Pathogenesis of Influenza A/H5N1 virus infection in ferrets differs between intranasal and intratracheal routes of inoculation

Most patients infected with highly pathogenic avian influenza A/H5N1 virus develop severe pneumonia resulting in acute respiratory distress syndrome, with extrarespiratory disease as an uncommon complication. Intranasal inoculation of ferrets with influenza A/H5N1 virus causes lesions in both the respiratory tract and extrarespiratory organs (primarily brain). However, the route of spread to extrarespiratory organs and the relative contribution of extrarespiratory disease to pathogenicity are largely unknown. In the present study, we characterized lesions in the respiratory tract and central nervous system (CNS) of ferrets (n = 8) inoculated intranasally with influenza virus A/Indonesia/5/2005 (H5N1). By 7 days after inoculation, only 3 of 8 ferrets had a mild or moderate bronchointerstitial pneumonia. In contrast, all 8 ferrets had moderate or severe CNS lesions, characterized by meningoencephalitis, choroiditis, and ependymitis, and centered on tissues adjoining the cerebrospinal fluid. These findings indicate that influenza A/H5N1 virus spread directly from nasal cavity to brain, and that CNS lesions contributed more than pulmonary lesions to the pathogenicity of influenza A/H5N1 virus infection in ferrets. In comparison, intratracheal inoculation of ferrets with the same virus reproducibly caused severe bronchointerstitial pneumonia. The method of virus inoculation requires careful consideration in the design of ferret experiments as a model for influenza A/H5N1 in humans.     

Emergent viruses: SARS-associate coronavirus and H5N1 influenza virus

Two viral agents with RNA genome are responsible for emerging illnesses: influenza virus A/H5N1 and Severe Acute Respiratory Syndrome virus (SARS). For the diagnosis of SARS virus infection, an epidemiological investigation is necessary to know whether the patient has been exposed to a risk in a country where the SARS virus is circulating or whether the patient had worked in a laboratory handling SARS virus. The detection of SARS virus is possible in various clinical samples (including urine) by viral culture or RT-PCR. The handling of those samples and RNA extraction must be performed in a BSL3 laboratory. The SARS virus RT-PCR is poorly sensitive, therefore the test should be performed on samples collected consecutively for several days. In front of a suspicion of A/H5N1, similar procedures are recommended. An epidemiologic investigation is necessary to specify whether the patient stayed in a country where A/H5N1 virus was circulating. Clinical samples needed for a specific diagnosis are: nasopharyngeal, throat-swab or fecal samples, cerebrospinal fluid and blood. The presence of A/H5N1 virus is confirmed by viral isolation or RNA detection by RT-PCR. RNA extraction must be performed in a BSL3 laboratory. For diagnosis of A/H5N1 virus infection, RT-PCR test amplifies specifically a fragment of H5 gene (Hemagglutinin). In french laboratories of medical virology, procedures are ready to diagnose the first case of A/H5N1 virus infection and cases of reemerging SARS virus infection.     

Persistence of the influenza virus in children with congenital CNS pathology

Sick infants born to mothers who experienced influenza during pregnancy were examined. The cerebrospinal fluid, serum and blood cells were collected from such children with signs of congenital immune deficiency and progressive pathology of the central nervous system. None of the specimens yielded infectious influenza virus, but by means of molecular hybridization virus-specific genetic sequences were found in small amounts in the cerebrospinal fluid and serum and in high concentrations in blood cells. Persistence of genes NP, M and H1 of influenza A/H1N1 virus was observed in the blood cells of one infant for 83 days (the observation period). At the same time, the lack of antibodies to viral M protein in serum of this baby was demonstrated by the immune blotting method.     

