H5N8亚型高致病性禽流感病毒的全球流行概况

H5N8亚型高致病性禽流感病毒(highly pathogenic avian influenza virus,HPAIV)随候鸟的迁徙活动及商业贸易活动现已蔓延至亚洲、欧洲、非洲、美洲等国家和地区.2014-2015和2016-2019年H5N8亚型HPAIV已引发两波全球疫情,现正经历第三波疫情,导致家禽及野生鸟类...     

Spread of H5N1 avian influenza virus: an ecological conundrum

The role of wild birds in the spread of influenza H5N1 virus remains speculative and the ecology of influenza A viruses in nature is largely unstudied. There is an urgent need for multidisciplinary studies to explore the ecology of avian influenza viruses in wild birds and the environment to support ecological interpretation of the source of disease outbreaks in poultry.     

H5N1 avian influenza in China

H5N1 highly pathogenic avian influenza virus was first detected in a goose in Guangdong Province of China in 1996. Multiple genotypes of H5N1 viruses have been identified from apparently healthy waterfowl since 1999. In the years 2004–2008, over 100 outbreaks in domestic poultry occurred in 23 provinces and caused severe economic damage to the poultry industry in China. Beginning from 2004, a culling plus vaccination strategy has been implemented for the control of epidemics. Since then, over 35420000 poultry have been depopulated, and over 55 billion doses of the different vaccines have been used to control the outbreaks. Although it is logistically impossible to vaccinate every single bird in China due to the large poultry population and the complicated rearing styles, there is no doubt that the increased vaccination coverage has resulted in decreased disease epidemic and environmental virus loading. The experience in China suggests that vaccination has played an important role in the protection of poultry from H5N1 virus infection, the reduction of virus load in the environment, and the prevention of H5N1 virus transmission from poultry to humans. Supported by the Key Animal Infectious Disease Control Program of the Ministry of Agriculture, the Chinese National S&T Plan(Grant No. 2004BA519A-57), National Key Basic Research and Development Program of China (Grant Nos: 2005CB523005, 2005CB523200).     

NASBA快速检测禽流感H5亚型病毒

采用建立的依赖核酸序列的扩增(Nucleicacidsequencebasedamplification,NASBA)对禽流感病毒3株H5亚型、1株H1、H3、H6亚型、3株禽流感H9亚型、5株不同宿主来源的新城疫病毒、鸭肝炎病毒、鸭瘟病毒、SPF鸡胚尿囊液及禽流感(H9)疫苗、新城疫疫苗、传染性法氏囊病疫苗、传染性支气管炎疫苗进行检测,结果NASBA(H5试剂)仅检测到禽流感病毒H5亚型,表明方法的特异性强。采用已知禽流感病毒A/Chicken/HK/1000/97(H5N1)的鸡胚尿囊液(ELD5010-7.5/mL),经10倍连续稀释,将经典的鸡胚病原分离法和NASBA进行比较,二种方法的灵敏度相当。用A/Chicken/HK/1000/97(H5N1)病毒人工感染SPF鸡、商品鸡,采用NASBA和病原分离法同时对人工感染鸡的粪拭子、血液进行了动态检测;采集感染死亡鸡的组织脏器,共检测了101个组织脏器,两种方法的符合率为90%(87/97)。     

Human avian influenza A (H5N1) virus infection in China

Highly pathogenic influenza A (H5N1) virus causes a widespread poultry deaths worldwide. The first human H5N1 infected case was reported in Hong Kong Special Administrative Region of China in 1997. Since then, the virus re-emerged in 2003 and continues to infect people worldwide. Currently, over 400 human infections have been reported in more than 15 countries and mortality rate is greater than 60%. H5N1 viruses still pose a potential pandemic threat in the future because of the continuing global spread and evolution. Here, we summarize the epidemiological, clinical and virological characteristics of human H5N1 infection in China monitored and identified by our national surveillance systems. Chinese Nature Science Foundation Key Project (Grant No. 30599433), Chinese Basic Science Research Program (973)Key Project (Grant No. 2005CB523006)     

重组毕赤酵母高密度发酵表达H5N1禽流感病毒糖蛋白

在10L发酵罐中,对高致病性禽流感病毒H5N1糖蛋白HA1在重组毕赤酵母中的表达发酵工艺进行了研究。通过分批补料培养方法探讨不同培养温度、诱导温度、补料方式、微量元素等因素对菌体的生长以及重组蛋白表达和活性的影响。结果表明,菌种培养和诱导温度均为25oC时,菌体的生长、分泌表达量和与广谱中和抗体的反应活性较好;微量元素是影响重组HA1蛋白生物活性的重要因素;通过优化高密度发酵工艺,H5N1病毒糖蛋白HA1在发酵罐中的表达量比摇瓶培养提高10.5倍,达到约120mg/L,为大规模制备高致病性禽流感病毒的HA1蛋白奠定了基础。     

