荧光定量RT-PCR方法检测感染小鼠不同组织中的H5N1禽流感病毒

目的测定H5N1禽流感病毒感染小鼠各脏器组织中的病毒核酸含量,了解病毒复制与疾病进程的关系,为H5N1禽流感病毒致病机理与防治研究提供技术支持。方法利用建立的荧光定量RT-PCR法对H5N1禽流感病毒感染BALB/c小鼠的不同脏器组织中病毒核酸进行定量检测,观察H5N1禽流感病毒在不同组织中的复制情况。结果利用荧光定量RT-PCR法可从H5N1禽流感病毒感染小鼠后第3d的肺脏、脑、脾脏、肾脏和肝脏组织中检测到不同含量的病毒核酸;而在感染后4d的肺脏、脑和脾脏等组织中的病毒核酸含量开始下降;在感染后第6d,H5N1禽流感病毒核酸在肺脏和脑组织中大量存在外,其余组织中均有少量病毒核酸。结论H5N1流感病毒感染小鼠肺脏和脑组织中病毒核酸含量较高,说明病毒主要在小鼠的肺脏和脑组织中复制。由于荧光定量RT-PCR法可对感染小鼠不同脏器组织中的病毒核酸进行准确定量,可为H5N1禽流感病毒所致疾病的早期诊断、流行病学和致病机理研究提供技术支持。     

H5N1禽流感病毒感染孕鼠的研究

目的用H5N1禽流感病毒感染昆明孕鼠,检测病毒在感染孕鼠各组织脏器中的复制及分布情况,并证明病毒能否通过孕鼠的胎盘垂直传染给胎鼠。方法用虎源H5N1亚型禽流感病毒滴鼻感染妊娠10-12 d的昆明孕鼠,观察孕鼠感染后的临床症状。接种病毒后第3、4、5、6和7 d分别处死3只孕鼠,取孕鼠的肺、脑、脾、肾、子宫、胎盘及胎鼠,利用RT-PCR、Real-time PCR和病毒分离方法检测各组织中的病毒核酸和病毒滴度,并进行病理组织学与免疫组织化学检测。结果昆明孕鼠接种病毒后第3 d,即可在肺、脑、脾、肾、子宫及胎盘组织中检测出H5N1禽流感病毒核酸,并从子宫、胎盘分离出H5N1禽流感病毒;感染后第6 d,从胎鼠体内检测到病毒核酸并分离出H5N1禽流感病毒。结论H5N1亚型禽流感病毒可以感染孕鼠,在孕鼠子宫和胎盘复制,感染后期可通过胎盘屏障传给胎鼠。     

Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo

Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium’s protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.Acute lung injury is a continuum of clinical and radiographic changes, terminating at its most severe, with acute respiratory distress syndrome. Infection with highly pathogenic avian influenza (HPAI) viruses of the H5N1 and more recent H7N9 subtypes often leads to acute lung injury whereas seasonal influenza viruses and the 2009 pandemic H1N1 influenza viruses do so more rarely. The underlying mechanisms of influenza-related acute lung injury remain unclear, and effective therapies are lacking. Viruses that are highly pathogenic to humans (e.g., H5N1 viruses) may differ intrinsically from the less pathogenic (LP) (e.g., seasonal H1N1) viruses in their replication competence, cell tropism, and/or cytokine dysregulation (1, 2). Early treatment of H5N1 disease with the antiinfluenza drug oseltamivir is helpful but does not ensure a favorable outcome (3). Thus, effective adjunctive therapies that do not compromise beneficial host defenses are needed (4).H5N1 (5) and H7N9 (6) influenza viruses target alveolar epithelial cells, which form the crucial gas exchange interface in the lung. These cells also help to maintain intraalveolar and intravascular fluid homeostasis by vectorial transport of sodium, chloride, and water from the apical to the basolateral surface of the alveolar epithelium [alveolar fluid clearance (AFC)]. Impaired AFC and increased alveolar protein permeability (APP) contribute to acute lung injury (7). Therapies that normalize alveolar fluid clearance are likely to be free of off-target effects, unlike immunomodulation, that may promote virus replication.Human bone marrow-derived multipotent mesenchymal stromal cells (MSCs) have applications in multiple clinical disorders, including sepsis, myocardial infarction, diabetes, and acute renal failure (8). Allogeneic MSC therapy has beneficial preclinical effects on endotoxin-, bacteria-, and ventilator-induced acute lung injury (9) via MSC secretion of the soluble paracrine growth factors angiopoietin-1 (Ang1) and keratinocyte growth factor (KGF) (9, 10). MSCs can also transfer mitochondria and microvesicles that modulate immunity and epithelial response to injury (11). Current clinical trials are testing MSCs as a therapy for sepsis and acute respiratory distress syndrome (12). However, little is known about the impact of MSCs on acute respiratory viral infections, including influenza, with the exception of a study in which MSCs failed to reduce influenza-induced lung injury in mice (13). Here, we showed that influenza A/H5N1 virus infection dysregulates AFC and APP in vitro by inducing infected cells to release soluble mediators that down-regulate alveolar sodium and chloride transporters. When we cocultured alveolar epithelium with MSCs, these injury mechanisms were prevented or reduced. We then treated mice infected with influenza A/H5N1 with MSCs and demonstrated a clinically significant reduction in lung pathology and increased survival in association with a modulation of these pathogenic mechanisms in vivo.     

The Influenza Virus H5N1 Infection Can Induce ROS Production for Viral Replication and Host Cell Death in A549 Cells Modulated by Human Cu/Zn Superoxide Dismutase (SOD1) Overexpression

Highly pathogenic H5N1 infections are often accompanied by excessive pro-inflammatory response, high viral titer, and apoptosis; as such, the efficient control of these infections poses a great challenge. The pathogenesis of influenza virus infection is also related to oxidative stress. However, the role of endogenic genes with antioxidant effect in the control of influenza viruses, especially H5N1 viruses, should be further investigated. In this study, the H5N1 infection in lung epithelial cells decreased Cu/Zn superoxide dismutase (SOD1) expression at mRNA and protein levels. Forced SOD1 expression significantly inhibited the H5N1-induced increase in reactive oxygen species, decreased pro-inflammatory response, prevented p65 and p38 phosphorylation, and impeded viral ribonucleoprotein nuclear export and viral replication. The SOD1 overexpression also rescued H5N1-induced cellular apoptosis and alleviated H5N1-caused mitochondrial dysfunction. Therefore, this study described the role of SOD1 in the replication of H5N1 influenza virus and emphasized the relevance of this enzyme in the control of H5N1 replication in epithelial cells. Pharmacological modulation or targeting SOD1 may open a new way to fight H5N1 influenza virus.     

Cytoprotection of heme oxygenase-1/carbon monoxide in lung injury

Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) have been the major cause of morbidity and mortality in intensive care units (ICU) over the past decades despite advances in therapeutic modalities. This syndrome is characterized by noncardiogenic pulmonary edema, and pulmonary and systemic inflammation resulting in respiratory failure (1, 2). Both exudative and proliferative organizing phases of ARDS/ALI have been described pathologically (3, 4). The exudative phase is often called diffuse alveolar damage (DAD) characterized by inflammation and hyaline membrane composed of fibrin and cellular debris (3--5). Pulmonary alveolar cell death is the major pathologic change during the exudative phase in DAD. Repair and remodeling of injured lung cells occur during the proliferative phase, characterized by hyperplasia of alveolar type II cells and fibroblast proliferation (3-5). A variety of cellular insults can cause ALI/ARDS including but not limited to sepsis, trauma, drugs, high concentration of oxygen therapy, and mechanical ventilation (1, 2, 5). The broad spectrum of insults that potentially cause ARDS highlights the complexity of pathogenesis of this syndrome.     

