Wild waterfowl migration and domestic duck density shape the epidemiology of highly pathogenic H5N8 influenza in the Republic of Korea

Highly pathogenic avian influenza (HPAI) viruses threaten human and animal health yet their emergence is poorly understood, partly because sampling of the HPAI Asian-origin H5N1 lineage immediately after its identification in 1996 was comparatively sparse. The discovery of a novel H5N8 virus in 2013 provides a new opportunity to investigate HPAI emergence in greater detail. Here we investigate the origin and transmission of H5N8 in the Republic of Korea, the second country to report the new strain. We reconstruct viral spread using phylogeographic methods and interpret the results in the context of ecological data on poultry density, overwintering wild bird numbers, and bird migration patterns. Our results indicate that wild waterfowl migration and domestic duck density were important to H5N8 epidemiology. Specifically, we infer that H5N8 entered the Republic of Korea via Jeonbuk province, then spread rapidly among western provinces where densities of overwintering waterfowl and domestic ducks are higher, yet rarely persisted in eastern regions. The common ancestor of H5N8 in the Republic of Korea was estimated to have arrived during the peak of inward migration of overwintering birds. Recent virus isolations likely represent re-introductions via bird migration from an as-yet unsampled reservoir. Based on the limited data from outside the Republic of Korea, our data suggest that H5N8 may have entered Europe at least twice, and Asia at least three times from this reservoir, most likely carried by wild migrating birds.     

2015年广州市3 447份外环境标本高致病性禽流感病毒监测结果分析

目的 分析2015年广州市禽类市场H5和H7高致病性禽流感病毒的污染情况,为人禽流感防控提供基础数据。方法 按随机抽样的方法,对广州市禽类市场进行相关标本采集,用实时荧光定量RT-PCR 方法检测禽流感病毒,阳性标本再进行H5和H7亚型禽流感病毒检测。结果 全年共采集标本3447份,A型、H5和H7亚型禽流感病毒阳性率分别为12.56%、0.75%和1.54%。中心城区和周边城区禽类市场均受到H5和H7亚型禽流感病毒的污染,病毒阳性率冬春季节大于夏秋季节。宰杀环节A型禽流感病毒检出率最高(15.16%),H5和H7亚型禽流感病检出率最高的标本均为地面涂抹拭子(阳性率为别为1.95%和3.41%)。结论 广州市禽类市场普遍存在H5和H7亚型禽流感病毒共污染的现象,尤其以冬春季节最为严重。在加强禽类市场全面清洗消毒的基础上,应重点关注宰杀环节和地表面环境等高危险因素,采取基于风险的针对性防控措施。     

广州市2014-2019年H5亚型禽流感病毒流行与基因遗传特征分析

目的 分析广州市H5亚型禽流感病毒流行特点及基因进化和变异特征,为禽流感的防控提供依据。方法 对2014-2019年广州市禽类市场外环境标本进行禽流感病毒核酸检测并分析H5亚型流行情况,随机选取48份（46份来源于环境和2份来源于病例）H5阳性标本进行HA和NA基因测序,应用生物信息学软件分析分子遗传特征。结果 2014-2019年监测的52 284份环境标本中,检出H5亚型阳性标本1 094份,阳性率为2.09%。遗传进化分析显示,HA基因属于Clade 2.3.4.4.C分支,NA基因主要归属于欧亚谱系H6N6进化分支,HA和NA基因的进化以时间聚类为特点,相近年份流行株聚成一个进化分支。分子特征显示,48份毒株裂解位点呈现高致病性分子特点,受体结合位点仍为禽源受体,但普遍发生S123P、S133A和T156A倾向结合人源受体的位点突变。抗原位点变异主要出现在B、E区,并表现时间分布的特点,同一年份流行株常发生与上一年份流行株不同的抗原位点变异。2017年开始所有毒株出现由氨基酸缺失导致的糖基化位点的增加（140-NHT）。结论 广州市禽类市场外环境H5亚型禽流感病毒长期流行,且阳性率占比逐渐升高。测序分析提示,广州市H5亚型禽流感病毒为Clade 2.3.4.4.C分支H5N6高致病性病毒,并且持续进化和变异,特别是普遍出现与人源受体结合能力增强的突变,需加强监测。     

