Little Evidence of Subclinical Avian Influenza Virus Infections among Rural Villagers in Cambodia

In 2008, 800 adults living within rural Kampong Cham Province, Cambodia were enrolled in a prospective cohort study of zoonotic influenza transmission. After enrollment, participants were contacted weekly for 24 months to identify acute influenza-like illnesses (ILI). Follow-up sera were collected at 12 and 24 months. A transmission substudy was also conducted among the family contacts of cohort members reporting ILI who were influenza A positive. Samples were assessed using serological or molecular techniques looking for evidence of infection with human and avian influenza viruses. Over 24 months, 438 ILI investigations among 284 cohort members were conducted. One cohort member was hospitalized with a H5N1 highly pathogenic avian influenza (HPAI) virus infection and withdrew from the study. Ninety-seven ILI cases (22.1%) were identified as influenza A virus infections by real-time RT-PCR; none yielded evidence for AIV. During the 2 years of follow-up, 21 participants (3.0%) had detectable antibody titers (≥1∶10) against the studied AIVs: 1 against an avian-like A/Migratory duck/Hong Kong/MPS180/2003(H4N6), 3 against an avian-like A/Teal/Hong Kong/w312/97(H6N1), 9 (3 of which had detectible antibody titers at both 12- and 24-month follow-up) against an avian-like A/Hong Kong/1073/1999(H9N2), 6 (1 detected at both 12- and 24-month follow-up) against an avian-like A/Duck/Memphis/546/74(H11N9), and 2 against an avian-like A/Duck/Alberta/60/76(H12N5). With the exception of the one hospitalized cohort member with H5N1 infection, no other symptomatic avian influenza infections were detected among the cohort. Serological evidence for subclinical infections was sparse with only one subject showing a 4-fold rise in microneutralization titer over time against AvH12N5. In summary, despite conducting this closely monitored cohort study in a region enzootic for H5N1 HPAI, we were unable to detect subclinical avian influenza infections, suggesting either that these infections are rare or that our assays are insensitive at detecting them.     

Highly Pathogenic Avian Influenza Virus among Wild Birds in Mongolia

Mongolia combines a near absence of domestic poultry, with an abundance of migratory waterbirds, to create an ideal location to study the epidemiology of highly pathogenic avian influenza virus (HPAIV) in a purely wild bird system. Here we present the findings of active and passive surveillance for HPAIV subtype H5N1 in Mongolia from 2005–2011, together with the results of five outbreak investigations. In total eight HPAIV outbreaks were confirmed in Mongolia during this period. Of these, one was detected during active surveillance employed by this project, three by active surveillance performed by Mongolian government agencies, and four through passive surveillance. A further three outbreaks were recorded in the neighbouring Tyva Republic of Russia on a lake that bisects the international border. No HPAIV was isolated (cultured) from 7,855 environmental fecal samples (primarily from ducks), or from 2,765 live, clinically healthy birds captured during active surveillance (primarily shelducks, geese and swans), while four HPAIVs were isolated from 141 clinically ill or dead birds located through active surveillance. Two low pathogenic avian influenza viruses (LPAIV) were cultured from ill or dead birds during active surveillance, while environmental feces and live healthy birds yielded 56 and 1 LPAIV respectively. All Mongolian outbreaks occurred in 2005 and 2006 (clade 2.2), or 2009 and 2010 (clade 2.3.2.1); all years in which spring HPAIV outbreaks were reported in Tibet and/or Qinghai provinces in China. The occurrence of outbreaks in areas deficient in domestic poultry is strong evidence that wild birds can carry HPAIV over at least moderate distances. However, failure to detect further outbreaks of clade 2.2 after June 2006, and clade 2.3.2.1 after June 2010 suggests that wild birds migrating to and from Mongolia may not be competent as indefinite reservoirs of HPAIV, or that HPAIV did not reach susceptible populations during our study.     

禽流感病毒与人流感的关系

禽流感病毒 (AIV)是甲 (A)型流感病毒 ,常引起禽类全身性感染或主要限于呼吸器官传染病 ,带来巨大的经济损失并严重威胁人类健康。对AIV的基因组、所编码的蛋白质及其功能、AIV毒力变异的分子基础、禽流感疫苗以及AIV与人流感的关系等进行概述。     

A Prospective Study of Romanian Agriculture Workers for Zoonotic Influenza Infections

Background

In this prospective study we sought to examine seroepidemiological evidence for acute zoonotic influenza virus infection among Romanian agricultural workers.

