H5、H7和H9亚型禽流感病毒四重荧光RT-PCR检测方法的建立与应用

旨在提高对禽流感病毒（avian influenza virus，AIV）检测效率，及时发现疫病。本研究通过分析M基因以及H5、H7和H9亚型的HA基因序列保守区域，设计并合成了相关探针和引物，建立了禽流感病毒（AIV）四重荧光RT-PCR检测方法，该方法可在检测禽流感病毒（AIV）的同时，确定病原是否为H5、H7和H9亚型。结果显示，该检测方法耗时短、特异性好、检测下限达到10^-5 ng·μL^-1。采用该方法检测临床采集的13个活禽交易市场的384份禽咽喉和泄殖腔双拭子样品，经检测，其中有60份样品为流感病毒阳性，且全部是H9亚型，该结果与行业标准方法（NY/T 772—2013）检测结果一致，κ值为1（P<0.001）。本方法能实现对禽流感病毒及H5、H7和H9亚型的高通量快速检测，将在AIV快速检测中发挥重要作用。     

Molecular epidemiology of avian influenza viruses circulating among healthy poultry flocks in farms in northern Vietnam

Repeated epizootics of highly pathogenic avian influenza (HPAI) virus subtype H5N1 were reported from 2003 to 2005 among poultry in Vietnam. More than 200 million birds were killed to control the spread of the disease. Human cases of H5N1 infection have been sporadically reported in an area where repeated H5N1 outbreaks among birds had occurred. Subtype H5N1 strains are established as endemic among poultry in Vietnam, however, insights into how avian influenza viruses including the H5N1 subtype are maintained in endemic areas is not clear. In order to determine the prevalence of different avian influenza viruses (AIVs), including H5N1 circulating among poultry in northern Vietnam, surveillance was conducted during the years 2006-2009. A subtype H5N1 strain was isolated from an apparently healthy duck reared on a farm in northern Vietnam in 2008 and was identified as an HPAI. Although only one H5N1 virus was isolated, it supports the view that healthy domestic ducks play a pivotal role in maintaining and transmitting H5N1 viruses which cause disease outbreaks in northern Vietnam. In addition, a total of 26 AIVs with low pathogenicity were isolated from poultry and phylogenetic analysis of all the eight gene segments revealed their diverse genetical backgrounds, implying that reassortments have occurred frequently among strains in northern Vietnam. It is, therefore, important to monitor the prevalence of influenza viruses among healthy poultry between epidemics in an area where AIVs are endemic.     

1株H6N5亚型禽流感病毒A/duck/Yangzhou/013/2008的全基因测序及遗传进化分析

在对华东地区家养水禽中流感病毒的带毒状况进行流行病学监测的过程中,采用常规的血清学试验和特异性RT-PCR方法,分离鉴定出1株H6N5亚型禽流感病毒A/duck/Yangzhou/013/2008(简称Dk/YZ/013/08)。为了探讨该亚型病毒在流感病毒生态分布中的作用,作者对Dk/YZ/013/08进行了全基因序列测定,并结合Gen-Bank中已收录的所有H6N5亚型病毒的基因组序列及其它参考序列进行了遗传进化分析。结果表明Dk/YZ/013/08的血凝素基因(HA)与近年中国台湾分离的鸭源毒株A/duck/Kingmen/E322/2004(H6N2)的核苷酸一致性最高(94%),推导的氨基酸剪切位点序列为"P-Q-I-E-T-R-G",为典型低致病性禽流感病毒的特征序列;神经氨酸酶基因(NA)与瑞士分离株A/mallard/Switzerland/WV4060167/2006(H3N5)的亲缘关系最近(核苷酸一致性96.9%);而碱性聚合酶2(PB2)基因则与A/duck/Zhejiang/11/2000(H5N1)的遗传距离最近,可能由H5N1亚型流感病毒提供,提示该毒株可能是一株重组病毒。     

鸭源禽流感病毒的分离和鉴定

从南京鸡鸭加工厂、孝陵卫鸭场及迈皋桥鸭场共采制１１３个泄殖腔拭子,用鸡胚尿囊腔接种传代法分离到４株禽流感病毒（ＡＩＶ）,每个样品均有ＮＤＶ混感。纯化后的分离株利用电镜负染技术观察到典型的ＡＩＶ粒子。琼扩试验证明４个分离株均为Ａ型流感病毒。经血凝素亚型分析,４株均属于Ｈ９亚型。致病性试验结果表明,４株鸭源禽流感病毒对鸡均表现为较低的致病力。本研究提示环境（特别是水体）保毒是ＡＩＶ得以长期存在和传播的重要因素和媒介。     