An inactivated H5N2 vaccine reduces transmission of highly pathogenic H5N1 avian influenza virus among native chickens

Vaccination against H5N1 highly pathogenic avian influenza in endemically affected areas is a potentially attractive option for local prevention and control. In Indonesia the majority of local outbreaks have occurred in back yard flocks with native chickens, and it is therefore of interest to determine whether these birds can be protected against infection by vaccination. To this end two transmission experiments were carried out with H5N1 virus (A/chicken/Legok/2003) in vaccinated and unvaccinated native chickens. The vaccine contained an inactivated heterologous H5N2 strain (A/turkey/England/N28/73 H5N2). Birds were vaccinated at 4 and 7 weeks of age and challenged at 10 weeks of age. During 10 days post-challenge tracheal and cloacal swabs were taken for virus isolation, and serum blood was collected regularly to measure haemaglutinin inhibiting (HI) antibody responses. The results show that transmission of H5N1 virus was rapid and efficient in unvaccinated birds, that infection and transmission were completely prevented in vaccinated birds, and that vaccinated birds that were exposed to unvaccinated inoculated birds were still protected from infection. These findings indicate that vaccination with a heterologous H5N2 vaccine is able to prevent virus transmission in flocks of native chickens.     

高致病性H7N9禽流感的治疗和预防

自2013年春我国上海市发生第一例高致病性H7N9禽流感以来,禽流感的治疗和预防已经成为广大医疗工作者研究的课题。     

Novel reassortant highly pathogenic H5N6 avian influenza viruses in poultry in China

We characterized two novel highly pathogenic H5N6 influenza viruses isolated from Chinese poultry in 2013. Genomic analysis showed that both isolates were reassortants, and derived their genes from H5 and H6 subtype viruses found in poultry in China. The virulence of the two isolates was examined in chickens and mice, and both isolates were found to be highly pathogenic in chickens and only moderately virulent for mice. Our results show that continued circulation of these viruses could endanger both avian species and humans.     

人感染禽流感病毒的传播

禽流感病毒属于A型流感病毒,其感染导致的传染病一般只在禽类间传播,然而1997年以来,存在于家禽中的一些禽流感病毒已经突破了动物种间屏障,能够直接从禽类传播给人类,导致亚洲及全球范围内很多感染病例的死亡,存在潜在大流行的威胁。此文对1997年以来禽流感病毒感染人类的状况进行分析,为今后新型禽流感暴发的预防和控制提供参考。     

A recombinant vesicular stomatitis virus replicon vaccine protects chickens from highly pathogenic avian influenza virus (H7N1)

Highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 cause fatal disease in poultry (fowl plague) but also have zoonotic potential. Currently commercially available vaccines often do not provide sufficient protection and do not allow easy discrimination between vaccinated and infected birds. Therefore, vaccination of domestic poultry against H5 and H7 HPAIV is not allowed in many countries, or is only possible after special permission has been provided. We generated a recombinant marker vaccine based on non-transmissible vesicular stomatitis virus (VSV) expressing the HA antigen of HPAIV A/FPV/Rostock/34 (H7N1) in place of the VSV G gene. This virus, VSV*ΔG(HA), was propagated on a helper cell line providing VSV G in trans. Since no progeny virus was produced after infection of non-complementing cells, the vector was classified as biosafety level 1 organism (“safe”). Chickens were immunized via the intramuscular route. Following booster vaccination with the same replicons high titers of serum antibodies were induced, which neutralized avian influenza viruses of subtypes H7N1 and H7N7 but not H5N2. Vaccinated chickens were protected against a lethal dose of heterologous HPAIV A/chicken/Italy/445/99 (H7N1). Secretion of challenge virus was short-term and significantly reduced. Finally, it was possible to discriminate vaccinated chickens from infected ones by a simple ELISA assay. We propose that VSV replicons have the potential to be developed to high-quality vaccines for protection of poultry against different subtypes of avian influenza viruses.     