Methods

Sera were drawn upon enrollment (2009) and again at 12 and 24 months from 312 adult agriculture workers and 51 age-group matched controls. Participants were contacted monthly for 24 months and queried regarding episodes of acute influenza-like illnesses (ILI). Cohort members meeting ILI criteria permitted respiratory swab collections as well as acute and convalescent serum collection. Serologic assays were performed against 9 avian, 3 swine, and 3 human influenza viruses.

Results

During the two-year follow-up, a total of 23 ILI events were reported. Two subjects'' specimens were identified as influenza A by rRT-PCR. During the follow-up period, three individuals experienced elevated microneutralization antibody titers ≥1∶80 against three (one each) avian influenza viruses: A/Teal/Hong Kong/w312/97(H6N1), A/Hong Kong/1073/1999(H9N2), or A/Duck/Alberta/60/1976(H12N5). However, none of these participants met the criteria for poultry exposure. A number of subjects demonstrated four-fold increases over time in hemagglutination inhibition (HI) assay titers for at least one of the three swine influenza viruses (SIVs); however, it seems likely that two of these three responses were due to cross-reacting antibody against human influenza. Only elevated antibody titers against A/Swine/Flanders/1/1998(H3N2) lacked evidence for such confounding. In examining risk factors for elevated antibody against this SIV with multiple logistic regression, swine exposure (adjusted OR = 1.8, 95% CI 1.1–2.8) and tobacco use (adjusted OR = 1.8; 95% CI 1.1–2.9) were important predictors.

Conclusions

While Romania has recently experienced multiple incursions of highly pathogenic avian influenza among domestic poultry, this cohort of Romanian agriculture workers had sparse evidence of avian influenza virus infections. In contrast, there was evidence, especially among the swine exposed participants, of infections with human and one swine H3N2 influenza virus.     

Animal and Human Health Implications of Avian Influenza Infections

Avian influenza (AI) is a listed disease of the World Organisation for Animal Health (OIE) that has become a disease of great importance both for animal and human health. Until recent times, AI was considered a disease of birds with zoonotic implications of limited significance. The emergence and spread of the Asian lineage highly pathogenic AI (HPAI) H5N1 virus has dramatically changed this perspective; not only has it been responsible of the death or culling of millions of birds, but this virus has also been able to infect a variety of non-avian hosts including human beings. The implications of such a panzootic reflect themselves in animal health issues, notably in the reduction of a protein source for developing countries and in the management of the pandemic potential. Retrospective studies have shown that avian progenitors play an important role in the generation of pandemic viruses for humans, and therefore these infections in the avian reservoir should be subjected to control measures aiming at eradication of the Asian H5N1 virus from all sectors rather than just eliminating or reducing the impact of the disease in poultry. Dennis J. Alexander—Unaffiliated Consultant Virologist     

来自禽流感病毒与表观遗传学的挑战

近年来,高致病性禽流感病毒及表观遗传学是广受关注的生物医学领域。《 中国科学C辑：生命科学》 (Science in China Series C-Life Sciences)在2009年连续发表了两个专题,对两个研究领域的现状及发展态势,特别是当前一些具有挑战性的问题,进行了综合评述。表观遗传学专题包括三个论题：a. 迄今对组蛋白和非组蛋白甲基化的认识和了解,重点论述了组蛋白的甲基标记及去甲基化酶对组蛋白甲基化的动力学调控;b. miRNA的生物发生及其在转录后基因沉默的功能作用;c. 植物中RNA指导的DNA甲基化和去甲基化。禽流感病毒专题重点综合评述了高致病性人H5N1禽流感病毒研究领域的现状及其挑战,包括流行病学,疾病控制及病毒作用机理。有5篇评述全面总结了高致病性禽流感A(H5N1)感染,特别是人患禽流感,在中国大陆和香港特别行政区的发现及处置全过程,包括疫情沿起和传播,临床诊断,病毒特性,以及政府和公共卫生机构的政策和措施,为有效预防今后可能再度发生类似的疫情提供了可贵的实际经验。该专题还包括了4篇述评和研究论文,对病毒种间传播的宿主原因及感染人的分子病理学,流感病毒核蛋白(NP)的三维结构及其与RNA的相互作用,以及流感病毒RNA聚合酶PA亚基的三维结构与功能,进行了深度解析,并讨论了基于结构的抗流感药物研发前景．     

禽流感病毒实验检测研究进展

禽流感病毒(avian influenza viruses,AIV)给人类健康已带来严重威胁,而实验室快速、准确的诊断技术对禽流感的预防、控制及应急反应决策起着极其关键的作用,这使其成为了研究热点并取得了巨大进步。就禽流感的实验检测技术研究进展从病毒分离、免疫学诊断及分子诊断3个方面加以综述。     