抗禽流感H9亚型血凝素单克隆抗体的病毒中和作用

通过血凝抑制(HI)和鸡胚中和试验(VN)证实，本实验室制备的抗禽流感H9M2单抗11A5和11B2株可特异性地抑制H9亚型禽流感病毒的血凝特性，而与禽流感H5和H7亚型以及其他具有血凝性感染禽类的病毒(如新城疫等)不反应。两株单抗腹水HI效价均达到15Log2。中和试验表明：上述两株单抗均可有效抑制H9N2病毒在SPF鸡胚中的增殖，使病毒失去血凝活性．测得11A5和11B2株腹水对H9N2禽流感病毒的半数保护量分别为10^-3.35和10^-4.58。该单抗的研制成功对于进一步建立快速鉴别诊断禽流感H9亚型病毒具有重要意义。     

(Highly pathogenic) avian influenza as a zoonotic agent

Zoonotic agents challenging the world every year afresh are influenza A viruses. In the past, human pandemics caused by influenza A viruses had been occurring periodically. Wild aquatic birds are carriers of the full variety of influenza virus A subtypes, and thus, most probably constitute the natural reservoir of all influenza A viruses. Whereas avian influenza viruses in their natural avian reservoir are generally of low pathogenicity (LPAIV), some have gained virulence by mutation after transmission and adaptation to susceptible gallinaceous poultry. Those so-called highly pathogenic avian influenza viruses (HPAIV) then cause mass die-offs in susceptible birds and lead to tremendous economical losses when poultry is affected. Besides a number of avian influenza virus subtypes that have sporadically infected mammals, the HPAIV H5N1 Asia shows strong zoonotic characteristics and it was transmitted from birds to different mammalian species including humans. Theoretically, pandemic viruses might derive directly from avian influenza viruses or arise after genetic reassortment between viruses of avian and mammalian origin. So far, HPAIV H5N1 already meets two conditions for a pandemic virus: as a new subtype it has been hitherto unseen in the human population and it has infected at least 438 people, and caused severe illness and high lethality in 262 humans to date (August 2009). The acquisition of efficient human-to-human transmission would complete the emergence of a new pandemic virus. Therefore, fighting H5N1 at its source is the prerequisite to reduce pandemic risks posed by this virus. Other influenza viruses regarded as pandemic candidates derive from subtypes H2, H7, and H9 all of which have infected humans in the past. Here, we will give a comprehensive overview on avian influenza viruses in concern to their zoonotic potential.     

Highly pathogenic avian influenza virus subtype H5N1 in Mute swans in the Czech Republic

In order to determine the actual prevalence of avian influenza viruses (AIV) in wild birds in the Czech Republic extensive surveillance was carried out between January and April 2006. A total of 2101 samples representing 61 bird species were examined for the presence of influenza A by using PCR, sequencing and cultivation on chicken embryos. AIV subtype H5N1 was detected in 12 Mute swans (Cygnus olor). The viruses were determined as HPAI (highly pathogenic avian influenza) and the hemagglutinin sequence was closely similar to A/mallard/Italy/835/06 and A/turkey/Turkey/1194/05. Following the first H5N1 case, about 300 wild birds representing 33 species were collected from the outbreak region and tested for the presence of AIV without any positive result. This is the first report of highly pathogenic avian influenza subtype H5N1 in the Czech Republic. The potential role of swan as an effective vector of avian influenza virus is also discussed.     

Characterization of H5N1 influenza viruses isolated from migratory birds in Qinghai province of China in 2006

Avian influenza H5N1 viruses pose a significant threat to human health because of their ability to infect humans directly. In the paper, three highly pathogenic H5N1 influenza viruses were isolated from three species of migratory birds in Qinghai Province of China in 2006. The analysis of the genome sequences indicated that the three isolates shared high homology with each other (94% to 99%). Three isolates shared a common ancestor and were closest to strains isolated from Qinghai and Siberia in 2005, but distinct from poultry viruses found in Southeast Asia. In experimental infection, all three viruses were highly pathogenic to chickens and mice. The results suggest that highly pathogenic avian influenza H5N1 viruses still exist in the migratory birds and could spread to other regions with wild bird migration.     