Longitudinal 2 years field study of conventional vaccination against highly pathogenic avian influenza H5N1 in layer hens

A licensed, inactivated vaccine based on a low pathogenic avian influenza virus strain (H5N2) was evaluated in layer hens kept under field conditions during a 2-year period. Vaccine efficacy was investigated by specific antibodies and by challenge-contact experiments using highly pathogenic avian influenza viruses (HPAIV) H5N1. Basic immunization with two applications induced clinical protection. Virus excretion by vaccinated hens was significantly reduced compared to non-vaccinated controls; transmission to non-vaccinated and vaccinated contact birds was not fully interrupted. Vaccination efficacy is influenced by several factors including antigenic relatedness between vaccine and field strains, but also by species, age and type of commercial uses of the host. Limitations and risks of HPAIV vaccination as silent spread of HPAIV and emergence of escape mutants must be considered a priori and appropriate corrective measures have to be installed.     

Free-grazing ducks and highly pathogenic avian influenza, Thailand

Thailand has recently had 3 epidemic waves of highly pathogenic avian influenza (HPAI); virus was again detected in July 2005. Risk factors need to be identified to better understand disease ecology and assist HPAI surveillance and detection. This study analyzed the spatial distribution of HPAI outbreaks in relation to poultry, land use, and other anthropogenic variables from the start of the second epidemic wave (July 2004-May 2005). Results demonstrate a strong association between H5N1 virus in Thailand and abundance of free-grazing ducks and, to a lesser extent, native chickens, cocks, wetlands, and humans. Wetlands used for double-crop rice production, where free-grazing duck feed year round in rice paddies, appear to be a critical factor in HPAI persistence and spread. This finding could be important for other duck-producing regions in eastern and southeastern Asian countries affected by HPAI.     

Long lasting immunity in chickens induced by a single shot of influenza vaccine prepared from inactivated non-pathogenic H5N1 virus particles against challenge with a highly pathogenic avian influenza virus

An influenza vaccine was prepared from inactivated whole particles of the non-pathogenic strain A/duck/Hokkaido/Vac-1/04 (H5N1) virus using an oil adjuvant containing anhydromannitol-octadecenoate-ether (AMOE). The vaccine was injected intramuscularly into five 4-week-old chickens, and 138 weeks after vaccination, they were challenged intranasally with 100 times 50% chicken lethal dose of the highly pathogenic avian influenza (HPAI) virus A/chicken/Yamaguchi/7/04 (H5N1). All 5 chickens survived without exhibiting clinical signs of influenza, although 2 days post-challenge, 3 vaccinated chickens shed limited titres of viruses in laryngopharyngeal swabs.     

人感染H7N9禽流感的流行特征

目的：对我国发生人感染 H7N9禽流感的流行病学特点进行分析,为H7N9禽流感的预防控制提供依据。方法人感染 H7N9禽流感确诊132例的病例相关数据来源于2013年3－5月中国国家卫生和计划生育委员会网站（http：／／www ．chinapop ．gov ．cn／yjb／h7n9／list ．shtml）和我国台湾地区卫生署疾管局网站（http：／／www ． cdc ．gov ．tw／）,通过Excel 2007软件进行统计分析,总结其发病地点、时间、患者性别和年龄分布、临床症状轻重等因素。结果132例H7N9禽流感确诊病例基本为散发状态,绝大多数发生在华东地区,浙江、上海和江苏3省市较多,各占34．85％、25．00％、20．45％;病例数在前3周呈上升趋势,第3周峰值达47例,随后逐渐下降,至第6周后基本无新增确诊病例;男女性别比为2．34∶1,男性病例远多于女性;年龄分布在4～91岁,以中老年人为主,＞45岁确诊病例占75．76％;确诊病例大部分表现为重症或病死,该两者占76．52％,其中死亡38例,病死率达28．79％。结论人感染H7N9禽流感确诊病例的流行病学特征与其他禽流感例如 H5N1显然不同,具体原因有待于进一步研究确定。     

