H5N1型禽流感病毒动物模型的研究现状

H5N1亚型禽流感病毒在全球多个国家肆虐,已严重威胁人类健康。禽流感病毒动物模型的建立可以为研究病毒的突变、传染性和发病机制提供良好的技术平台。本综述概括了H5N1对几种哺乳动物:食蟹猴、雪貂、小鼠、大鼠、沙鼠、家猫等的致病性,为以后理想动物模型的建立和研究提供一定的帮助。     

单克隆抗体治疗日本乙型脑炎病毒感染恒河猴的实验研究

用不同剂量的日本乙型脑炎病毒(JEV)SA14株鼠脑悬液从静脉、腹腔及皮下等不同途径感染6月龄4公斤左右JEV抗体阴性的恒河猴,发现用1ml1×10~8TCID_(50)病毒经皮下感染组的动物,发病比较规律、症状比较典型。感染后第6～8天体温突然升高至39℃以上,最高者可达41.5℃,兴奋性增高,腱反射亢进,并且日渐削瘦,第12天左右出现肢体痉挛性麻痹,肌张力增高;第20天后用呼吸及循环衰竭而死亡。但用1ml及0.5ml同一病毒悬液分别经腹腔及静脉途径感染的恒河猴,仅有发烧,而无其余症状。故治疗试验中选用皮下途径感染动物。     

禽流感病毒H5N1 单克隆抗体的生物学特性鉴定

目的:探讨禽流感病毒单克隆抗体的生物学特性。方法:制备H5N1禽流感病毒的单克隆抗体,鉴定其亚型、效价、血凝抑制活性以及与其他亚型流感病毒的交叉反应性。免疫印迹方法验证单抗与H5N1抗原的结合,人体正常组织芯片的免疫组织化学染色法观察单抗的组织学反应。结果:发现两株禽流感病毒H5N1的单抗(H5-32和H5-35)分别与人体的肾、胰腺组织存在阳性结合。结论:禽流感病毒H5N1与人体组织间可能存在某种关联,这或许可为禽流感病毒感染和致病研究提供参考。     

利用反向遗传学技术构建H5N9重组流感病毒株

目的 利用反向遗传学技术构建来源人感染禽流感病毒H5N1和H7N9 HA和NA基因的H5N9亚型禽流感病毒.方法 全基因合成A/Beijing/01/2003(H5N1)禽流感病毒HA基因片段和A/Zhejiang/DTID-ZJU10/2013(H7N9)禽流感病毒NA基因片段,插入到pHW2000载体,与携带有A/Puerto Rico/8/34(H1N1)的6个内部基因的pHW2000重组质粒一起转染293T和MDCK混合细胞,拯救H5N9重组病毒.结果 核酸测序、HA和NA基因转录和表达检测、细胞病变分析确定利用该反向遗传学系统可以成功拯救H5N9病毒.重组H5N9病毒在MDCK细胞上复制增殖能力低于相同方法拯救H1N1病毒.结论 利用反向遗传学技术成功构建一株H5N9重组病毒.     

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.     

2004-2012年我国禽流感流行情况

禽流感病毒(AIV)是一类可以引起禽类感染和(或)致病的A型流感病毒,它能引起大多数家禽、野生水禽的感染.部分禽流感病毒已经跨种属传播感染人群,1997年香港第一次报道禽流感H5N1亚型病毒能感染人类,不断出现的H9N2、H7N2、H7N3、H7N7等禽流感亚型感染人类的报道,并显示不同程度的致病性和病死率[1-3].2013年3月在中国首次报道新的基因重组H7 N9亚型禽流感病毒直接感染人类,导致重症肺炎引起死亡[4].     

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。     

抗禽流感(H5N1)免疫球蛋白制备及体外抑制甲型流感病毒FM1株的试验研究

目的:制备抗禽流感(H5N1)卵划黄免疫球蛋白(IgY),探讨其体外阻断甲型流感病毒FM1株的作用.方法:对经过禽流感疫苗免疫后产生并富集在卵黄中的卵黄免疫球蛋白进行提纯,用辛酸-硫酸氨二步盐析-凝胶色谱法进行纯化,利用细胞病变(CPE)抑制实验,在狗肾细胞系(MDCK)检测抗禽流感(H5N1)免疫球蛋白无毒浓度(TD0)范围及阻断甲型流感病毒FM1株攻击MDCK细胞的最小阻断浓度.结果:抗禽流感免疫球蛋白对DMCK的无毒浓度为1.764 mg/ml,当浓度为0.082 8mg/ml对流感病毒仍有阻断作用.结论:利用辛酸-硫酸氨二步盐析-凝胶色谱法成功制备卵黄免疫球蛋白,抗禽流感(H5N1)免疫球蛋白体外对流感病毒有较强的抑制作用.     