The variable codons of H5N1 avian influenza A virus haemagglutinin genes

We investigated the selection pressures on the haemagglutinin genes of H5N1 avian influenza viruses using fixed effects likelihood models. We found evidence of positive selection in the sequences from isolates from 1997 to 2007, except viruses from 2000. The haemagglutinin sequences of viruses from southeast Asia, Hong Kong and mainland China were the most polymorphic and had similar nonsyn-onymous profiles. Some sites were positively selected in viruses from most regions and a few of these sites displayed different amino acid patterns. Selection appeared to produce different outcomes in vi-ruses from Europe, Africa and Russia and from different host types. One position was found to be positively selected for human isolates only. Although the functions of some positively selected posi-tions are unknown, our analysis provided evidence of different temporal, spatial and host adaptations for H5N1 avian influenza viruses.     

Highly pathogenic H5N1 influenza virus causes coagulopathy in chickens

Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus-infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.     

Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger

In early 2013, a Bengal tiger (Panthera tigris) in a zoo died of respiratory distress. All specimens from the tiger were positive for HPAI H5N1, which were detected by real-time PCR, including nose swab, throat swab, tracheal swab, heart, liver, spleen, lung, kidney, aquae pericardii and cerebrospinal fluid. One stain of virus, A/Tiger/JS/1/2013, was isolated from the lung sample. Pathogenicity experiments showed that the isolate was able to replicate and cause death in mice. Phylogenetic analysis indicated that HA and NA of A/Tiger/JS/1/2013 clustered with A/duck/Vietnam/OIE-2202/2012 (H5N1), which belongs to clade 2.3.2.1. Interestingly, the gene segment PB2 shared 98% homology with A/wild duck/Korea/CSM-28/20/2010 (H4N6), which suggested that A/Tiger/JS/1/2013 is a novel reassortant H5N1 subtype virus. Immunohistochemical analysis also confirmed that the tiger was infected by this new reassortant HPAI H5N1 virus. Overall, our results showed that this Bengal tiger was infected by a novel reassortant H5N1, suggesting that the H5N1 virus can successfully cross species barriers from avian to mammal through reassortment.     

Quantum‐dots‐based fluoroimmunoassay for the rapid and sensitive detection of avian influenza virus subtype H5N1

The continuous spread of highly pathogenic avian influenza virus (AIV) subtype H5N1 is threatening the poultry industry and human health worldwide. Rapid and sensitive diagnostic methods are required for the H5N1 surveillance. In this study, the fluorescent (FL) probe of CdTe quantum dots (QDs) was designed using covalently linked rabbit anti‐AIV H5N1 antibody. Based on these QD–antibody conjugates, a novel sandwich FL‐linked immunosorbent assay (sFLISA) was developed for H5N1 viral antigen detection. The sFLISA allowed for H5N1 viral antigen determination in a linear range of 8.0 × 10^?3 to 5.1 × 10^?1 μg mL^?1 with the limit of detection (LOD) of 1.5 × 10^?4 μg mL^?1. In comparison with virus isolation for 103 clinic samples, the sensitivity and specificity of sFLISA were found to be 93.6 and 91.1% respectively. The sFLISA supplied a novel approach to rapid and sensitive detection of AIV subtype H5N1 and showed great potential for biological applications in immunoassays. Copyright © 2009 John Wiley & Sons, Ltd.     

Genomic signature analysis of the recently emerged highly pathogenic A(H5N8) avian influenza virus: implying an evolutionary trend for bird-to-human transmission

In early 2014, a novel subclade (2.3.4.4) of the highly pathogenic avian influenza (HPAI) A(H5N8) virus caused the first outbreak in domestic ducks and migratory birds in South Korea. Since then, it has spread to 44 countries and regions. To date, no human infections with A(H5N8) virus have been reported, but the possibility cannot be excluded. By analyzing the genomic signatures of A(H5N8) strains, we found that among the 47 species-associated signature positions, three positions exhibited human-like signatures (HLS), including PA-404S, PB2-613I and PB2-702R and that mutation trend of host signatures of avian A(H5N8) is different before and after 2014. About 82% of A(H5N8) isolates collected after January of 2014 carried the 3 HLS (PA-404S/PB2-613I/PB2-702R) in combination, while none of isolates collected before 2014 had this combination. Furthermore, the HA protein had S137A and S227R substitutions in the receptor-binding site and A160T in the glycosylation site, potentially increasing viral ability to bind human-type receptors. Based on these findings, the newly emerged HPAI A(H5N8) isolates show an evolutionary trend toward gaining more HLS and, along with it, the potential for bird-to-human transmissibility. Therefore, more extensive surveillance of this rapidly spreading HPAI A(H5N8) and preparedness against its potential pandemic are urgently needed.     

