Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses

In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin(HA) cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.     

Assessment of vaccination strategies against highly pathogenic avian influenza in China

Vaccination for highly pathogenic avian influenza (HPAI) has been implemented in China for a decade, however, the virus is still present in poultry. A series of recombinant vaccines, Re-1 to Re-7, have been developed and used, and Re-8 will also be used in clinical settings to prevent the prevailing flu strains. The question remains, when can China eradicate the disease? Here, we review the epidemiology of H5 HPAI along with the development, usage and problems of vaccines. Further suggestions for controlling the disease in China are provided.     

A highly conserved neutralizing epitope on group 2 influenza A viruses

Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. The identification of V(H)1-69 antibodies that broadly neutralize almost all influenza A group 1 viruses constituted a breakthrough in the influenza field. Here, we report the isolation and characterization of a human monoclonal antibody CR8020 with broad neutralizing activity against most group 2 viruses, including H3N2 and H7N7, which cause severe human infection. The crystal structure of Fab CR8020 with the 1968 pandemic H3 hemagglutinin (HA) reveals a highly conserved epitope in the HA stalk distinct from the epitope recognized by the V(H)1-69 group 1 antibodies. Thus, a cocktail of two antibodies may be sufficient to neutralize most influenza A subtypes and, hence, enable development of a universal flu vaccine and broad-spectrum antibody therapies.     

基于GIS的禽流感发生格局研究

    基于GIS空间数据处理技术,综合风险评估的理论和方法探询我国禽流感发生的风险格局.结果表明,我国禽流感疫情在经过2004年以来的综合防治以及2005年以来的免疫政策,目前疫情发生强度虽有所减弱,但疫情扩散和病毒变异风险依然存在:①我国以散养和小规模养殖模式为主体的养殖结构使禽流感发生的接触和暴露风险增大,特别是在候鸟迁徙区域的西南流域温地、西藏流域湿地、新疆流域湿地、甘蒙流域湿地、长江流域湿地以及黑龙江流域湿地,这些湿地区域在候鸟迁徙、带毒扩散上的廊道作用将逐渐显现;②目前我国禽流感疫情发生的聚集型态势明显.区域尺度疫点格局分析表明,在云南、辽宁、新疆、内蒙、广西和西藏的禽流感疫情发生的空间相关性显著,为聚集型分布,格局分析同时表明,目前我国禽流感发生已经形成了以西南一华南流域湿地、长江流域湿地、西藏流域湿地、新疆流域湿地、甘蒙流域湿地和黑龙江流域湿地等为中心的多点位发生态势;③全国尺度疫点格局分析表明,我国2004--2006年禽流感发生疫点的空间分布为聚集型,R统计特征值为0.43126(0.05水平),标准化ZR为7.69399,95%的置信区间内说明我国目前疫情已经跨越了零星发生的态势,聚集分布的空间特征使我国禽流感在一定时间段内复发成为可能.     

高致病性禽流感抗体水平监测及免疫程序制定

采用血凝抑制试验对贵阳市家禽高致病性禽流感免疫状况及试验鸡母源抗体和首免后免疫抗体的消长动态进行了检测,结果表明,贵阳市高致病性禽流感抗体几何平均效价为3．38210g2;试验鸡母源抗体群体几何平均效价及免疫保护率11、16日龄分别为2^4.3、2^3.41及45％、20％;免疫抗体群体几何平均效价及免疫保护率51、58日龄分别为2^5、2^2.25及66．7％、0％。该地区应及时加强免疫,首免时间和二免时间分别定为15、51日龄。     