山西省首例高致病性禽流感A/H5N1病毒感染患者的救治体会

目的 探索高致病性禽流感A/H5N1病毒感染(简称人禽流感)病例的临床特点及治疗体会.方法 对2009年1月17口山西省太原市第四人民医院收治的1例人禽流感患者的临床表现、实验室检查、影像学改变及治疗方法等进行总结.结果 患者鼻咽抽取物A/H5N1病毒经逆转录聚合酶链反应(RT-PCR)、实时PCR榆测A/H5N1病毒核酸阳性,确诊为人禽流感病例.患儿女,2岁11个月,体重11 kg.患儿平素体健,发病的有活禽屠宰市场环境暴露史.以发热、咳嗽、呼吸困难为主要症状;肺部病灶进展快,病程第8天肺部病变迅速进展,x线胸片呈"白肺",病情迅速进展为ARDS,静脉应用甲泼尼龙165 mg(15 mg·kg-1·d-1)及无创通气治疗,病程第9天病情有所好转,肺部病灶明显吸收;病程第11天开始给予奥司他韦及100 ml高滴度A/H5N1病毒疫苗免疫血浆,后患者病情日见好转,氧合指数及各种酶学指标恢复正常,肺内病灶明显吸收;病程第28大患者好转出院.结论 人禽流感发现晚、病情重、进展快,临床主要表现为严重的ARDS;有效的氧疗及呼吸支持是治疗的基础;糖皮质激素对肺部病灶的吸收有一定疗效,使用剂量有待进一步探讨;A/H5N1病毒疫苗免疫血浆治疗可能对改善患者的预后有一定帮助.     

恒河猴H5N1禽流感病毒性肺炎模型建立及其发病机制

目的 探讨H5N1病毒(AF148678/ACGoose/Guangdong/11961H5N1)感染恒河猴诱发自初期至恢复期过程肺炎的机制。方法 以H5N1 病毒液经鼻滴入恒河猴,染毒后第0 h、12 h、1 d、2 d、3 d、4 d、6 d、10 d、12 d和14 d采血作血液学和流感特异抗体检测。染毒后第1、3、6、14 d分别剖杀1 只恒河猴,取气管支气管淋巴结、肺、心、肝、肾、大脑及小脑进行病毒分离、滴定、病理及免疫组化检测并观察感染后临床表现。结果 猴感染后出现发热、呼吸困难,食欲下降等。病毒仅在肺组织复制,主要侵犯下呼吸道的肺泡上皮细胞和肺巨噬细胞。感染后第1~3 d引起暂时性的严重肺炎,主要为中性粒细胞浸润;第6 d后逐渐恢复,以巨噬细胞浸润为主;第14 d后以T淋巴细胞浸润为主,肺组织呈恢复期状态。结论 下呼吸道是H5N1病毒感染主要攻击的对象,这可能是H5N1病毒在人与人之间传递的障碍之一,其发病可能经过病毒侵入、复制阶段及免疫损伤阶段。     

Viral Load and Patterns of SARS-CoV-2 Dissemination to the Lungs,Mediastinal Lymph Nodes,and Spleen of Patients with COVID-19 Associated Lymphopenia

Lymphopenia is a frequent hematological manifestation, associated with a severe course of COVID-19, with an insufficiently understood pathogenesis. We present molecular genetic immunohistochemical, and electron microscopic data on SARS-CoV-2 dissemination and viral load (VL) in lungs, mediastinum lymph nodes, and the spleen of 36 patients who died from COVID-19. Lymphopenia <1 × 10⁹/L was observed in 23 of 36 (63.8%) patients. In 12 of 36 cases (33%) SARS-CoV-2 was found in lung tissues only with a median VL of 239 copies (range 18–1952) SARS-CoV-2 cDNA per 100 copies of ABL1. Histomorphological changes corresponding to bronchopneumonia and the proliferative phase of DAD were observed in these cases. SARS-CoV-2 dissemination into the lungs, lymph nodes, and spleen was detected in 23 of 36 patients (58.4%) and was associated with the exudative phase of DAD in most of these cases. The median VL in the lungs was 12,116 copies (range 810–250281), lymph nodes—832 copies (range 96–11586), and spleen—71.5 copies (range 0–2899). SARS-CoV-2 in all cases belonged to the 19A strain. A immunohistochemical study revealed SARS-CoV-2 proteins in pneumocytes, alveolar macrophages, and bronchiolar epithelial cells in lung tissue, sinus histiocytes of lymph nodes, as well as cells of the Billroth pulp cords and spleen capsule. SARS-CoV-2 particles were detected by transmission electron microscopy in the cytoplasm of the endothelial cell, macrophages, and lymphocytes. The infection of lymphocytes with SARS-CoV-2 that we discovered for the first time may indicate a possible link between lymphopenia and SARS-CoV-2-mediated cytotoxic effect.     