Perspectives from Spanish infectious diseases professionals on 2009 A (H1N1) influenza: the third half

The first influenza pandemic in more than 40 years was declared in 2009. We aimed to evaluate the beliefs of Spanish infectious diseases professionals regarding several aspects of 2009 A (H1N1) influenza once the epidemic waned. An online survey was designed and distributed among members of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC). The survey considered hospital organization and preparedness planning and conduct, as well as the opinion of the infectious diseases professionals regarding several key issues. Between 7 March and 22 March 2010, 303 responses, corresponding to 12.8% of the SEIMC membership, were received. Of the respondents, 48.2% were microbiologists and 42.3% were clinicians dealing with infectious diseases. Forty-one per cent of respondents did not believe that 2009 A (H1N1) influenza had a more severe presentation than other seasonal influenzas. Only 5% fully agreed that 2009 A (H1N1) influenza had a more severe presentation. Influenza planning was available in 69.7% of represented institutions before the arrival of 2009 A (H1N1) influenza, and was considered to be useful, to different extents, by most professionals. In most institutions (88.3%), a multidisciplinary team was created to coordinate local pandemic influenza actions. The most successful protocols were those provided by regional healthcare authorities, followed by those from the CDC. The most problematic issues regarding 2009 A (H1N1) influenza were the management of patients in the emergency room and the vaccination and awareness of healthcare professionals (HCPs) regarding infection control. Microbiological diagnosis and the availability of antivirals were the least problematic areas. Although the majority of surveyed infectious diseases professionals did not believe that 2009 A (H1N1) influenza had an especially severe presentation, most of them agreed with the way that this epidemic was managed in their institutions.     

96例甲型H1N1流感的临床分析

目的 通过回顾性分析96例甲型HlNl流感的流行病学及临床特点,提高对甲型H1Nl流感的诊断和治疗水平.方法 收集2009年8月20日至9月23日于杭州市第六人民医院住院治疗的96例甲型H1N1流感患者的临床资料,对其流行病学及临床特点进行分析.结果 患者平均年龄（26.52±10.62）岁,64例有甲流接触史.以发热100%、咳嗽86.4%、咽痛66.6%和肌肉酸痛32.3%为主要临床表现.结论 甲型H1N1流感临床表现与季节性流感很难区分,需结合流行病学调查与实验室检查及时诊断隔离治疗.     

流感及H5N1亚型禽流感病毒液相检测芯片的制备

目的 建立利用液相芯片技术检测甲、乙型流感和H5N1亚型高致病性禽流感病毒的方法,并对该方法进行评价。方法 对GenBank中甲型流感病毒的NP、乙型流感病毒的HA以及高致病性禽流感病毒（H5N1）的H5、N1基因片段序列进行同源性比对,根据保守序列,设计针对各基因的简并引物和寡核苷酸探针,制备探针偶联微球,将样本核酸多重PCR扩增产物与微球进行杂交,以Bio-Plex液相芯片检测系统进行芯片检测。结果 该方法可以对甲型流感病毒的NP基因、乙型流感病毒的HA基因以及高致病性禽流感病毒（H5N1）的H5、N1基因同时进行检测,病毒核酸的最低检出量为1pg,检测特异性高。结论 成功构建了甲、乙型流感病毒和H5N1亚型高致病性禽流感病毒液相芯片检测系统,为流感、禽流感的快速检测、诊断奠定了基础。     

Recruitment,activation and proliferation of CD8+ memory T cells in an immunoprivileged site

The capacity of a memory cytotoxic T lymphocyte (CTL) population to protect against viral infections is well established, but the processes underlying this protection are less well understood. We have used heterotypic intranasal immunization with influenza A/X31 (H3N2) to protect against a subsequent infection with the neurovirulent influenza A/WSN (H1N1) in either the cerebrospinal fluid or the immunoprivileged brain parenchyma. Viral clearance from both sites was associated with a local infiltration and proliferation of A/WSN-specific CD8⁺ T cells. Infection in the cerebrospinal fluid elicited a proliferative response in the draining lymph nodes, an anti-H1N1 serum antibody response and an increase in the extracerebral A/WSN-specific CTL precursor frequency. In contrast, infection in the brain parenchyma elicited no lymph node proliferative response or serum antibody response and caused a transient decrease in the extracerebral CTL precursor frequency. Thus the memory CTL population protected against an intracerebral viral infection independent of any immune response occurring in systemic lymphoid tissue.     

Comparative analytical sensitivities of six rapid influenza A antigen detection test kits for detection of influenza A subtypes H1N1, H3N2 and H5N1.