Genetically Diverse Low Pathogenicity Avian Influenza A Virus Subtypes Co-Circulate among Poultry in Bangladesh

Influenza virus surveillance, poultry outbreak investigations and genomic sequencing were assessed to understand the ecology and evolution of low pathogenicity avian influenza (LPAI) A viruses in Bangladesh from 2007 to 2013. We analyzed 506 avian specimens collected from poultry in live bird markets and backyard flocks to identify influenza A viruses. Virus isolation-positive specimens (n = 50) were subtyped and their coding-complete genomes were sequenced. The most frequently identified subtypes among LPAI isolates were H9N2, H11N3, H4N6, and H1N1. Less frequently detected subtypes included H1N3, H2N4, H3N2, H3N6, H3N8, H4N2, H5N2, H6N1, H6N7, and H7N9. Gene sequences were compared to publicly available sequences using phylogenetic inference approaches. Among the 14 subtypes identified, the majority of viral gene segments were most closely related to poultry or wild bird viruses commonly found in Southeast Asia, Europe, and/or northern Africa. LPAI subtypes were distributed over several geographic locations in Bangladesh, and surface and internal protein gene segments clustered phylogenetically with a diverse number of viral subtypes suggesting extensive reassortment among these LPAI viruses. H9N2 subtype viruses differed from other LPAI subtypes because genes from these viruses consistently clustered together, indicating this subtype is enzootic in Bangladesh. The H9N2 strains identified in Bangladesh were phylogenetically and antigenically related to previous human-derived H9N2 viruses detected in Bangladesh representing a potential source for human infection. In contrast, the circulating LPAI H5N2 and H7N9 viruses were both phylogenetically and antigenically unrelated to H5 viruses identified previously in humans in Bangladesh and H7N9 strains isolated from humans in China. In Bangladesh, domestic poultry sold in live bird markets carried a wide range of LPAI virus subtypes and a high diversity of genotypes. These findings, combined with the seven year timeframe of sampling, indicate a continuous circulation of these viruses in the country.     

NASBA——一种新型禽流感病毒检测方法

NASBA(nucleic acid sequence-based amplification)是一项持续等温的核酸扩增技术,特别适合于以RNA为模版的扩增,与其它常用禽流感病毒检测方法(病毒培养法、免疫学方法和PCR)相比,具有灵敏度高、特异性强、操作简便等特点。就NASBA的操作原理及其在禽流感病毒检测中的成功应用进行综述。NASBA不仅成为禽流感病毒检测的有力工具,而且对于其它恶性传染病的监测、检测同样具有重要价值和意义。     

流感/禽流感病毒致病性研究进展

流感病毒是引起流行性感冒的病原体,它属正粘病毒科(Orthomyxoviridae),是具有包膜和分节段的单链负RNA病毒.甲、乙型流感病毒基因组均含8个节段,而丙型流感病毒仅含7个节段.根据流感病毒核蛋白(NP)和基质蛋白(MP)抗原特性及其基因特征的不同,流感病毒分为甲(A)、乙(B)、丙(C)三型.     

禽流感病毒新型疫苗的研究进展

疫苗免疫是禽流感防控的主要措施之一,随着生物技术的不断发展,基因工程亚单位疫苗、活载体疫苗、DNA疫苗等新型疫苗得以研究和开发,这为禽流感的防控提供了新的手段。新型疫苗除具有传统疫苗的保护效果外,在生物安全和普遍防控等方面也具有广泛的优势,是禽流感疫苗发展的新方向。     

黄酮类化合物对禽流感病毒的抑制作用

采用鸡胚培养法探讨黄酮类化合物对禽流感H5N1亚型病毒的抑制作用。实验分三种给药方式:即直接作用、先感染病毒后用药和先加药后感染。第一种给药方式说明黄酮类化合物可以直接灭活H5N1病毒;第二种给药方式说明黄酮类化合物可通过抑制流感病毒唾液酶的活性,从而抑制病毒粒子的复制;第三种给药方式反映一定浓度的药物可以阻断病毒对细胞的吸附作用。结果表明,黄酮类化合物对禽流感病毒的预防及治疗均有显著效果。     

禽流感病毒神经氨酸酶蛋白的研究进展

禽流感病毒在世界范围内广泛流行,给养禽业造成了巨大的经济损失,有效的疫苗接种策略可以帮助预防和控制该疾病.抗原转换和漂移是流感病毒发生变异的两种主要机制.NA蛋白是流感病毒的表面纤突之一属于Ⅱ型糖蛋白,在病毒成熟时发挥神经氨酸酶的作用进而有利于病毒的成熟和释放,并在调控受体结合、病毒出芽等方面发挥着重要的作用.抑制NA蛋白活性具有预防和治疗疾病作用的事实证实NA蛋白是一个抗病毒的有效靶点.此外基于NA蛋白设计流感疫苗,具有免疫保护期更持久和保护范围更广的优势.     