Immunologic evaluation of 10 different adjuvants for use in vaccines for chickens against highly pathogenic avian influenza virus

Avian influenza viruses (AIV) are a threat to poultry production worldwide. Vaccination is utilized as a component of control programs for both high pathogenicity (HP) and low pathogenicity (LP) AIV. Over 95% of all AIV vaccine used in poultry are inactivated, adjuvanted products. To identify the best formulations for chickens, vaccines were prepared with beta-propiolactone (BPL) inactivated A/British Columbia/314514-1/2004 H7N3 LP AIV using ten commercially available or experimental adjuvants. Each vaccine formulation was evaluated for immunogenicity in chickens. Challenge studies with an antigenically homologous strain of HPAIV were conducted to compare protection against mortality and measure reductions in virus levels in oral swabs. The four best adjuvants from the studies with BPL inactivated antigen were selected and tested identically, but with vaccines prepared from formalin inactivated virus. Mineral and vegetable oil based adjuvants generally induced the highest antibody titers with 100% seroconversion by 3 weeks post vaccination. Chitosan induced positive antibody titers in 100% of the chickens, but the titers were significantly lower than those of most of the oil based adjuvants. Antibody levels from calcium phosphate and alginate adjuvanted groups were similar to those of non-adjuvanted virus. All groups that received adjuvanted vaccines induced similar levels of protection against mortality (0–20%) except the groups vaccinated with calcium phosphate adjuvanted vaccines, where mortality was similar (70%) to groups that received non-adjuvanted inactivated virus or no vaccine (60–100% mortality). Virus shedding in oral swabs was variable among the treatment groups. Formalin inactivated vaccine induced similar antibody titers and protection against challenge compared to BPL inactivated vaccine groups. These studies support the use of oil adjuvanted vaccines for use in the poultry industry for control for AIV.     

Short- and long-term protective efficacy against clade 2.3.4.4 H5N2 highly pathogenic avian influenza virus following prime-boost vaccination in turkeys

Highly pathogenic avian influenza virus (HPAIV) infections are frequently associated with systemic disease and high mortality in domestic poultry, particularly in chickens and turkeys. Clade 2.3.4.4 represents a genetic cluster within the Asian HPAIV H5 Goose/Guangdong lineage that has transmitted through migratory birds and spread throughout the world. In 2014, clade 2.3.4.4 strains entered the U.S. via the Pacific flyway, reassorted with local strains of the North American lineage, and produced novel HPAIV strains of the H5N1, H5N2, and H5N8 subtypes. By 2015, the H5N2 HPAIVs disseminated eastwards within the continental U.S. and Canada and infected commercial poultry, causing the largest animal health outbreak in recent history in the U.S. The outbreak was controlled by traditional mass depopulation methods, but the outbreak was of such magnitude that it led to the consideration of alternative control measures, including vaccination. In this regard, little information is available on the long-term protection of turkeys vaccinated against avian influenza. In this report, a vaccination study was carried out in turkeys using 3 prime-boost approaches with a combination of 2 different vaccines, an alphavirus-based replicon vaccine and an adjuvanted-inactivated reverse genetics vaccine. Vaccine efficacy was assessed at 6 and 16 weeks of age following challenge with a prototypic novel clade 2.3.4.4 H5N2 HPAIV. All three vaccines protocols were protective with significantly reduced virus shedding and mortality after challenge at 6 weeks of age. In contrast, significant variations were seen in 16-week old turkeys after challenge: priming with the alphavirus-based replicon followed by boost with the adjuvanted-inactivated vaccine conferred the best protection, whereas the alphavirus-based replicon vaccine given twice provided the least protection. Our study highlights the importance of studying not only different vaccine platforms but also vaccination strategies to maximize protection against HPAIV especially with regards to the longevity of vaccine-induced immune response.     