低致病性与高致病性H7N9亚型禽流感病毒分子特征及受体结合能力分析

对2017年从鸡病料中分离到的2株H7N9亚型禽流感病毒(Avian influenza virus,AIV)A/chicken/Hubei/093/2017(H7N9)和A/chicken/Hubei/207/2017(H7N9)(CK93和CK207)进行了全基因组测序。对血凝素(Hemagglutinin,HA)和神经氨酸酶(Neuraminidase,NA)序列保守性、HA糖基化位点、NA耐药位点和6个内部基因的关键氨基酸位点进行了分析,同时测定了2株毒株的受体结合能力。结果显示,CK93毒株HA裂解位点为PEIPKGR↓GLF,为低致病性AIV特征;而CK207毒株裂解位点为PKPKRTAR↓GLF,为高致病性AIV特征。HA第226位氨基酸在CK93毒株为亮氨酸(226L),而在CK207毒株为谷氨酰胺(226Q)。但受体结合能力测定发现,CK93和CK207都具有结合α-2,3唾液酸受体和α-2,6唾液酸受体的特性,说明Q226L并非影响病毒受体结合特性的决定性因素。本研究结果提示应加强对H7N9亚型禽流感病毒变异监测预防其突变可能造成流行的风险。     

我国高致病性禽流感疫情及其应对机制

为研究我国高致病性禽流感疫情的蔓延特点、变化趋势和应对策略,预防禽流感疫情的大爆发,利用世界卫生组织公布的2003—2013年有关禽流感疫情数据,以及国家卫生部发布的有关疫情公告,进行统计与分析。结果表明:在WHO报告的15个发生H5N1禽流感疫情的国家中,我国发生确诊病例42人、死亡28人,总感染人数和死亡人数均位于第四,具有发生时间长、死亡率高的特点。H7N9疫情发生突然,爆发期约1个月,截至2013年底,共发生感染病例141人,死亡45人,疫情波及12个省(市)的42个地市。我国在完善应急预案制度基础上,建立了公开、及时、透明的禽流感疫情信息通报机制,同时将H7N9禽流感纳入国家乙级传染病监控体系,诊疗水平不断提高。     

Emergence of H5N1 highly pathogenic avian influenza in Democratic People's Republic of Korea

In the past decade, there has been extensive global surveillance for highly pathogenic avian influenza (HPAI) infection in both animals and humans, however, few studies on epidemiology of avian influenza in Democratic People's Republic of Korea (DPRK) were published. During the period 2013–2014, HPAI H5N1 viruses were detected with outbreaks in domestic poultry in DPRK. Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to clade 2.3.2.1c with high homology. The HPAI H5N1 virus found in ducks at the Tudan Duck Farm in 2013 was might introduced by migratory birds and then led to the outbreaks on neighboring chicken farms in 2014. These data provide direct evidence for the transmission of avian influenza viruses from wild birds to waterfowl to terrestrial birds. Therefore, the monitoring and control of influenza virus in ducks must be given top priority, which are essential components to prevent and control HPAI.     

Evolution of human influenza A viruses over 50 years: rapid, uniform rate of change in NS gene

Variation in influenza A viruses was examined by comparison of nucleotide sequences of the NS gene (890 bases) of 15 human viruses isolated over 53 years (1933 to 1985). Changes in the genes accumulate with time, and an evolutionary tree based on the maximum parsimony method can be constructed. The evolutionary rate is approximately 2 X 10(-3) substitution per site per year in the NS genes, which is about 10(6) times the evolutionary rate of germline genes in mammals. This uniform and rapid rate of evolution in the NS gene is a good molecular clock and is compatible with the hypothesis that positive selection is operating on the hemagglutinin (or perhaps some other viral genes) to preserve random mutations in the NS gene.     

Evolution of cell recognition by viruses

Evolution of receptor specificity by viruses has several implications for viral pathogenesis, host range, virus-mediated gene targeting, and viral adaptation after organ transplantation and xenotransplantation, as well as for the emergence of viral diseases. Recent evidence suggests that minimal changes in viral genomes may trigger a shift in receptor usage for virus entry, even into the same cell type. A capacity to exploit alternative entry pathways may reflect the ancient evolutionary origins of viruses and a possible role as agents of horizontal gene transfers among cells.     

