SERS-based immunomagnetic bead for rapid detection of H5N1 influenza virus

The surface-enhanced Raman scattering (SERS) has recently drawn attention in the detection of respiratory viruses, but there have been few reports of the direct detection of viruses. In this study, a sandwich immunomagnetic bead SERS was established for the rapid diagnosis of the H5N1 influenza virus. The detection limit was estimated to be 5.0 × 10⁻⁶ TCID₅₀/ml. The method showed excellent specificity with no cross-reaction with H1N1, H5N6 or H9N2. The H5N1 influenza virus detection accuracy of the SERS method was 100% in chicken embryos. The results hold great promise for the utilization of SERS as an innovative approach in the diagnosis of influenza virus.     

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 infection with highly pathogenic H5N1 influenza virus

Highly pathogenic H5N1 influenza A viruses have spread relentlessly across the globe since 2003, and they are associated with widespread death in poultry, substantial economic loss to farmers, and reported infections of more than 300 people with a mortality rate of 60%. The high pathogenicity of H5N1 influenza viruses and their capacity for transmission from birds to human beings has raised worldwide concern about an impending human influenza pandemic similar to the notorious H1N1 Spanish influenza of 1918. Since many aspects of H5N1 influenza research are rapidly evolving, we aim in this Seminar to provide an up-to-date discussion on select topics of interest to influenza clinicians and researchers. We summarise the clinical features and diagnosis of infection and present therapeutic options for H5N1 infection of people. We also discuss ideas relating to virus transmission, host restriction, and pathogenesis. Finally, we discuss vaccine development in view of the probable importance of vaccination in pandemic control.     

H5N1亚型禽流感病毒诱导的机体免疫损伤分析

目的 进一步了解H5N1禽流感病毒的致病机制,同时对H5N1禽流感病人的临床救治方案提供科学依据.方法 采集2011年深圳市1例H5N1禽流感病例病程中多份血液样本,应用流式细胞术和酶联免疫法,检测该病人外周血中各亚群淋巴细胞及细胞因子水平.结果 H5N1禽流感患者外周血中各亚群淋巴细胞均出现绝对数量减少,CD4+/CD8+出现倒置.同时发现血清中高水平的IL-6、IL-10、TNF、IFN-γ、IL-12、IP-10和MCP-1.结论 H5N1禽流感病毒会对机体的免疫系统造成严重伤害,同时会刺激机体过度表达高水平的细胞因子,从而导致典型的免疫应答介导的病理损伤.     

人禽流感死亡病例不同组织器官中H5N1病毒的检测分析

目的了解H5N1病毒在人禽流感死亡病例组织器官中的分布和含量。方法在BSL-3实验室，测定H5N1禽流感病毒分离株的TCID50，并采用荧光定量PCR制作标准曲线；将人禽流感死亡病例的尸解标本，包括心、肝、脾、肺、肾、脑等组织标本研磨处理后，进行荧光定量PCR检测，并根据标准曲线推算H5N1禽流感病毒的病毒载量。结果心、肝、肾、脑组织标本中H5N1禽流感病毒检测为阴性，在肺、脾组织标本中检测到H5N1病毒，病毒载量分别为10^6.3TCID50／ml和10^4.55TCID50／ml。结论除呼吸系统外，在脾组织中也存在H5N1禽流感病毒。     

H5N1高致病性禽流感病毒研究进展

流行性感冒病毒属正粘病毒科,根据内部核蛋白（NP）和基质蛋白（MP）抗原性的不同,将流感病毒分为A型、B型和c型3型。A型流感病毒的基因组是由8个分节段的单负链RNA组成,编码11种蛋白质,表面糖蛋白为HA和NA,根据HA和NA的抗原差异分为16种HA亚型（H1一H16）和9种NA亚型（N1～N9）。     

