2015—2016年济南市活禽市场禽流感病毒监测分析

目的 了解济南市活禽市场外环境样本中甲型禽流感病毒动态分布状况,分析禽流感病毒污染特征.方法 2015年11月至2016年10月,选择济南市所属槐荫区活禽批发市场与历城区活禽零售市场进行外环境样本采集,样本类型包括禽处理台表面擦拭物、笼具表面擦拭物、粪便标本、清洗禽类的污水、禽类饮用水等.应用荧光定量RT-PCR方法对样本进行甲型流感病毒核酸检测,阳性样本进一步进行H5、H7及H9亚型检测.采用x2检验比较不同类型活禽市场、不同类型样本各亚型禽流感病毒核酸阳性率差异.结果 全年共采集2943份禽流感外环境样本,甲型禽流感病毒核酸阳性样本检出率为13.73%(404份),H5、H7与H9亚型检出率分别为4.52%(133份)、0.07%(2份)和8.49%(250份).五种不同类型样本中,甲型禽流感病毒阳性率最高的为禽处理台表面擦拭物(21.88%),最低的为禽类粪便(9.64%)(x2=38.535,P<0.001).但在活禽批发市场中,甲型禽流感病毒阳性率最高的样本为禽类饮用水(46.60%),零售市场中阳性率最高的样本为禽处理台表面擦拭物(18.65%).环境样本中甲型禽流感病毒的波动呈季节性趋势,冬春季节阳性率较高,最高为12月份,5—9月呈低发平缓趋势.结论 济南市活禽市场环境存在禽流感病毒的污染,并以H5和H9亚型为主.活禽市场中禽类饮用水与零售市场中禽类处理台面的病毒检出率最高.     

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.     

我国分离人H5N1禽流感病毒血凝素基因特性的研究

目的 分析我国分离的人高致病性禽流感H5N1病毒的抗原性以及基因特性.方法 对所分离的H5N1病毒的血凝素基因和神经氨酸酶基因进行序列测定.结果 对血凝素基因的序列测定结果表明从我国分离的H5N1病毒具有较高的同源性,但是与越南、泰国等国家分离的H5N1病毒明显不同.序列测定的结果同时表明无论是受体特异性还是连接肽都是禽源的.结论 目前我国分离的H5N1病毒属于同一个组,与泰国、越南分离毒株不同,并且发现病毒的特性仍然是禽源的,没有发现从禽到人的重组或者重配。     

高致病性禽流感H5N1病毒研究进展

1997年8月发现首例人禽流感病例以来,不断有人禽流感病例发生,截至2007年3月1日,WHO报道的经实验室确证的H5N1禽流感感染者共277例,死亡167例,死亡率超过50%。目前WHO对禽流感的预警是3级,即是一种新的流感亚型,已经在人类中引起严重疾病,但还没有在人类中持续传播流行。本文就目前H5N1禽流感病毒来源,跨越物种传播的机制,致病力决定因素及人间传播能力等几个关键的基础问题研究进展进行综述。     

高致病性禽流感H5N1病毒研究进展

1997年8月发现首例人禽流感病例以来,不断有人禽流感病例发生,截至2007年3月1日,WHO报道的经实验室确证的H5N1禽流感感染者共277例,死亡167例,死亡率超过50％。目前WHO对禽流感的预警是3级,即是一种新的流感亚型,已经在人类中引起严重疾病,但还没有在人类中持续传播流行。本文就目前H5N1禽流感病毒来源,跨越物种传播的机制,致病力决定因素及人间传播能力等几个关键的基础问题研究进展进行综述。     

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。     

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.     

Pathogenicity of the Korean H5N8 highly pathogenic avian influenza virus in commercial domestic poultry species

In 2014, the highly pathogenic avian influenza (HPAI) virus H5N8 triggered outbreaks in wild birds and poultry farms in South Korea. In the present study, we investigated the pathogenicity of the H5N8 HPAI virus, belonging to the clade 2.3.4.4, in different species of poultry. For this, we examined clinical signs and viral shedding levels following intranasal inoculation of the virus in 3-week-old commercial layer chickens and quails, 10-week-old Korean native chickens, and 8-week-old Muscovy ducks. Intranasal inoculation with 10^6.0 viruses at 50% egg-infective dose resulted in 100% mortality in the layer chickens (8/8) and quails (4/4), but 60% and 0% deaths in the Korean native chickens (3/5) and Muscovy ducks (0/4), respectively. In addition, transmission of the inoculated virus to contact-exposed birds was evident in all the species used in this study. Based on our results, we conclude that the H5N8 HPAI virus has lower pathogenicity and transmissibility in poultry species compared with previously reported H5N1 HPAI viruses.     

Genetic characterization of two low pathogenic avian influenza virus H5N1 isolates from Ontario, Canada

Genomes of two low pathogenic H5N1 avian influenza (LPAI) viruses, A/Turkey/ON/84/1983 and A/Mallard/ON/499/2005 from Ontario, Canada were cloned and genetically characterized. Phylogenetic analysis showed that the Canadian isolates cluster with other North American AIVs and are distinct from the Euro-Asian H5N1 isolates. Individual gene comparisons demonstrated that the Ontario isolates were most similar to the viruses isolated from around the same time period and geographical area. A long deletion of 22 amino acids was identified in the stalk region of NA of A/Turkey/ON/84/1983 isolate, a characteristic mutation related to its adaptation to domestic birds. To our knowledge A/Turkey/ON/84/1983 genomic sequence is the first and only available entire genomic sequence of a H5N1 AIV from domestic birds in Canada and USA. This work is a joint collaboration between the principal investigators Davor Ojkic and Shayan Sharif.     