Experimental infection of cattle with highly pathogenic avian influenza virus (H5N1)

Four calves were experimentally inoculated with highly pathogenic avian influenza virus A/cat/Germany/R606/2006 (H5N1) isolated from a cat in 2006. All calves remained healthy, but several animals shed low amounts of virus, detected by inoculation of nasal swab fluid into embryonated chicken eggs and onto MDCK cells. All calves seroconverted.     

一例H5N1人禽流感病例感染源调查分析

目的 明确1例人禽流感H5N1确诊病例的感染来源.方法 通过现场流行病学调查、密切接触者医学观察、高危从业人员症状监测、实验室检测和溯源调查等方法，判断感染来源.结果 2012年5月17-19日该患儿随母在广州A市场购买活鸭，现场经历屠宰过程.23、24日发病后在当地医院就诊，诊断为呼吸道感染；26日抵香港后诊断为人禽流感病例.23名密切接触者和34名市场禽类销售屠宰从业人员未出现流感样症状.患儿住家附近B市场2名禽类销售人员H9型血清抗体阳性，阳性率为6.06％(2/33).A、B两市场砧板、禽笼等环境标本H5、H9型阳性率分别为9.8％(5/51)和2.0％(1/51).B市场一份砧板H5型阳性标本与患儿标本同属2.3.2.1分支.B市场阳性档口的进货来源与A市场一致.结论 该禽流感病例的感染来源为广州市肉菜市场禽类销售档口.     

Birds and influenza H5NI virus movement to and within North America

Highly pathogenic avian influenza (HPAI) H5N1 expanded considerably during 2005 and early 2006 in both avian host species and geographic distribution. Domestic waterfowl and migratory birds are reservoirs, but lethality of this subtype appeared to initially limit migrant effectiveness as introductory hosts. This situation may have changed, as HPAI H5N1 has recently expanded across Eurasia and into Europe and Africa. Birds could introduce HPAI H5N1 to the Western Hemisphere through migration, vagrancy, and importation by people. Vagrants and migratory birds are not likely interhemispheric introductory hosts; import of infected domestic or pet birds is more probable. If reassortment or mutation were to produce a virus adapted for rapid transmission among humans, birds would be unlikely introductory hosts because of differences in viral transmission mechanisms among major host groups (i.e., gastrointestinal for birds, respiratory for humans). Another possible result of reassortment would be a less lethal form of avian influenza, more readily spread by birds.     

一起水禽H5N1疫情暴发后人群感染风险评估

目的 评估动物禽流感疫情暴发后人群感染的风险,探讨禽流感传播的可能性.方法 采用现场流行病学调查、分子流行病学、血清学研究及应急监测方法 ,对病、死禽的所有密切接触者进行医学观察;采用红细胞凝集抑制实验、实时荧光逆转录-聚合酶链式反应(RT-PCR)、基因测序方法 ,检测全部密切接触者的血清抗体,采集4个疫点环境标本检测禽流感H5核酸.结果 检测4个疫点环境标本22份,H5核酸阳性1份,序列分析与广州市2006年人禽流感病毒株A/China/GD01/2006(H5N1)的同源性为95.9%;检测疫区及周边2个农贸市场活禽交易场所环境标本62份,H5核酸均阴性;采集密切接触者的血样68份、咽拭子68份,禽流感H9抗体阳性6份,H5抗体、H5核酸均阴性,医学观察7 d,未发现禽流感感染者;应急监测区报告流感样患者337例,经排查未发现可疑禽流感患者.结论 此起水禽H5N1暴发未造成扩散,也未出现人感染病例,表明此次疫情的禽流感病毒H5N1对人的传播能力尚不强,引起人群感染的风险较低.