"Constanze": a trinational project on avian influenza in wild birds at Lake Constance

When highly pathogenic avian influenza H5N1 (HPAI H5N1) arrived at Lake Constance in February 2006, little was known about its ecology and epidemiology in wild birds. In order to prevent virus transmission from wild birds to poultry, the adjacent countries initiated the tri-national, interdisciplinary research program ?Constanze? to investigate avian influenza infections in water birds at Lake Constance. In collaboration with government agencies scientists examined the prevalence of AI virus in the region of Lake Constance for a period of 33 months, compared the effectiveness of different surveillance methods and analysed the migration behaviour of water birds. Although virus introduction from regions as far as the Ural Mountains seemed possible based on the migration behaviour of certain species, no influenza A viruses of the highly pathogenic subtype H5N1 (HPAIV) was found. However, influenza A viruses of different low pathogenic subtypes were isolated in 2.2 % of the sampled birds (swabs). Of the different surveillance methods utilised in the program the sampling of so called sentinel birds was particularly efficient.     

Avian influenza A viruses in birds of the order Psittaciformes: reports on virus isolations, transmission experiments and vaccinations and initial studies on innocuity and efficacy of oseltamivir in ovo

Birds of the order Psittaciformes are - besides chickens, turkeys and other birds - also susceptible to infection with avian influenza A viruses (AIV) and succumb following severe disease within one week. Published data prove that various parakeets, amazons, cockatoos, African grey parrots and budgerigars (genera Barnardius, Psittacula, Cacatua, Eolophus, Amazona, Myiopsitta, Psittacus and Melopsittacus) were found dead following natural infections. Natural infections of highly pathogenic avian influenza viruses (HPAIV) of the haemagglutinin subtypes H5 and H7 cause severe disease and high rates of mortality. Experimental transmission studies with AlVs of the subtypes H5 and H7 confirm these data. Viruses of the subtypes H3N8, H4N6, H4N8, H11N6 and H11N8 may cause also clinical signs and occasionally losses in naturally infected psittacine birds. Clinical signs and losses were also noted following experimental infection of budgerigars with a H4N6 virus. In the EU and in other countries, vaccination of exposed exotic and rare birds and poultry is a possible and an acceptable measure to provide protection. Currently, the EU Commission accepts inactivated adjuvanted vaccines whereas in some other countries recently developed vector vaccines are applied. However, birds remain susceptible during the time interval between application of any vaccine and the development of immunity. This critical period can be bridged with antiviral drugs. Our in ovo studies demonstrate that the neuraminidase inhibitor oseltamivir is non-toxic for chicken embryos at concentrations of 0.1, 1.0 and 10.0 mg/kg body weight. These dosages prevented entirely the replication of a HPAIV of the subtype H7N1 when this drug is given shortly prior to, simultaneously or soon after inoculation of chicken embryos with this AIV. Thus, we speculate that exposed valuable birds such as psittacines at risk can be successfully treated.     

H9N2亚型禽流感病毒抗原性变异的研究

对1998—2002年间在河南省豫北地区分离到的5株H9N2亚型禽流感病毒的抗原性变异进行了研究。经HI试验、鸡胚中和试验、细胞中和试验及攻毒保护试验证明，5株H9N2亚型间已经发生了抗原性漂移。98A5和99S毒株间的保护力接近100％，HI试验、鸡胚中和试验、细胞中和试验的相关性均在0．74以上。表明2毒株间的抗原性相近；用00Y毒株攻击其他4株免疫的鸡，其保护率仅为60％～80％；而02Y株对除00Y株外的4株的免疫保护率分别为60％、75％、80％、100％，与分离年代呈负相关性，HI、鸡胚中和试验、细胞中和试验也取得类似结果，说明2000年后的毒株间已发生抗原性变异。     

Australian surveillance for avian influenza viruses in wild birds between July 2005 and June 2007