关于WHO 2005年以来高致病性H5N1禽流感病毒的临床和治疗进展

自1997年中国香港特别行政区报道首例人感染H5N1禽流感病毒以来,截至2008年9月10日,这场在禽类中史无前例地持续流行、造成了人类感染并具有高病死率的疫情已经波及到15个国家和地区,总发病387例,死亡245例,其中我国疫情30例,病死率超过60%,防控形势十分严峻.本文旨在整理分析2005年以来世界卫生组织和全球对禽流感病毒临床防治研究成果及人感染H5N1禽流感病例报道的最新信息,力求提供一个防治人禽流感方面的较为全面而清晰的介绍.     

Immunofluorescence determination of the pathogenesis of infection with influenza virus in mice following exposure to aerosolized virus.

The pathogenesis of infection with influenza A virus in mice was studied by exposure of specific pathogen-free mice to aerosols of influenza virus and by monitoring of mortality, viral titers in lung homogenates, and presence of viral antigens in respiratory cells as determined by immunofluorescence. In two experiments with different death rates (100% and 43%), viral antigen accumulated in the epithelial cells lining the airways, in alveolar macrophages, in alveolar cells, and in visceral pleura. By enumeration of the number of airways, alveolar macrophages, and alveolar cells containing influenza viral antigens at different intervals after exposure to the viral aerosol, it was determined that viral replication occurred initially in the epithelial cells lining the airways and later extended to the alveolar macrophages and alveolar cells. This semiquantitative survey of the dynamics of influenza viral infection by aerosol indicated that the viral infection in mice was a descending process.     

H5N1 influenza virus-induced mediators upregulate RIG-I in uninfected cells by paracrine effects contributing to amplified cytokine cascades

Highly pathogenic avian influenza H5N1 viruses cause severe disease in humans, and dysregulation of cytokine responses is believed to contribute to the pathogenesis of human H5N1 disease. However, mechanisms leading to the increased induction of proinflammatory cytokines by H5N1 viruses are poorly understood. We show that the innate sensing receptor RIG-I is involved in interferon regulatory factor 3 (IRF3), NF-κB nuclear translocation, p38 activation, and the subsequent interferon (IFN) β, IFN-λ1, and tumor necrosis factor α induction during H5N1 infection. Soluble mediators from H5N1-infected human macrophages upregulate RIG-I, MDA5, and TLR3 to much higher levels than those from seasonal H1N1 in uninfected human macrophages and alveolar epithelial cells via paracrine IFNAR1/JAK but not IFN-λ receptor signaling. Compared with H1N1 virus-induced mediators, H5N1 mediators markedly enhance the cytokine response to PolyIC and to both seasonal and H5N1 virus infection in a RIG-I-dependent manner. Thus, sensitizing neighboring cells by upregulation of RIG-I contributes to the amplified cytokine cascades during H5N1 infection.     