湖北省首例人感染H5N6禽流感病例流行病学调查

目的 对湖北省首例人感染H5N6禽流感病例的流行病学资料进行分析,探讨病例发现过程、调查处理措施、实验室检测方法,为防控人感染H5N6禽流感疫情提供依据.方法 采用描述性流行病学方法,分析湖北省首例人感染H5N6禽流感病例诊治过程、密切接触者信息,开展现场流行病学调查,并采集病例、密切接触者、活禽市场外环境标本进行实验室检测分析.结果 患者2016年4月9日发病,体温40℃,有活禽市场暴露史.发病早期采集的下呼吸道标本(痰液、气管分泌液)为H5N6禽流感病毒核酸阳性,而上呼吸道标本H5N6禽流感病毒核酸检测均为阴性.患者无外出史和H5N6禽流感病例接触史.患者经两个月的治疗痊愈出院.密切接触者58人均未出现发热和呼吸道感染症状.采集病例经常路过的两家活禽市场和一家土鸡专卖店外环境标本共36份,其中检出H5N6禽流感病毒核酸1 1份,阳性率为30.56％.结论 该病例为湖北省首例人感染H5N6禽流感病例,属本地感染的散发病例,未出现人传人.传播途径可能为:活禽市场通过禽-环境-人的途径传播.在病例诊断中,下呼吸道标本(尤其是痰液、气管分泌液)具有重要意义.另外,尽早应用奥司他韦对病例的成功救治起到了重要的作用.     

单克隆抗体治疗日本乙型脑炎病毒感染恒河猴的实验研究

恒河猴感染日本乙型脑炎病毒(JEV)后,出现脑炎的一系列症状与体征,终因呼吸及循环衰竭而死亡。病毒感染猴体温升至39℃以上后的第2天,注射单克隆抗体(McAb)制剂进行治疗。结果McAb经肌肉、静脉、硬膜下加肌肉等不同途径注射后,均显示良好的疗效,对恒河猴的保护率达100％;其中以硬膜下加肌肉途径注射McAb的效果最佳。多次注射McAb,未发现任何不良反应与毒副作用;提示将鼠源性McAb用于灵长类安全可靠,若过渡到临床使用,是可行的。     

An avian influenza vaccine for humans targeting the polymerase B2 protein inside the capsid instead of hemagglutinin or neuramidase on the virus surface

Vaccines for avian influenza typically are aimed at hemagglutinin or neuramidase on the outside of the virus capsid. A major problem with such an approach is that the genes coding for these proteins have a very rapid mutation rate, forcing commercial producers to wait for mutations to occur before developing effective new versions of standard vaccines. However, a recent study has revealed that the 1918 flu virus, like the H5N1 avian flu virus, has an E627K mutation in its polymerase B2 component, which is located inside the virus capsid. Other research has indicated that this mutation strongly influences the virulence of H5N1. It seems reasonable to believe that the constancy, over more than 80 years, of the E627K mutation could be exploited to begin developing a vaccine now, rather than waiting for new mutations. Consequently, a publicly available database at the National Center for Biotechnology Information (NCBI) website, and the SYFPEITHI online computer algorithm, were used to generate a hypothesis about a peptide-based vaccine targeted at the E627K mutation in PB2 of the avian influenza virus. It was found that the peptide sequence, DTVQIIKLL, present in the PB2 protein of the H5N1 virus, would be expected to bind to HLA-A26 restricted immune system cell surface receptors. Hence, the bound peptide might be capable of stimulating protection from cytotoxic T lymphocytes. Should the present hypothesis be confirmed in laboratory studies, and an effective vaccine developed for individuals expressing the HLA-A26 receptor; further research would be indicated. This research would be aimed at determining whether molecular modifications to the DTVQIIKLL peptide could make it effective with other members of the HLA-A1 supertype to which HLA-A26 belongs. In addition to allowing vaccine development to begin now, this peptide-based approach would have the advantage of avoiding the use of dangerous, live, avian influenza virus during mass production.     

Molecular evolution of the six internal genes of H5N1 equine influenza A virus

Phylogenetic and evolutionary patterns of the six internal genes of an equine H5N1 influenza A virus isolated in Egypt on 2009 were analyzed using direct sequencing. All of the internal genes of the equine H5N1 strain showed a genetic pattern potentially related to Eurasian lineages. Variable dendrogram topologies revealed an absence of reassortment in the equine strain while confirming its close relatedness to other Egyptian H5N1 strains from human and avian species. The equine strain is characterized by a variety of amino acid substitutions in six internal proteins compared to the available Egyptian H5N1 strains. Interestingly, the equine strain displayed amino acids in the PB2, PA, M2 and NS2 proteins that are unique among the available H5N1 sequences in the flu database, and their potential effect on virulence needs to be further investigated.     