Vaccination of gallinaceous poultry for H5N1 highly pathogenic avian influenza: Current questions and new technology

Vaccination of poultry for avian influenza virus (AIV) is a complex topic as there are numerous technical, logistic and regulatory aspects which must be considered. Historically, control of high pathogenicity (HP) AIV infection in poultry has been accomplished by eradication and stamping out when outbreaks occur locally. Since the H5N1 HPAIV from Asia has spread and become enzootic, vaccination has been used on a long-term basis by some countries to control the virus, other countries have used it temporarily to aid eradication efforts, while others have not used it at all. Currently, H5N1 HPAIV is considered enzootic in China, Egypt, Viet Nam, India, Bangladesh and Indonesia. All but Bangladesh and India have instituted vaccination programs for poultry. Importantly, the specifics of these programs differ to accommodate different situations, resources, and industry structure in each country. The current vaccines most commonly used are inactivated whole virus vaccines, but vectored vaccine use is increasing. Numerous technical improvements to these platforms and novel vaccine platforms for H5N1 vaccines have been reported, but most are not ready to be implemented in the field.     

Living poultry markets in rural area: Human infection with H7N9 virus re-emerges in Zhejiang Province,China, in winter 2014

BackgroundAvian influenza A H7N9 virus, previously undetected in humans, has caused infections in many areas in China since February 2013. Here we report the re-emergence of a case of H7N9 in rural Jiaxing city, Zhejiang Province, in the winter of 2014.ObjectivesTo understand (1) the clinical syndrome, epidemiological and virological characteristics of this case; (2) the importance of controlling live poultry markets (LPMs) in rural areas.Study designThere is one patient and 16 contacts, including 4 family members living in the same household, and 12 medical personnel. Pharyngeal swabs and serum samples were collected from the patient and her contacts. Environment samples were also obtained from the local LPMs. We conducted detailed clinical and epidemiological investigations and laboratory work, including viral RNA extraction, RT-PCR detection and sequencing. Characteristic and phylogenetic analyses were performed using the obtained sequences.ResultsH7N9s were detected in environmental samples collected in LPMs in Jiaxing, Zhejiang. Unknown mutations were discovered in amino acids in the sample from the patient. The strain from the patient was in a clade different from isolates obtained in 2013 in phylogenetic trees of HA, NA and PB2.ConclusionsA severe case of H7N9 was identified in early winter, 2014. Epidemiological and clinical tests were consistent with patterns reported previously, while laboratory findings showed the virus to be different. Live poultry markets in rural Zhejiang Province are in need of closer supervision and enhanced management.     

Generation and characterisation of monoclonal antibodies against influenza virus A, subtype H5N1

The emergence of highly pathogenic avian influenza A virus (HPAIV) subtype H5N1 in 1997 has since resulted in large outbreaks in poultry and in transmission from poultry to humans, mostly in southeast Asia, but also in several European countries. Effective diagnosis and control measures are essential for the management of HPAIV infections. To develop a rapid diagnostic test, a panel of murine monoclonal antibodies (mAbs) against influenza virus A subtype H5 was generated. Eleven mAbs were produced and characterised according to their reactivity by indirect and sandwich ELISA and western blotting against different H5 subtypes representing past and viruses currently circulating. Ten out of 11 mAbs reacted strongly with the haemagglutinin (HA) protein of H5 viruses, whereas one mAb reacted with the M1 protein. Targeted HA protein epitopes seemed to be conformational. One hybridoma clone binds to a linear epitope of the M1 protein. One specific mAb reacts with HPAIV H5 in the immunofluorescence test, and two antibodies neutralised H5 viruses. On the basis of the results, the set of seven mAbs is appropriate for developing diagnostic tests. With the generated mAbs, a sandwich ELISA was developed recognising all H5N1 strains tested but no other influenza viruses. With this ELISA, as little as 0.005 HA units or 0.1 ng/ml H5N1 was detected, surpassing other ELISA tests. The novel reagents have the potential to improve significantly available rapid antigen detection systems.     

Dogs are highly susceptible to H5N1 avian influenza virus

Replication of avian influenza viruses (AIVs) in dogs may facilitate their adaptation in humans; however, the data to date on H5N1 influenza virus infection in dogs are conflicting. To elucidate the susceptibility of dogs to this pathogen, we infected two groups of 6 beagles with 10⁶ 50% egg-infectious dose of H5N1 AIV A/bar-headed goose/Qinghai/3/05 (BHG/QH/3/05) intranasally (i.n.) and intratracheally (i.t.), respectively. The dogs showed disease symptoms, including anorexia, fever, conjunctivitis, labored breathing and cough, and one i.t. inoculated animal died on day 4 post-infection. Virus shedding was detected from all 6 animals inoculated i.n. and one inoculated i.t. Virus replication was detected in all animals that were euthanized on day 3 or day 5 post-infection and in the animal that died on day 4 post-infection. Our results demonstrate that dogs are highly susceptible to H5N1 AIV and may serve as an intermediate host to transfer this virus to humans.