水禽源流感病毒的禽类感染谱

用两株水禽源流感病毒番鸭源ZM株、鹅源SG株人工感染不同禽类,探讨毒株对不同禽类的致病情况及禽类的易感程度。结果表明：鸡、鹌鹑、鹧鸪对两毒株均具有高度的易感性;水禽类的鹅、番鸭也较为易感,而肉鸭和绍鸭有一定的抵抗力,说明鸭的易感性与品种有关;鸽的易感性较弱。     

Highly conserved protective epitopes on influenza B viruses

Identification of broadly neutralizing antibodies against influenza A viruses has raised hopes for the development of monoclonal antibody-based immunotherapy and "universal" vaccines for influenza. However, a substantial part of the annual flu burden is caused by two cocirculating, antigenically distinct lineages of influenza B viruses. Here, we report human monoclonal antibodies, CR8033, CR8071, and CR9114, that protect mice against lethal challenge from both lineages. Antibodies CR8033 and CR8071 recognize distinct conserved epitopes in the head region of the influenza B hemagglutinin (HA), whereas CR9114 binds a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses. These antibodies may inform on development of monoclonal antibody-based treatments and a universal flu vaccine for all influenza A and B viruses.     

Recombination of influenza A viruses of human and animal origin

Simultaneous infection of the allantoic sac of the chick embryo with influenza A/equine 1/56 and any of three recombinants derived from human influenza viruses produced stable hybrids with antigens from each parent strain. These hybrids contain the hemagglutinin protein of the equine virus and the neuraminidase of the human strains. The experiments demonstrate genetic homology of human and equine influenza A viruses and suggest the possibility of their recombination in nature.     

预防高致病性禽流感的环境控制措施

禽流感是禽流行性感冒的简称,被国际兽医局定为A类传染病,又称真性鸡瘟或欧洲鸡瘟.此病可通过呼吸道、消化道、皮肤损伤和眼结膜等多种途径传播,人员、飞鸟和往来车辆是传播本病毒的重要媒介.2003年底至2004年初亚洲一些国家如韩国、日本、泰国、越南、老挝、巴基斯坦等先后爆发了大规模的禽流感,并波及到我国一些地区.几个国家因此病死、宰杀、销毁的家禽已超过5亿只,给亚洲家禽养殖造成了严重的灾难,带来了巨大的损失,越南和泰国等国家已出现人感染禽流感病毒而死亡的病例.由于禽流感主要依靠水平传播,如空气、粪便、饲料和饮水等,且对于高致病性禽流感没有有效的治疗措施,做好综合防治、做好养禽场的环境控制,切断病毒的水平传播途径,是预防禽流感的关键环节和有效措施.     

禽流感高免卵黄抗体的研制与应用

用禽流感Ｈ９亚型油乳剂灭活苗多次免疫产蛋鸡,获得高免蛋,并禽流感高免卵黄液,在发病初期给禽流感病鸡肌注高免卵黄液,获得了满意的治疗效果。     

应用RT-PCR和病毒学方法检测低致病力禽流感感染的研究

分别应用病毒分离和鉴定试验以及禽流感病毒(AIV)型特异性RT-PCR技术,对疑似H9亚型AIV感染的10份病死鸡组织病料进行了实验室诊断。结果有7份病例用上述两种方法检测均为阳性;2份病料仅分离到H9亚型AIV;1份病例仅H9亚型RT-PCR阳性。研究表明,对于H9亚型AIV临床组织样品的检测,病毒分离试验的敏感度高于RT-PCR。当检测因运输或消毒药物作用而导致病料中病毒死亡的H9亚型AI临床病例时,RT-PCR有明显的优势。将RT-PCR和病毒分离鉴定两种方法相结合,可提高临床样本的H9亚型AIV的检出率。     

高致病性禽流感疫情风险下养殖户经济损失评价

2004年我国爆发了大范围的高致病性禽流感疫情,为保障养殖业生产安全和社会稳定,政府采取对疫点和疫区所有禽只进行扑杀并无害化处理的措施,有效防止了疫情的进一步蔓延,但也给养殖户造成了严重的经济损失。政府部门就此给予了一定的补偿,然而目前的补偿标准存在诸多的不合理性,没有切实考虑到养殖户的实际利益损失。在剖析现行补偿标准缺陷的基础上,借鉴发达国家的扑杀补偿措施,分析我国禽流感疫情给养殖户造成的经济损失构成及其计算方法,以期为相关政策的制定提供参考依据。