Persistence of tick-borne encephalitis virus IV. Virus localization after intracerebral inoculation

Tick-borne encephalitis (TBE) virus was isolated from the brains and spinal cords, blood, livers, lymph nodes and kidneys from Macaca rhesus monkeys showing acute and subacute fatal encephalitis. In subacute encephalitis, virus titres in the CNS were lower than in acute disease (3.0--6.2 against 3.8--8.3 log LD50/ml). TBE virus localization in chronic encephalitis was largely the same as in acute and subacute disease. In monkeys with a chronic course and stable paralysis of the upper extremity, infectious TBE virus was isolated on day 383 from subcortical ganglia and spinal cord. In lymph nodes and spleen, it could be detected only by a combination of methods (co-cultivation in association with fluorescent antibody technique and complement-fixation test, explantation of organ fragments) more sensitive than is the inoculation of mice with organ homogenates. TBE virus was detected by the same methods on day 90 in the CNS and internal organs of a monkey with chronic encephalitis in the stage of remission.     

An update on swine-origin influenza virus A/H1N1: a review

Influenza viruses cause annual epidemics and occasional pandemics that have claimed the lives of millions. The emergence of new strains will continue to pose challenges to public health and the scientific communities. The recent flu pandemic caused by a swine-origin influenza virus A/H1N1 (S-OIV) presents an opportunity to examine virulence factors, the spread of the infection and to prepare for major influenza outbreaks in the future. The virus contains a novel constellation of gene segments, the nearest known precursors being viruses found in swine and it probably arose through reassortment of two viruses of swine origin. Specific markers for virulence can be evaluated in the viral genome, PB1-F2 is a molecular marker of pathogenicity but is not present in the new S-OIV. While attention was focused on a threat of an avian influenza H5N1 pandemic emerging from Asia, a novel influenza virus of swine origin emerged in North America, and is now spreading worldwide. However, S-OIV demonstrates that even serotypes already encountered in past human pandemics may constitute new pandemic threats. There are concerns that this virus may mutate or reassort with existing influenza viruses giving rise to more transmissible or more pathogenic viruses. The 1918 Spanish flu pandemic virus was relatively mild in its first wave and acquired more virulence when it returned in the winter. Thus preparedness on a global scale against a potential more virulent strain is highly recommended. Most isolates of the new S-OIVs are susceptible to neuraminidase inhibitors, and currently a vaccine against the pandemic strain is being manufactured and will be available this fall. This review summarizes the current information on the new pandemic swine-origin influenza virus A/H1N1.     

Extrapulmonary tissue responses in cynomolgus macaques (Macaca fascicularis) infected with highly pathogenic avian influenza A (H5N1) virus

The mechanisms responsible for virulence of influenza viruses in humans remain poorly understood. A prevailing hypothesis is that the highly pathogenic virus isolates cause a severe cytokinemia precipitating acute respiratory distress syndrome and multiple organ dysfunction syndrome. Cynomolgus macaques (Macaca fascicularis) infected with a human highly pathogenic avian influenza (HPAI) H5N1 virus isolate (A/Vietnam/1203/2004) or reassortants of human influenza virus A/Texas/36/91 (H1N1) containing genes from the 1918 pandemic influenza A (H1N1) virus developed severe pneumonia within 24 h postinfection. However, virus spread beyond the lungs was only detected in the H5N1 group, and signs of extrapulmonary tissue reactions, including microglia activation and sustained up-regulation of inflammatory markers, most notably hypoxia inducible factor-1α (HIF-1α), were largely limited to this group. Extrapulmonary pathology may thus contribute to the morbidities induced by H5N1 viruses.     

Human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals

Viral attachment to the host cell is critical for tissue and species specificity of virus infections. Recently, pattern of viral attachment (PVA) in human respiratory tract was determined for highly pathogenic avian influenza virus of subtype H5N1. However, PVA of human influenza viruses and other avian influenza viruses in either humans or experimental animals is unknown. Therefore, we compared PVA of two human influenza viruses (H1N1 and H3N2) and two low pathogenic avian influenza viruses (H5N9 and H6N1) with that of H5N1 virus in respiratory tract tissues of humans, mice, ferrets, cynomolgus macaques, cats, and pigs by virus histochemistry. We found that human influenza viruses attached more strongly to human trachea and bronchi than H5N1 virus and attached to different cell types than H5N1 virus. These differences correspond to primary diagnoses of tracheobronchitis for human influenza viruses and diffuse alveolar damage for H5N1 virus. The PVA of low pathogenic avian influenza viruses in human respiratory tract resembled that of H5N1 virus, demonstrating that other properties determine its pathogenicity for humans. The PVA in human respiratory tract most closely mirrored that in ferrets and pigs for human influenza viruses and that in ferrets, pigs, and cats for avian influenza viruses.     