Histopathological findings in a critically ill patient with avian influenza A (H7N9)

To date, data regarding the pulmonary histopathology of human H7N9 disease are scarce. We herein describe a patient with a severe case of avian influenza A (H7N9). A chest computerized tomography (CT) scan showed diffuse ground-glass opacities and consolidation throughout the lungs. A resection of pulmonary bullae in the right middle lobe was performed by video-assisted thoracic surgery (VATS) based on the extracorporeal membrane oxygenation (ECMO) supportive technique on the 23^rd day after the onset of symptoms because of a right pneumothorax persistent air leak. The histopathological findings of the resected lung tissue revealed pneumocyte hyperplasia and fibroproliferative changes along with diffuse alveolar damage. Bronchoalveolar lavage fluid (BALF) specimens for influenza A (H7N9) virus were continuously positive for more than three weeks, despite oseltamivir treatment, and continuous viral replication significantly prolonged the course of the disease. The patient’s clinical status continuously deteriorated, with the development of refractory hypoxemia due to progressive and rapid lung fibrosis, which was confirmed by the final histological changes observed from a limited post-mortem biopsy of lung tissue. Pre-terminally, he developed multi-organ failure and died on the 39^th day after symptom onset, despite corticosteroid treatment.     

Preventive Activity against Influenza (H1N1) Virus by Intranasally Delivered RNA-Hydrolyzing Antibody in Respiratory Epithelial Cells of Mice

The antiviral effect of a catalytic RNA-hydrolyzing antibody, 3D8 scFv, for intranasal administration against avian influenza virus (H1N1) was described. The recombinant 3D8 scFv protein prevented BALB/c mice against H1N1 influenza virus infection by degradation of the viral RNA genome through its intrinsic RNA-hydrolyzing activity. Intranasal administration of 3D8 scFv (50 μg/day) for five days prior to infection demonstrated an antiviral activity (70% survival) against H1N1 infection. The antiviral ability of 3D8 scFv to penetrate into epithelial cells from bronchial cavity via the respiratory mucosal layer was confirmed by immunohistochemistry, qRT-PCR, and histopathological examination. The antiviral activity of 3D8 scFv against H1N1 virus infection was not due to host immune cytokines or chemokines, but rather to direct antiviral RNA-hydrolyzing activity of 3D8 scFv against the viral RNA genome. Taken together, our results suggest that the RNase activity of 3D8 scFv, coupled with its ability to penetrate epithelial cells through the respiratory mucosal layer, directly prevents H1N1 virus infection in a mouse model system.     

Dhori virus (Orthomyxoviridae: Thogotovirus) infection in mice: a model of the pathogenesis of severe orthomyxovirus infection

After intranasal, subcutaneous, or intraperitoneal infection with Dhori virus (DHOV), adult mice developed a fulminant and uniformly fatal illness with many of the clinical and pathologic findings seen in mice infected with H5N1 highly pathogenic avian influenza A virus. Histopathologic findings in lungs of DHOV-infected mice consisted of hemorrhage, inflammation, and thickening of the interstitium and the alveolar septa and alveolar edema. Extra-pulmonary findings included hepatocellular necrosis and steatosis, widespread severe fibrinoid necrosis in lymphoid organs, marked lymphocyte loss and karyorrhexis, and neuronal degeneration in brain. Similar systemic histopathologic findings have been reported in the few fatal human H5N1 cases examined at autopsy. Because of the relationship of DHOV to the influenza viruses, its biosafety level 2 status, and its similar pathology in mice, the DHOV-mouse model may offer a low-cost, relatively safe, and realistic animal model for studies on the pathogenesis and management of H5N1 virus infection.     

Acute respiratory distress syndrome induced by avian influenza A (H5N1) virus in mice