A/H6N1亚型禽流感病毒致病性评估及与2009 A/H1N1亚型流感病毒在哺乳动物体内的遗传兼容性

目的探讨人、禽流感病毒在哺乳动物体内的遗传兼容性,为下一步研究H6亚型禽流感病毒重配和致病性变异的分子机制奠定基础。方法野鸭源A/H6N1亚型禽流感病毒A/Mallard/SanJiang/275/2007以101EID50～106EID50的攻毒剂量经鼻内途径感染小鼠,通过临床症状观察、病毒滴定和病理切片观察进行病毒学和组织学两方面检测对小鼠的致病性;同时,将此病毒与2009年A/H1N1流感病毒A/Changchun/01/2009(H1N1)混合感染豚鼠,分析两株病毒在哺乳动物体内的遗传兼容性。每天采集豚鼠鼻洗液并用噬斑纯化技术获得重配病毒,对获得的重配病毒进行全基因组序列的测定。结果 H6N1亚型禽流感病毒能直接感染小鼠,但对小鼠不致死。106EID50的攻毒剂量可有效感染小鼠,攻毒后第5天,小鼠表现出被毛较粗乱、活动减少、体重下降、呼吸急促的临床症状,但至攻毒后第10天开始康复,而对照组(MOCK)小鼠在14 d的观察期内无明显临床症状。病毒滴定结果表明,该病毒主要在小鼠肺脏和鼻甲骨中复制,病毒滴度可达104.5EID50/mL。病理学观察发现感染小鼠肺泡壁增厚,有大量炎性细胞浸润,纤维蛋白渗出并伴有轻微出血;在A/H6N1和A/H1N1混合感染豚鼠的重配实验中,经过三轮噬斑纯化从豚鼠鼻洗液中分离到6株重配病毒,说明A/H6N1亚型禽流感病毒与A/H1N1亚型流感病毒具有很好的遗传兼容性,能在豚鼠体内能发生重配。结论野鸭源A/H6N1亚型流感病毒无需适应就能够感染哺乳动物;该病毒与A/H1N1流感病毒具有很好的遗传兼容性,在哺乳动物体内能够发生基因重配,产生新的重配病毒,其公共卫生意义应引起高度关注。     

H5N1亚型禽流感病毒进化的研究进展

由H5N1流感病毒引起的高致病性禽流感,在禽类之间广泛传播。当人类接触这些禽类时,可能会被感染并产生严重的呼吸道症状,且死亡率高达60%。血凝素(hemagglutinin,HA)是H5N1病毒中和抗体的主要抗原,为了便于对病毒的HA突变进行研究,根据HA遗传基因的差异远近,所有的H5病毒株都被划分在20个分支内。对于H5N1病毒进化的研究在禽流感疫苗的研制、禽流感大流行的预防等方面均具有重要意义。现对禽流感、H5N1病毒特征、血凝素的结构功能、H5N1病毒的分支以及病毒进化的研究进行概述。     

Interspecies transmission and host restriction of avian H5N1 influenza virus

Long-term endemicity of avian H5N1 influenza virus in poultry and continuous sporadic human infections in several countries has raised the concern of another potential pandemic influenza. Suspicion of the avian origin of the previous pandemics results in the close investigation of the mechanism of interspecies transmission. Entry and fusion is the first step for the H5N1 influenza virus to get into the host cells affecting the host ranges. Therefore receptor usage study has been a major focus for the last few years. We now know the difference of the sialic acid structures and distributions in different species, even in the different parts of the same host. Many host factors interacting with the influenza virus component proteins have been identified and their role in the host range expansion and interspecies transmission is under detailed scrutiny. Here we review current progress in the receptor usage and host factors. Supported by the National Basic Research Program of China (Grant Nos. 2005CB523001, 2005CB523002), National Key Technologies Research & Development Program (Grant 2006BAD06A01/2006BAD06A04); US National Institutes of Health (NIH) (Grant 3 U19 AI051915-05S1), the National Natural Science Foundation of China (Grant 30599434). GAO FG is a distinguished young investigator of the NSFC (Grant No. 30525010).     