Development of a consensus microarray method for identification of some highly pathogenic viruses

Some highly pathogenic viruses, such as Chikungunya virus, Japanese encephalitis virus, Yellow fever virus, Dengue virus, Hanta virus, SARS‐CoV, and H5N1 avian influenza virus can cause severe infectious diseases. However, the consensus method for detecting these viruses has not been well established. A rapid and sensitive microarray approach for detection of these viruses and a panel of specific probes covering nine genera and 16 virus species were designed. 70‐mer oligonucleotides were used at the genus level and 50‐mer oligonucleotides were at the species level, respectively. To decrease the interference of the host genome in hybridization, the consensus genus primers were designed and used to reverse transcribe only virus genome. The synthesis of the second strand was carried out with a random primer sequence (5′‐GTTTCCCAGTAGGTCTCNNNNNNNN‐3′). The amplified products were labeled and processed for microarray analyses. This microarray‐based method used the highly conserved consensus primers to synthesize specifically the virus cDNA and could identify effectively Chikungunya virus, Japanese encephalitis virus, Yellow fever virus, Dengue virus, Tick borne encephalitis virus, and H5N1 avian influenza virus. Using this method, one unknown virus isolated from pig brain in Shanxi Province, China was identified. This method may have an important potential application for the diagnosis of virus infection. J. Med. Virol. 81:1945–1950, 2009. © 2009 Wiley‐Liss, Inc.     

Asia: avian influenza H5N1

The emergence of the first human cases of avian influenza in Hong Kong in 1997 has raised fears of a new pandemic originating from the Asian continent. Despite unprecedented international mobilization, first to stop then to limit its diffusion, the highly pathogenic avian virus A/H5N1 has successfully spread in Asia, Europe and Africa by successive epizootic outbreaks affecting migratory birds and poultry, Transmission from animals to humans is uncommon but severe with a fatality rate exceeding 60% in confirmed cases. Nearly half of the countries struck by the disease is in Asia. With 87% of confirmed cases and 91% of deaths, Asia is also one of the continent most heavily affected. Like the neighbouring countries in South-East Asia, Cambodia has been repeatedly hit by avian flu in the recent years. The measures implemented to prevent the spread of A/H5N1 virus in farms, to improve the behaviour of farmers and to clean up the poultry markets emphasize the multiple difficulties to control this zoonosis. Meanwhile, influenza surveillance is being carried out in humans, based on a reporting system and a network of sentinel hospitals connected to the Institut Pasteur du Cambodge. Thus, Cambodia takes an active part in the wide international network which should quickly detect any mutational event among avian flu viruses that might lead to the emergence of a pandemic.     

一株鹅H5N1亚型流感病毒基因特性的分析

目的 弄清了Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）毒株对鹅致病的分子生物学基础 ，研究香港区人群中发生的禽（Ｈ５Ｎ１）流感的病因，方法 病毒ＲＮＡ经逆转录合成ｃＤＮＡ经聚合酶链反应（ＰＣＲ）扩增，产物纯化，采用双脱链末端终止法测定核苷酸序列，结果 Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）与Ａ／ＨＫ／１５６／９７（Ｈ５Ｎ１）毒株ＲＮＡ４核苷酸序列有２２个位点不同（同源性为９８．８％）无任何掉失或插入。它与人和     

Induction of cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza virus infection in mice by inoculation of apathogenic H5N1 influenza virus particles inactivated with formalin

We investigated whether a vaccine derived from an apathogenic reassortant type A H5N1 influenza strain could induce immune responses in vivo that mediated protection from highly pathogenic avian influenza virus infection in mice. After two subcutaneous immunizations with formalin-inactivated H5N1 whole virus particles (whole particle vaccine), significant killing specific for cells presenting a nucleoprotein peptide from the vaccine strain of the virus was observed. Similar vaccination with viruses treated with ether and formalin, which are commonly used for humans as ether-split vaccines, induced little or no cytotoxic T-cell response. Furthermore, whole particle vaccines of the apathogenic H5N1 strain were more effective than ether-split vaccines at inducing antibody production able to neutralize a highly pathogenic H5N1 strain. Finally, whole particle vaccines of H5N1 protected mice against infection by an H5N1 highly pathogenic avian influenza virus more effectively than did ether-split vaccines. These results suggest that formalin-inactivated virus particles of apathogenic strains are effective for induction of both cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza viruses in vivo, resulting in protection from infection by a highly pathogenic H5N1 virus.