高滴度感染力H5N1亚型禽流感病毒假病毒颗粒的制备

利用一个瞬时共转染系统,将H5N1亚型禽流感病毒的血凝素（Hemagglutinin,HA）蛋白与神经氨酸酶（Neuraminidase,NA）蛋白整合到鼠白血病病毒假病毒颗粒表面,包装成表达HA与NA的假病毒颗粒,通过透射电子显微镜形态学观察、感染滴度分析、血凝试验和中和试验研究其生物学特性。研究获得了高滴度感染力的H5N1假病毒颗粒（H5N1 Pseudotyped particle,H5N1Pp）,H5N1Pp的感染力滴度为1E8 Pp/mL,形态、血凝活性及中和特性均与野生H5N1病毒相似,结果为H5N1病毒受体、HA与NA的功能、中和抗体、抗病毒药物开发研究的开展建立了平台。     

H5N1型禽流感病毒感染非人灵长类动物的观察

目的观察H5N1型禽流感病毒对中国非人灵长类动物的易感性并建立动物模型。方法将病毒通过滴鼻接种实验猴,观察感染后动物的临床症状,采血、咽拭子及各器官组织进行血清学、病原学及病理学检查,记录抗体变化、病毒分离情况及病理学改变。结果感染后动物表现轻度食欲下降、一过性体温升高及外周血白细胞减少,肺组织病毒分离及RT-PCR阳性,病理检查感染急性期动物肺组织表现为间质性肺炎,肺泡间隔增宽,充血出血明显,肺泡受压变形,间质及肺泡内有大量炎细胞浸润,符合病毒性肺炎的改变,感染后14 d动物血清IgG抗体水平较感染前升高4倍。结论H5N1病毒可感染非人灵长类动物,可以作为感染模型进行H5N1病毒的发病机制、疫苗评价、药物筛选等研究。     

Evidence for the Convergence Model: The Emergence of Highly Pathogenic Avian Influenza (H5N1) in Viet Nam

Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (EID), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza (HPAI) H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs.     

H5N1禽流感的威胁与全球应对

当前H5N1禽流感在迁徙禽类、家禽中的暴发,以及越来越多的人感染病例的发生,使流感全球大流行的可能性持续存在。简要综述了H5N1禽流感在鸟类和其他动物中的暴发情况,H5N1禽流感的人感染病例,以及全球禽流感应对计划及疫苗、药物、病原体基础研究的进展。     

禽流感H5N1亚型病毒感染恒河猴模型的建立及禽流感发病机制初探

[目的]建立禽流感H5N1病毒感染恒河猴的动物模型,探讨禽流感在哺乳类动物的发病机制。[方法]通过“环甲膜穿刺术”经气管注射鸡胚培养的禽流感H5N1病毒(AF148678;ACGoose/Guangdong/11961H5N1)感染恒河猴,观察恒河猴染毒后出现的临床体征,用显微计数法检测外周血白细胞的动态变化,用ELISA检测禽流感病毒特异性抗体变化规律,用流式细胞仪检测外周血T淋巴细胞及其亚群的动态变化。在染毒后第1天、第3天、第10天和第14天分别剖杀染毒组恒河猴1只,HE染色观察主要组织器官的病理变化,用病毒分离、免疫组化和RT-PCR三种方法分析禽流感病毒侵袭机体的特点。[结果]临床症状和体征:急性起病,表现为发热,呼吸困难,精神状态下降,活动度明显减少,食欲下降,咳嗽,紫绀等,肺部听诊双肺可闻及干、湿音。1、病理特点:以肺部损伤为主,伴多器官病变。肺部的病变主要表现为弥漫性肺泡损伤,先后经历渗出期、增生期和纤维化期;在肝脏、肾脏和中枢神经系统中也观察到变性、坏死等病理变化。2、病毒侵袭机体的特点:病毒只在呼吸系统中复制,不在呼吸道以外的组织器官中复制;肺内支气管上皮细胞、肺泡上皮细胞和肺巨噬细胞是禽流感病毒侵犯的主要细胞类型。3、外周血象特点:外周血白细胞总数、淋巴细胞数出现短暂的下降,中性粒细胞数先升后降,但均于感染第7天后逐渐恢复到正常水平。4、抗体变化特点:感染后第7~11天,抗体水平持续快速升高;感染第11天后,抗体水平呈逐渐缓慢升高趋势(观察到染毒后50天为止)。5、细胞免疫特点:细胞免疫功能受损,表现为CD3+T淋巴细、CD3+CD4+T淋巴细胞和CD3+CD8+T淋巴细胞均出现短暂的下降,但这种细胞免疫功能受损是可逆的,到感染第7天后逐渐恢复回升至正常。[结论]1、恒河猴感染后的临床特点、病理变化、外周血象、免疫反应等均与人禽流感严重病例相类似,表明该模型是成功的,可为禽流感病毒在人体内致病机理的研究以及抗禽流感病毒的药物和疫苗评价提供最近似于人类的动物模型。2、综合本研究的实验结果,我们认为,H5N1禽流感毒主要攻击的对象为呼吸系统,不在呼吸道以外的组织器官中复制。禽流感病毒感染引起的急性弥漫性肺损伤是发病的中心环节,其发病可能经过病毒侵入、复制阶段,免疫损伤阶段和多器官功能损伤阶段。     