BACKGROUND: Rapid and simple methods for diagnosing human influenza A (H5N1) disease urgently needed. The limited data so far suggest that the currently available rapid antigen detection kits have poor clinical sensitivity for diagnosis of human H5N1 disease. OBJECTIVES: To compare the analytical sensitivity of six commercially available rapid antigen detection kits for the detection of "human" (subtypes H1N1, H3N2) and "avian" (subtype H5N1) influenza A viruses. STUDY DESIGN: Six commercially available test kits for the detection of influenza A were investigated. Analytic sensitivity for the detection of two contemporary H1N1, two H3N2 and three H5N1 viruses was determined using virus culture as a reference method. RESULTS AND CONCLUSIONS: Each test kit detected the H5N1 virus subtypes as efficiently as they detected conventional human viruses of subtypes H1N1 or H3N2. However, limits of detection of influenza viruses of all subtypes by antigen detection kits were >1000-fold lower than virus isolation. Thus, the reportedly poor clinical sensitivity of these antigen detection kits for diagnosis of patients with H5N1 disease is not due to a difference of sensitivity for detecting avian influenza H5N1 compared to human influenza viruses.     

我国青年人群血清中流感抗体的连续三年监测

目的：了解青年人群血清中抗流感病毒的抗体水平和病毒变异程度。方法：应用微量血凝素抑制试验，对从1997—2000年在我国8个地区，每年春、秋两季，共采集3000份血清标本进行抗流感病毒抗体水平的检测。结果：发现人群中抗流感病毒抗体的水平，在春、秋两季间波动；抗甲3型流感病毒(H3N2)的抗体水平，从1999—2000年保持了高水平；而抗甲1型流感病毒(H1N1)的抗体水平，从1998年以来呈悌形下降，易感人群由15％上升到75％。结论：近3年中流感病毒甲3型、甲1型和乙型均曾在我国局部流行，甲1型流感病毒(H1N1)发生了明显变异，甲3型流感病毒(H3N2)将可能发生变异。     

Direct detection of highly pathogenic avian influenza A/H5N1 virus from mud specimens

Contaminated mud and soil may play roles as reservoirs and sources of transmission for avian influenza A virus. However, the persistence of highly pathogenic avian influenza (HPAI) H5N1 virus in soil or mud has not been well documented, and specific methods of H5N1 virus detection in mud and soil specimens have not been described. The aim of this work was to evaluate the capacities of five different commercial kits and one elution-concentration technique to extract nucleic acids from H5N1 virus and to detect infectious viral particles in experimentally infected mud specimens. The viral RNA detection thresholds for the QIAamp kit, Trizol LS and the MagNA Pure LC kit were 5 × 10(2)RNA copies per gram of mud. Trizol reagent and the RNA PowerSoil? kit were unsuccessful in recovering any viral RNA from mud. When the elution-concentration technique was performed prior to nucleic acid extraction, the performance of the MagNA Pure kit increased to a level that allowed the detection of H5N1 nucleic acids in naturally contaminated environmental samples that had previously tested negative after direct extraction using commercial kits. The levels of detection of infectious virus after inoculation into embryonated eggs were higher in concentrates than in eluates.     

人感染高致病性禽流感病毒H5N1核酸检测方法的建立及应用

目的 建立人感染高致病性禽流感病毒H5N1的核酸检测方法,用于人感染高致病性禽流感病毒疑似病例临床标本的检测.方法 针对甲型流感病毒保守基因M设计RT-PCR和real-time PCR引物检测是否为甲型流感病毒,同时针对H5N1禽流感病毒设计针对H5和N1基因的特异性RT-PCR和real-time PCR引物作亚型检测,建立禽流感H5N1病毒RT-PCR和real-time PCR检测方法.结果 本研究建立的RT-PCR和real-time PCR方法可以特异性地检测H5N1病毒,并且与人流感病毒H1、H3没有交叉反应.RT-PCR检测方法灵敏度可到1TCID50,real-time PCR灵敏度可达0.01TCID50.利用上述方法检测人感染高致病性禽流感病毒H5N1疑似病例临床标本,从42例不明原因肺炎病例中检测出阳性标本13例.结论 本研究建立的RT-PCR和real-time PCR方法可以用于人感染高致病性禽流感病毒H5N1临床标本的实验室检测.     