禽流感病毒H1、H3、H5、N2亚型多重RT-PCR检测方法的初步研究

目的根据禽流感病毒H1、H3、H5亚型的HA基因和N2型NA基因的保守序列,设计出四对RT-PCR引物,建立一步法多重RT-PCR对禽流感H1、H3、H5、N2四亚型进行快速检测。方法利用所设计的四对引物,通过对该方法扩增条件的优化,成功建立快速检测禽流感病毒H1、H3、H5、N2四亚型的一步法多重RT-PCR。利用禽流感H1、H3、H5、N2四亚型毒株和其它相关标准毒株进行敏感性和特异性试验。结果与结论所建立的一步法多重RT-PCR具有较高的特异性和敏感性,与禽流感其它亚型和NDV、IBV、ARV?IBDV的核酸均无交叉反应。用该方法检测现场样品395份(4省20多个地区),结果与经典检测方法一致。     

A sensitive retroviral pseudotype assay for influenza H5N1‐neutralizing antibodies

Background The World Health Organisation (WHO) recommended the development of simple, safe, sensitive and specific neutralization assays for avian influenza antibodies. We have used retroviral pseudotypes bearing influenza H5 hemagglutinin (HA) as safe, surrogate viruses for influenza neutralization assays which can be carried out at Biosafety Level 2. Results Using our assay, sera from patients who had recovered from infection with influenza H5N1, and sera from animals experimentally immunized or infected with H5 tested positive for the presence of neutralizing antibodies to H5N1. Pseudotype neutralizing antibody titers were compared with titers obtained by hemagglutinin inhibition (HI) assays and microneutralization (MN) assays using live virus, and showed a high degree of correlation, sensitivity and specificity. Conclusions The pseudotype neutralization assay is as sensitive as horse erythrocyte HI and MN for the detection of antibodies to H5N1. It is safer, and can be applied in a high‐throughput format for human and animal surveillance and for the evaluation of vaccines.     

Isolation of H1N1 influenza virus in munich

Summary During an outbreak of a mild upper respiratory tract infection in a university children's hospital in Munich, an H1N1 influenza virus was isolated. Serological analysis of the isolate showed that antigenically the virus resembled the USSR/90/77 strain of influenza A virus which has been isolated in many parts of the world during the last two years.

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Isolierung eines H1N1-Influenzavirus in München

Zusammenfassung Während der Epidemie einer milden Infektion des oberen Respirationstraktes in einer Universitäts-Kinderklinik in München wurde ein Influenzavirus vom Typ H1N1 isoliert. Die serologische Analyse des Virusisolats ergab eine Antigenität, wie sie dem USSR/90/77-Stamm der Influenza-A-Viren entspricht. Dieser Virusstamm konnte in vielen Teilen der Erde während der vergangenen zwei Jahre isoliert werden.

     

Systemic influenza virus H5N1 infection in cats after gastrointestinal exposure

Please cite this paper as: Vahlenkamp et al. (2010) Systemic influenza virus H5N1 infection in cats after gastrointestinal exposure. Influenza and Other Respiratory Viruses 4(6), 379–386. Background  Highly pathogenic avian influenza virus (HPAIV) H5N1 infections in felids have been reported in several countries. Feeding on infected birds has been suggested as potential source of infection. Objectives  The study aimed to verify gastrointestinal infection as possible portal of entry for HPAIV H5N1 in cats. Methods  Four cats were infected oculo‐nasopharyngeally with 10⁶ 50% egg infectious dose (EID₅₀) of HPAIV H5N1 A/cat/Germany/R606/2006. Two cats were infected intravenously with 10⁶ EID₅₀ and two cats were inoculated orally with 10⁷ EID₅₀ HPAIV embedded in gelatine capsules to mimic gastrointestinal exposure and to avoid virus contact to oropharyngeal or respiratory tissues. Cats were monitored for 6 days by physical examination, virus excretion, and peripheral blood lymphocyte counts. Blood chemical parameters (including AST, ALT, CPK, and TBIL) and viral excretion using pharyngeal and rectal swabs were analyzed. Results  Infected cats showed elevated body temperature up to 41·3°C starting from day 1 or 2 p.i. All infected cats excreted virus in pharyngeal swabs within 2 days p.i. co‐inciding with the development of clinical signs (anorexia, depression, and labored breathing) irrespective of the infection route. Virus dissemination occurred through cell‐free and cell‐associated viremia. Infected cats developed lymphopenia, hepatic necrosis, pneumonia, and significantly elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine phosphokinase (CPK), and TBIL. Conclusions  The experiments show that the gastrointestinal tract can serve as portal for the entry of HPAIV H5N1 into cats. Infection routes used did not influence viral tissue tropism and course of disease.     