Experimental Infections of Dogs with Type C Influenza Virus

A study was performed to determine if type C influenza infection could be established in dogs as a model for human cases. Mongrel dogs were infected with the Ann Arbor/1/50 strain of type C influenza virus and were examined for clinical symptoms, virus isolation and antibody response. After the first exposure to the virus, all infected animals developed nasal discharge and some of them also showed swelling of the eyelids, and suffusion of the eyes with tears and eye mucus, within 1 to 4 days. The animals showed an increase in hemagglutination-inhibiting (HI) serum antibody, and recovery of the agent from the nasal swabs was successful. The symptoms lasted for as long as 10 days in most infected dogs, which was comparable to our human cases reported previously (Katagiri, S., Ohizumi, A., and Homma, M. 1983. J. Infect. Dis. 48 : 51–56). After the second and third virus exposures at intervals of 50 days, all animals developed the same symptoms as those described above and the rise in antibody titer was evident. The virus could be recovered from four of the six dogs 2 to 5 days after the second exposure and from one dog as late as 10 days after the third exposure. Increases in antibody titer in the IgM fraction were observed after every infection. In control dogs which were mock-infected with UV-inactivated virus, no symptoms were evident and recovery of the virus was not successful although an increase in HI serum antibody titer was seen. These results show that mongrel dogs are sensitive to type C influenza virus and that repeated infections characteristic of human influenza C can be experimentally produced in dogs.     

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(Nucleic acid sequence-based amplification,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).     

整合禽流感病毒血凝素糖蛋白的假型鼠白血病病毒

本研究通过一个瞬时转染系统将H5N1亚型鹅源禽流感病毒囊膜表面的血凝素(HA)糖蛋白整合到鼠白血病病毒(MuLV)颗粒表面并进行了感染性测定。将包含HA基因的真核表达质粒pcDNA-HA与MuLV假病毒构建体系的两种质粒pHIT60(包括MuLV的结构蛋白基因,即gag和pol)和pHIT111(为MuLV的基因组,还包括一个报告基因LacZ)瞬时共转染转化了SV40大T抗原的人胚肾细胞293T,48小时后收集假病毒上清进行了一系列鉴定。将假病毒上清超速离心后用抗H5亚型禽流感病毒的多抗通过Western-blot证实HA 蛋白能够在此假病毒颗粒表面表达,表明HA能够整合到此病毒粒子表面。通过感染293T、COS 7和NIH3T3 三种不同的靶细胞,均能检测到LacZ基因的表达,证实所构建的假病毒粒子具有感染性。本研究成功构建了具有感染性的MuLV-HA假病毒,为研究鹅源禽流感病毒侵入细胞的机理及其组织嗜性的变异提供一种新方法。     

整合禽流感病毒血凝素糖蛋白的假型鼠白血病病毒

本研究通过一个瞬时转染系统将H5N1亚型鹅源禽流感病毒囊膜表面的血凝素(HA)糖蛋白整合到鼠白血病病毒(MuLV)颗粒表面并进行了感染性测定.将包含HA基因的真核表达质粒pcDNA-HA与MuLV假病毒构建体系的两种质粒pHIT60(包括MuLV的结构蛋白基因,即gag和pol)和pHIT111(为MuLV的基因组,还包括一个报告基因LacZ)瞬时共转染转化了SV40大T抗原的人胚肾细胞293T,48小时后收集假病毒上清进行了一系列鉴定.将假病毒上清超速离心后用抗H5亚型禽流感病毒的多抗通过Western-blot证实HA 蛋白能够在此假病毒颗粒表面表达,表明HA能够整合到此病毒粒子表面.通过感染293T、COS-7和NIH3T3三种不同的靶细胞,均能检测到LacZ基因的表达,证实所构建的假病毒粒子具有感染性.本研究成功构建了具有感染性的MuLV-HA假病毒,为研究鹅源禽流感病毒侵入细胞的机理及其组织嗜性的变异提供一种新方法.     

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)。