Development of two types of rapid diagnostic test kits to detect the hemagglutinin or nucleoprotein of the swine-origin pandemic influenza A virus H1N1

Since its emergence in April 2009, pandemic influenza A virus H1N1 (H1N1 pdm), a new type of influenza A virus with a triple-reassortant genome, has spread throughout the world. Initial attempts to diagnose the infection in patients using immunochromatography (IC) relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1 pdm. Here, we prepared monoclonal antibodies that react with H1N1 pdm but not seasonal influenza A (H1N1 and H3N2) or B viruses. Using two of these antibodies, one recognizing viral hemagglutinin (HA) and the other recognizing nucleoprotein (NP), we developed kits for the specific detection of H1N1 pdm and tested them using clinical specimens of nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illnesses. The specificities of both IC test kits were very high (93% for the HA kit, 100% for the NP kit). The test sensitivities for detection of H1N1 pdm were 85.5% with the anti-NP antibody, 49.4% with the anti-HA antibody, and 79.5% with a commercially available influenza A virus detection assay. Use of the anti-NP antibody could allow the rapid and accurate diagnosis of H1N1 pdm infections.     

流感病毒H1N1和H5N1感染小鼠小胶质细胞与星形胶质细胞诱导趋化因子转录水平上调

目的:探讨胶质细胞感染流感病毒后的天然免疫反应,检测流感病毒H1N1和H5N1体外感染小鼠小胶质细胞和星形胶质细胞,是否会诱导胶质细胞趋化因子转录水平的变化及其规律.方法:从新生小鼠大脑皮质分离培养神经胶质细胞,并进一步纯化小胶质细胞和星形胶质细胞,经纯度鉴定后,用感染复数为2的流感病毒H1N1和H5N1进行体外感染,8小时后用免疫荧光检测流感病毒核蛋白(NP)的表达,以确认感染细胞比例.在感染早期(6小时)和感染中期(24小时)分别提取细胞RNA,检测趋化因子转录水平的变化.结果:分离得到小鼠的小胶质细胞和星形胶质细胞,病毒感染后超过95%的细胞可以被感染,感染后的小胶质细胞与星形胶质细胞的CCI-3、CCL-5、CXCL-2、CXCL-9和CXCL-10的转录水平发生不同程度的上调,其中CXCL-10的上调幅度最为明显,禽流感病毒H5N1感染能诱导更强烈的上调反应.结论:流感病毒H1N1和H5N1感染小鼠小胶质细胞与星形胶质细胞,可诱导趋化因子转录水平上调.     

Avian influenza A (H5N1).

Since their reemergence in 2003, highly pathogenic avian influenza A (H5N1) viruses have reached endemic levels among poultry in several southeast Asian countries and have caused a still increasing number of more than 100 reported human infections with high mortality. These developments have ignited global fears of an imminent influenza pandemic. The current knowledge of the virology, clinical spectrum, diagnosis and treatment of human influenza H5N1 virus infections is reviewed herein.     

Evaluation of twenty rapid antigen tests for the detection of human influenza A H5N1, H3N2, H1N1, and B viruses

Twenty rapid antigen assays were compared for their ability to detect influenza using dilutions of virus culture supernatants from human isolates of influenza A H5N1 (clade 1 and 2 strains), H3N2 and H1N1 viruses, and influenza B. There was variation amongst the rapid antigen assays in their ability to detect different influenza viruses. Six of the 12 assays labeled as distinguishing between influenza A and B had comparable analytical sensitivities for detecting both influenza A H5N1 strains, although their ability to detect influenza A H3N2 and H1N1 strains varied. The two assays claiming H5 specificity did not detect either influenza A H5N1 strains, and the two avian influenza‐specific assays detected influenza A H5N1, but missed some influenza A H3N2 virus supernatants. Clinical trials of rapid antigen tests for influenza A H5N1 are limited. For use in a pandemic where novel influenza strains are circulating (such as the current novel influenza A H1N1 09 virus), rapid antigen tests should ideally have comparable sensitivity and specificity for the new strains as for co‐circulating seasonal influenza strains. J. Med. Virol. 81:1918–1922, 2009. © 2009 Wiley‐Liss, Inc.     