H5N1重组禽流感病毒样颗粒免疫原性研究

目的对构建的H5N1重组禽流感病毒样颗粒(VLPs)进行初步免疫原性探讨,并与H5N1全病毒灭活疫苗(WIV)进行体液免疫和细胞免疫的比较。方法在0周和3周分别以纯化H5N1重组禽流感病毒样颗粒、H5N1全病毒灭活疫苗及pH7.2 PBS腿部肌肉注射BALB/c小鼠,于不同时间收集血清,以血凝抑制试验(HI)和血清IgG抗体酶联免疫吸附试验(ELISA)评估体液免疫,CD4+、CD8+T细胞亚群及酶联免疫斑点试验(ELISPOT)评估细胞免疫,并以同型毒株滴鼻攻击,观察小鼠存活率。结果病毒样颗粒各组和全病毒灭活疫苗免疫后小鼠血清ELISA IgG效价均有升高;中和抗体效价除病毒样颗粒120 ng/只免疫剂量外其他免疫小鼠HI效价均达1︰40;小鼠脾CD4+T淋巴细胞亚群分类:全病毒灭活疫苗组(600μg/只)为36.56%;病毒样颗粒组(120 ng/只,600 ng/只,2 500 ng/只)分别为26.58%,32.20%,29.25%;PBS组为26.65%;CD8+T淋巴细胞亚群分类:全病毒灭活疫苗组(600 ng/只)为10.78%;病毒样颗粒组(120 ng/只,600 ng/只,2 500 ng/只)分别为1 3.53%,14.24%,1 3.35%;PBS组为10.69%。ELISPOT试验统计学数据显示,病毒样颗粒和全病毒灭活疫苗的小鼠脾单个核细胞分泌IFN-γ细胞与PBS组有显著性差异;小鼠保护性试验结果显示,除病毒样颗粒120 ng/只免疫剂量小鼠的存活率为87.5%外,其他病毒样颗粒实验组小鼠均为100%,PBS对照组为12.5%。结论 H5N1重组禽流感病毒样颗粒能诱导体液免疫和细胞免疫,并能抵御同型病毒株的攻击,可作为H5N1人用禽流感的候选疫苗。     

An H5N1 highly pathogenic avian influenza virus isolated from a local tree sparrow in Indonesia

The isolation of an H5N1 influenza A virus from a tree sparrow (Passer montanus) captured in East Java, Indonesia in 2010 is reported here. Its hemagglutinin and neuraminidase were genetically similar to those of human isolates from 2006-2007 in Indonesia. The finding of a tree sparrow H5N1 virus that possesses genetically similar surface molecules to those of human viruses highlights the importance of monitoring resident wild birds, as well as migratory birds, for pandemic preparedness.     

禽流感病毒H5N1全基因组克隆与分析

为明确广东地区分离的一株禽流感病毒H5N1的遗传背景,建立流感病毒反向遗传的平台。对该株禽流感病毒进行了空斑纯化与组织细胞培养,检测其在MDCK细胞中的增殖特性;利用H5N1病毒通用引物,通过RT-PCR对该病毒全基因组的8条片段进行全长克隆及测序分析;将H5N1的8条全长基因组片段分别插入反向遗传通用载体中,构建禽流感病毒H5N1的感染性克隆。结果表明,该H5N1毒株在MDCK细胞中可不依赖胰酶进行有效增殖与复制,可使MDCK细胞出现典型细胞病变,具有高致病性禽流感病毒的细胞增殖特征。RT-PCR克隆得到该H5N1毒株的PB2、PB1、PA、HA、NP、NA、M和NS八条全长片段,经测序分析确认该毒株的基因序列,其内部编码序列出现多处突变,其中HA连接肽为多个连续碱性氨基酸,表明该毒株可不依赖胰酶进行有效复制,与细胞培养结果一致,未出现抗药性的遗传突变。PCR与测序证明,插入H5N1八个全长基因组片段的载体序列完全正确,表明成功构建了该毒株的感染性克隆。为明确病毒遗传信息,建立流感病毒反向遗传的平台,为进一步研究禽流感病毒相关疫苗提供了研究基础。     

Nucleic acid sequence-based amplification methods to detect avian influenza virus

Infection of poultry with highly pathogenic avian influenza virus (AIV) can be devastating in terms of flock morbidity and mortality, economic loss, and social disruption. The causative agent is confined to certain isolates of influenza A virus subtypes H5 and H7. Due to the potential of direct transfer of avian influenza to humans, continued research into rapid diagnostic tests for influenza is therefore necessary. A nucleic acid sequence-based amplification (NASBA) method was developed to detect a portion of the haemagglutinin gene of avian influenza A virus subtypes H5 and H7 irrespective of lineage. A further NASBA assay, based on the matrix gene, was able to detect examples of all known subtypes (H1-H15) of avian influenza virus. The entire nucleic acid isolation, amplification, and detection procedure was completed within 6h. The dynamic range of the three AIV assays was five to seven orders of magnitude. The assays were sensitive and highly specific, with no cross-reactivity to phylogenetically or clinically relevant viruses. The results of the three AIV NASBA assays correlated with those obtained by viral culture in embryonated fowl's eggs.