Objective   To identify and gain an understanding of the influenza viruses circulating in wild birds in Australia.
Design   A total of 16,303 swabs and 3782 blood samples were collected and analysed for avian influenza (AI) viruses from 16,420 wild birds in Australia between July 2005 and June 2007. Anseriformes and Charadriiformes were primarily targeted.
Procedures   Cloacal, oropharyngeal and faecal (environmental) swabs were tested using polymerase chain reaction (PCR) for the AI type A matrix gene. Positive samples underwent virus culture and subtyping. Serum samples were analysed using a blocking enzyme-linked immunosorbent assay for influenza A virus nucleoprotein.
Results   No highly pathogenic AI viruses were identified. However, 164 PCR tests were positive for the AI type A matrix gene, 46 of which were identified to subtype. A total of five viruses were isolated, three of which had a corresponding positive PCR and subtype identification (H3N8, H4N6, H7N6). Low pathogenic AI H5 and/or H7 was present in wild birds in New South Wales, Tasmania, Victoria and Western Australia. Antibodies to influenza A were also detected in 15.0% of the birds sampled.
Conclusions   Although low pathogenic AI virus subtypes are currently circulating in Australia, their prevalence is low (1.0% positive PCR). Surveillance activities for AI in wild birds should be continued to provide further epidemiological information about circulating viruses and to identify any changes in subtype prevalence.     

Review of the literature on avian influenza A viruses in pigeons and experimental studies on the susceptibility of domestic pigeons to influenza A viruses of the haemagglutinin subtype H7

The scientific literature of the past century is reviewed on fowl plague (presently termed highly pathogenic avian influenza, HPAI) in pigeons. HPAI viruses cause epidemic disease outbreaks with high rates of losses in many avian species, particularily in chickens and turkeys. Also susceptible to disease are quails, guinea fowl, ducks, geese, ostriches, passerine birds, and birds of prey whereas conflicting reports on the susceptibility of the domestic pigeon exist. Based on literature reports and on own experiments, and applying as criteria for judgements clinically overt forms of disease, virus multiplication plus shedding and seroconversion, it is concluded that domestic pigeons are only partially susceptible to influenza A viruses of the haemagglutinin subtype H7. Infection of pigeons with H7 viruses results only in some of them in signs, virus shedding and seroconversion. Using the same criteria, pigeons appear to be even less susceptible to infection with influenza A viruses of the H5 subtype. Only one of five publications describe in 1/19 pigeons exposed to H5 influenza A virus depression one day before death, and only 2/19 multiplied and excreted virus, and 1/19 developed circulating antibodies. Consequently, pigeons play only a minor role in the epidemiology of H5 influenza viruses. In contrast, following infection with influenza A virus of the subtype H7 clinical signs in pigeons consist of conjunctivitis, tremor, paresis of wings and legs, and wet droppings. H7-infected pigeons multiply and excrete H7 viruses and develop circulating antibodies. Albeit of the status of infection, free-flying domestic pigeons can act as mechanical vectors and vehicles for long-distance transmission of any influenza A virus if plumage or feet were contaminated.     

我国2006年野鸟H5N1 HPAIV表面基因进化特征

国家禽流感参考实验室在2006年,从来自青、藏两省(区)3种野鸟及辽宁省的2种死亡的野鸟体内共分离到14株H5N1亚型高致病力禽流感病毒(High pathogenic avian influenza virus,HPAIV),比较发现其HA、NA基因核苷酸的同源率在97.9%～99.9%之间。所有病毒的HA基因的裂解位点均具有HPAIV特有的连续碱性氨基酸-RRRKKR-,并具有近年来H5N1亚型流行株所特有的NA基因颈部49～68位20个氨基酸缺失及NS基因80～84位5个氨基酸的缺失;遗传进化分析表明2006年野鸟病毒由2005年野鸟病毒进化而来,并形成独立的进化分支;辽宁省近2年的4株野鸟病毒亲缘关系较近,说明引起2005年该省锦州地区H5N1 HPAI疫情的病毒在该地区的野鸟体内继续存在。     

The isolation of influenza virus from chickens in Israel and studies on its antigenic relationships with other native isolates of the same antigenicity by means of monoclonal antibodies

A hemagglutinating (HA) agent isolated from an outbreak of a respiratory disease in a kibbutz broiler farm was identified as influenza virus A/chicken/Degania, Israel/80(H7N2). Investigation using a panel of 5 monoclonal antibodies against H7 antigenic subtype has shown substantial difference of the isolate from the other H7-containing influenza viruses isolated in Israel. Antigenic relationships between the native H7-containing strains revealed by means of the monoclonal antibodies led to re-evaluation of the suggested views on local epizootiology and interspecies transfer of avian influenza.     