A nutritional supplement formula for influenza A (H5N1) infection in humans

By early February 2006, the World Health Organization had reported 165 human cases of H5N1 influenza since December 2003, with 88 fatalities. However, the avian H5N1 influenza virus apparently is not yet efficiently transmitted between humans. Though a near-term possibility of a global H5N1 influenza pandemic remains, currently there is no vaccine or anti-viral drug that is proven to be safe and effective in preventing or treating H5N1 influenza in humans. There is thus a compelling public interest in developing alternative prophylaxis and treatment strategies for H5N1 influenza, which would need to address the complex pathogenesis of H5N1 influenza that is responsible for its apparently unusually high virulence. The authors present here a significant body of medical and scientific evidence to support the prophylactic use of a carefully designed nutritional supplement formulation that may antagonize the major pathogenic processes of H5N1 influenza in humans. Through several independently-mediated mechanisms, the formulations may: (a) degrade H5N1 virulence by directly affecting the virus itself, (b) inhibit H5N1 viral replication by maintaining cellular redox equilibrium in host cells, (c) inhibit H5N1 replication by a blockade of the nuclear-cytoplasmic translocation of the viral ribonucleoproteins and reduced expression of late viral proteins related to the inhibition of protein kinase C activity and its dependent pathways, (d) down-regulate activation and proliferation of proinflammatory cytokines in respiratory epithelial cells and macrophages that are implicated in the pathogenesis of H5N1 influenza, and (e) protect the lungs and other vital organs from virus- and cytokine-induced oxidative stress by supplying and maintaining sufficient levels of exogenous and endogenous antioxidants. Key mediators in these processes include selenium, vitamin E, NAC/glutathione, resveratrol, and quercetin. Taken prophylactically, and throughout the duration and recovery of an H5N1 infection, the nutritional supplement formula may aid humans infected with H5N1 influenza to survive with a reduced likelihood of major complications, and may provide a relatively low-cost strategy for individuals as well as government, public-health, medical, health-insurance, and corporate organizations to prepare more prudently for an H5N1 pandemic. Some evidence also indicates that the supplement formulation may be effective as an adjunctive to H5N1 vaccine and anti-viral treatments, and should be tested as such.     

A multiplex real-time RT-PCR for detection and identification of influenza virus types A and B and subtypes H5 and N1

A multiplex real-time RT-PCR method for the simultaneous detection of influenza virus types A and B and identification of subtypes H5 and N1 in a single tube is described. The method was developed with four sets of primers and probes which were specific to influenza virus (sub)types A, B, H5, and N1, and evaluated by using a total of 40 influenza virus reference strains, including 17 avian influenza A (12 H5N1, 1 H1N1, 1 H3N2, 1 H4N6, 1 H7N3, and 1 H9N2), 18 human influenza A (11 H3N2, 6 H1N1 and 1 H5N1) and 5 influenza B viruses. The method exhibited a high specificity and sensitivity of approximately 10(1)-10(2)copies/microl for each (sub)type and a high reproducibility with intra- and inter-assay CV from 0.13 to 4.24%. In an analysis of 189 clinical samples from patients during the year 2004 and 2005, the method identified 81 positive samples (42.9%) and identified simultaneously 14 type B samples and 11 subtype N1 samples, in comparison only 46 positive samples (24.3%) identified by the conventional culturing method. The method would be a useful molecular diagnostic tool for large-scale screening of clinical samples for influenza virus.     

Isolation of influenza A viruses in 1981 in Leningrad with and antigenic formula (H0N1) related to strain A/PR8/34

wo strains of influenza A (H0N1) virus closely related to A/PR8/34 virus were isolated from the cerebrospinal fluid of children suffering from a disease diagnosed as purulent meningitis. Apart from similarities. Marked differences of the new strains from the laboratory A/PR8/34 virus were found which rules out the association of the isolates with contamination with the laboratory strains. Examinations of paired sera from the children, sources of the isolates, revealed no rise in titres of antihemagglutinating antibodies but established a 4-8-fold rise of titres of antineuraminidase antibodies to the A/PR8/34 virus which attests to infection of these children with influenza H0N1 viruses.