). The mice were additionally administered exogenous artificial surfactant in the presence or absence of a new NAI, laninamivir octanoate. Mouse survival, body weight and general condition were observed for up to 20 days after inoculation. Viral titer and cytokine/chemokine levels in the lungs, lung weight, pathological analysis, and blood O₂ and CO₂ pressures were evaluated. Infected mice treated with combination therapy of laninamivir octanoate with artificial surfactant showed a significantly higher survival rate compared with those that received laninamivir octanoate monotherapy (p = 0.003). However, virus titer, lung weight and cytokine/chemokine responses were not different between the groups. Histopathological examination, a hydrostatic lung test and blood gas analysis showed positive results in the combination therapy group.

Conclusions/Significance

Combination therapy of laninamivir octanoate with artificial surfactant reduces lethality in mice infected with influenza virus, and eventually suppresses DAD formation and preserves lung function. This combination could be effective for prevention of severe pneumonia secondary to influenza virus infection in humans, which is not improved by NAI monotherapy.     

The effect of pneumonia induced in mice with Mycoplasma pulmonis on resistance to subsequent bacterial infection and the effect of a respiratory infection with Sendai virus on the resistance of mice to Mycoplasma pulmonis.

The effect of pneumonia induced by Mycoplasma pulmonis in mice on the resistance of the lung to additional bacterial infection was examined. The effect of pneumonia induced by Sendai virus on the resistance of mice to M. pulmonis was also investigated and compared with the effect of Sendai virus on resistance to Staphylococcus aureus. Sendai virus infection decreased subsequent resistance to M. pulmonis in proportion to the virus dose. Decreased resistance to subsequent S. aureus and M. pulmonis infection was greatest at about the same time after inoculation of virus and was related to virus-induced lesions. Besides affecting the resistance of mice to subsequent mycoplasma infection, Sendai virus could enhance an existing mycoplasma infection. Pneumonia induced by M. pulmonis did not decrease resistance to subsequent bacterial infection. The mechanism whereby Sendai virus decreases host resistance is therefore similar for bacteria and mycoplasmas, but pneumonia induced by mycoplasmas does not have the same effect.     

高致病性禽流感（H5N1）实验流行病学初探——感染途径

目的观测不同感染途径实验性感染BALB／c小鼠后的感染状况,了解H5N1病毒的感染特点,为更深入的流行病学研究提供理论基础。方法分别通过口腔接种、污染饲料、腹腔注射、皮肤损伤途径感染6—8周龄BALB／c小鼠,定期安乐动物采血及各器官组织进行血清学、病原学、病理学检查,记录抗体变化、病毒分离情况及病理学改变。结果各途径感染小鼠感染后出现竖毛、弓背、觅食减少、体重减轻、精神呆滞及神经系统症状,肺组织病毒分离及RT-PCR阳性,病理表现间质性肺炎。感染后第7d动物血清中可检测到H5N1抗体。结论通过口腔接种、污染饲料、腹腔注射及皮肤损伤多种途径感染BALB／c小鼠,均能发生感染,提示呼吸道以外的感染方式不容忽视,有益于人类更好的防控禽流感;可通过多种方式建模,深入研究禽流感的发病机制。