Influenza virus surveillance in waterfowl in Pennsylvania after the H5N2 avian outbreak

During the latter stages of the lethal H5N2 influenza eradication program in domestic poultry in Pennsylvania in 1983-84, surveillance of waterfowl was done to determine if these birds harbored influenza viruses that might subsequently appear in poultry. From late June to November 1984, 182 hemagglutinating viruses were isolated from 2043 wild birds, primarily ducks, in the same geographical area as the earlier lethal H5N2 avian influenza outbreak. The virus isolates from waterfowl included paramyxoviruses (PMV-1, -4, and -6) and influenza viruses of 13 antigenic combinations. There was only one H5N2 isolate from a duck. Although this virus was antigenically related to the lethal H5N2 virus, genetic and antigenic analysis indicated that it could be discriminated from the virulent family of H5N2 viruses, and it did not originate from chickens. Many of the influenza viruses obtained from wild ducks were capable of replicating in chickens after experimental inoculation but did not cause disease. These studies show that many influenza A virus strains circulating in waterfowl in the vicinity of domestic poultry in Pennsylvania did not originate from domestic poultry. These influenza viruses from wild ducks were capable of infecting poultry; however, transmission of these viruses to poultry apparently was avoided by good husbandry and control measures.     

Molecular characterization of the hemagglutinin and neuraminidase genes of H5N1 influenza A viruses isolated from poultry in Vietnam from 2004 to 2005

Highly pathogenic H5N1 avian influenza A viruses have been spreading among domestic poultry, wild aquatic birds, and humans in many Asian countries since 2003. The largest number of patients, to date, infected with the H5N1 viruses are in Vietnam, where these viruses continue to cause outbreaks in domestic poultry. Here, we molecularly characterized the hemagglutinin and neuraminidase genes of nine H5N1 viruses isolated between January 2004 and August 2005 from domestic poultry in Vietnam. We found that several groups of highly pathogenic H5N1 avian influenza viruses are circulating among these birds, which suggests that H5N1 viruses of different lineages have been introduced into Vietnam multiple times.     

Pathogenicity of H5N1 influenza A viruses isolated in Vietnam between late 2003 and 2005

Since late 2003, highly pathogenic H5N1 influenza A viruses have spread among poultry and wild aquatic birds in Asian countries. Transmission of these viruses to humans can be lethal. Most human cases of infection with H5N1 viruses have occurred in Vietnam. Therefore, to understand the pathogenicity in mammals of these H5N1 viruses, we took viruses isolated from poultry (5 strains) and humans (2 strains) in Vietnam and tested their virulence in mice. The results showed that the H5N1 viruses from humans were pathogenic in mice and that one avian isolate was also pathogenic. These findings suggested that the H5N1 viruses circulating in poultry adapted during replication in humans or that strains pathogenic in mice were transmitted directly to humans.     

Genetic relationship of H3 subtype avian influenza viruses isolated from domestic ducks and wild birds in Korea and their pathogenic potential in chickens and ducks

The H3 subtype avian influenza virus (AIV) is one of the most frequently isolated subtypes in domestic ducks, live poultry markets, and wild birds in Korea. In 2002-2009, a total of 45 H3 subtype AIVs were isolated from the feces of clinically normal domestic ducks (n=28) and wild birds (n=17). The most prevalent subtypes in domestic ducks were H3N2 (35.7%), H3N6 (35.7%), H3N8 (25.0%), and H3N1 (3.6%, novel subtype in domestic duck in Korea). In contrast, H3N8 (70.6%) is the most prevalent subtype in wild birds in Korea. In the phylogenetic analysis, HA genes of the Korean H3 AIVs were divided into 3 groups (Korean duck, wild bird 1, and wild bird 2) and all viruses of duck origin except one were clustered in a single group. However, other genes showed extensive diversity and at least 17 genotypes were circulating in domestic ducks in Korea. When the analysis expanded to viruses of wild bird origin, the genetic diversity of Korean H3 AIVs became more complicated. Extensive reassortments may have occurred in H3 subtype influenza viruses in Korea. When we inoculated chickens and ducks with six selected viruses, some of the viruses replicated efficiently without pre-adaptation and shed a significant amount of viruses through oropharyngeal and cloacal routes. This raised concerns that H3 subtype AIV could be a new subtype in chickens in Korea. Continuous surveillance is needed to prepare the advent of a novel subtype AIV in Korea.