RATIONALE AND OBJECTIVE: The acute respiratory distress syndrome (ARDS) caused by avian influenza H5N1 viral infection has been reported in many humans since this virus was found to infect humans in Hong Kong in 1997, but no studies regarding an animal model of ARDS with H5N1 viral infection have been found in the literature. Here we present a mouse model of ARDS induced by H5N1 virus. METHODS: Six- to 8-wk-old BALB/c mice were inoculated intranasally (50 micro l) with 1 x 10(2) 50% mouse infectious doses of A/Chicken/Hebei/108/2002 (H5N1) virus. Lung injury was assessed by observation of lung water content and histopathology. Arterial blood gas, white blood cell count in bronchial alveolar lavage fluid, and tumor necrosis factor-alpha and interleukin-6 in bronchoalveolar lavage fluid and serum were measured at the indicated time points. RESULTS: Our data showed that H5N1 viral infection in mice resulted in typical ARDS, which was characterized by the following features: (1) about 80% of mice (13 of 16) dead on Days 6 to 8 postinoculation; (2) highly edematous lungs and dramatically increased lung wet:dry weight ratios and lung wet weight:body weight ratios; (3) inflammatory cellular infiltration, alveolar and interstitial edema, and hemorrhage in lungs; (4) progressive and severe hypoxemia; and (5) significant increase in neutrophils, tumor necrosis factor-alpha, and interleukin-6 in BALF. CONCLUSION: These results suggested that we successfully established a mouse model of ARDS with H5N1 viral infection, which may benefit further investigation into the pathogenesis of human ARDS induced by H5N1 virus.     

成都市首例人感染H9N2禽流感病例调查与分析

目的总结分析成都市首例人感染H9N2禽流感病例流行病学调查结果,为今后科学应对类似疾病提供参考。方法采用现场流行病学和实验室检测相结合的方法,收集病例临床和流行病学资料,采集并检测病例、相关环境等标本,分析可能的感染来源、流行病学特征和临床特征。结果病例发病前10天内有禽类接触史,其标本经检测为H9N2禽流感病毒阳性;病例长期患有慢性疾病,此次发病后进展较快,经积极治疗无效后死亡;其居住环境、家中剩余鸡肉和周边市场检出H9禽流感阳性。结论该病例感染来源为周边市场感染H9N2禽流感病毒的鸡;对严重基础疾病者,感染H9N2禽流感病毒可导致重症或诱发死亡。     

2009H1N1流感病毒对A549细胞和BEAS-2B细胞作用的初步研究

目的 探求2009H1N1流感病毒对A549细胞和BEAS-2B细胞作用,为研究2009H1N1流感病毒的致病机理提供线索。方法 不同来源(死亡、重症、普通病例分离)的2009H1N1流感病毒和季节性H1N1流感病毒分别感染A549和BEAS-2B细胞12、24、48、72 h后用流式细胞术检测细胞凋亡和细胞周期。结果 感染A549细胞12和24 h,普通病例分离的2009H1N1流感病毒组的细胞凋亡率最高(P<0.05),重症组的细胞凋亡率最低(P<0.05);48 h和72 h,死亡组细胞凋亡率最高(P<0.05)。感染BEAS-2B细胞12 h,重症组细胞凋亡率最高(P<0.05);48 h,死亡组和重症组细胞凋亡率高(P<0.05);72 h,死亡组和普通组细胞凋亡率高(P<0.05)。4株病毒主要将A549细胞阻滞在S期,将BEAS-2B细胞阻滞在G0/G1期。结论 在细胞凋亡和细胞周期的细胞学观察水平上2009H1N1流感病毒和季节性H1N1流感病毒之间存在差异,不同来源的2009H1N1流感病毒之间也存在差异。     

Case-control study of risk factors for avian influenza A (H5N1) disease, Hong Kong, 1997.

In May 1997, a 3-year-old boy in Hong Kong died of a respiratory illness related to influenza A (H5N1) virus infection, the first known human case of disease from this virus. An additional 17 cases followed in November and December. A case-control study of 15 of these patients hospitalized for influenza A (H5N1) disease was conducted using controls matched by age, sex, and neighborhood to determine risk factors for disease. Exposure to live poultry (by visiting either a retail poultry stall or a market selling live poultry) in the week before illness began was significantly associated with H5N1 disease (64% of cases vs. 29% of controls, odds ratio, 4.5, P=.045). By contrast, travel, eating or preparing poultry products, recent exposure to persons with respiratory illness, including persons with known influenza A (H5N1) infection, were not associated with H5N1 disease.