Preparation and evaluation of specific polyclonal antibodies to H5N1 subtype of the avian influenza virus in Egypt

Influenza A virus continue to cause widespread morbidity and mortality. The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Egypt is threatening poultry and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, polyclonal antibodies (PAbs) were developed against the H5N1 avian influenza virus (AIV) and used in an enzyme-linked immunosorbent assay (ELISA) to detect the H5 viral antigen. A group of rabbits were immunized with H5N1 vaccine to obtain PAbs as the detector antibody after conjugation with horse radish peroxidase and fluorescein isothiocyanate (FITC). The conjugated PAbs proved to be specific for the detection of AIV in field specimens, and results were confirmed using reference antisera obtained from Veterinary Lab in Weybridge. Specimens collected from different governorates of Egypt and tested positive for AIV by haemagglutination test were used to evaluate the produced PAbs. The detection limit of ELISA using the prepared peroxidase conjugated PAbs was 1:100,000, while the limit using fluorescein conjugated PAbs was 1:10,000. Extracts from pharyngeal-tracheal mucus of apparently healthy chickens mixed with H5 AIVs also yielded positive signals in ELISA. Such data suggest that these PAbs are useful in the surveillance and diagnosis of AIV in birds in Egypt.     

Influenza viruses and the evolution of avian influenza virus H5N1.

Although small in size and simple in structure, influenza viruses are sophisticated organisms with highly mutagenic genomes and wide antigenic diversity. They are species-specific organisms. Mutation and reassortment have resulted in newer viruses such as H5N1, with new resistance against anti-viral medications, and this might lead to the emergence of a fully transmissible strain, as occurred in the 1957 and 1968 pandemics. Influenza viruses are no longer just a cause of self-limited upper respiratory tract infections; the H5N1 avian influenza virus can cause severe human infection with a mortality rate exceeding 50%. The case death rate of H5N1 avian influenza infection is 20 times higher than that of the 1918 infection (50% versus 2.5%), which killed 675000 people in the USA and almost 40 million people worldwide. While the clock is still ticking towards what seems to be inevitable pandemic influenza, on April 17, 2007 the U.S. Food and Drug Administration (FDA) approved the first vaccine against the avian influenza virus H5N1 for humans at high risk. However, more research is needed to develop a more effective and affordable vaccine that can be given at lower doses.     

H1N1 influenza A virus induced atrioventricular block

We report a case of an 18-year-old female who presented with respiratory failure secondary to H1N1 infection, and who subsequently developed high-degree atrioventricular (AV) block. The conduction abnormalities persisted over 2 weeks following complete resolution of respiratory symptoms. A permanent pacemaker was implanted for safety and subsequent pacemaker follow-up suggested reversibility of the conduction abnormality. This case highlights the potential impact of the H1N1 influenza virus on the cardiac conduction system.     

H9N2 influenza A virus circulates in H5N1 endemically infected poultry population in Egypt

We describe the identification and characterization of the H9N2 influenza subtype reported in Egyptian broiler and broiler breeder farms for the first time. Circulation of this subtype in a highly pathogenic H5N1 influenza virus endemic population provides an opportunity for genetic reassortment and emergence of novel viruses.