Newcastle Disease Virus-Vectored H7 and H5 Live Vaccines Protect Chickens from Challenge with H7N9 or H5N1 Avian Influenza Viruses

Sporadic human infections by a novel H7N9 virus occurred over a large geographic region in China. In this study, we show that Newcastle disease virus (NDV)-vectored H7 (NDV-H7) and NDV-H5 vaccines are able to induce antibodies with high hemagglutination inhibition (HI) titers and completely protect chickens from challenge with the novel H7N9 or highly pathogenic H5N1 viruses, respectively. Notably, a baculovirus-expressed H7 protein failed to protect chickens from H7N9 virus infection.     

H5N1亚型禽流感病毒NS1基因在昆虫细胞中的表达

将H5N1亚型禽流感病毒（AIV）NS1基因插入到杆状病毒转移载体pFastBac1中,获得重组转移载体pFastBac1- NS1。将pFastBac1- NS1转化到DH10Bac感受态细胞中,筛选到重组转座子rBacmid-NS1。在脂质体转染试剂介导下将rBacmid-NS1转染对数生长期的Sf9昆虫细胞获得重组杆状病毒rBV-NS1。rBV-NS1感染Sf9细胞后,通过SDS-PAGE、Western blot和ELISA分析表明：获得了分子量为26ku的特异性NS1蛋白;并且该蛋白可与H5N1 AIV攻毒鸭的血清发生特异性免疫反应,而不能与H5N1AIV灭活疫苗免疫鸭的血清发生反应。试验结果表明：NS1在Sf9昆虫细胞中获得了高效表达,具有与天然蛋白相似的免疫活性,并可以作为区分免疫及自然感染个体的鉴别诊断抗原。本实验为建立禽流感病毒自然感染家禽与禽流感灭活苗免疫家禽的鉴别诊断方法奠定基础。     

The Evolutionary Pattern of Glycosylation Sites in Influenza Virus (H5N1) Hemagglutinin and Neuraminidase

Two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of influenza viruses play crucial roles in transfaunation, membrane fusion and the release of progeny virions. To explore the distribution of N-glycosylation sites (glycosites) in these two glycoproteins, we collected and aligned the amino acid sequences of all the HA and NA subtypes. Two glycosites were located at HA0 cleavage sites and fusion peptides and were strikingly conserved in all HA subtypes, while the remaining glycosites were unique to their subtypes. Two to four conserved glycosites were found in the stalk domain of NA, but these are affected by the deletion of specific stalk domain sequences. Another highly conserved glycosite appeared at the top center of tetrameric global domain, while the others glycosites were distributed around the global domain. Here we present a detailed investigation of the distribution and the evolutionary pattern of the glycosites in the envelope glycoproteins of IVs, and further focus on the H5N1 virus and conclude that the glycosites in H5N1 have become more complicated in HA and less influential in NA in the last five years.     

Prophylactic and Therapeutic Efficacy of Avian Antibodies Against Influenza Virus H5N1 and H1N1 in Mice

Background

Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY) found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1.

Methods and Findings

We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection.

Conclusions

The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the current H1N1 pandemic.     

H5N1亚型禽流感病毒基因重排后毒力的变化

[目的]为了探讨高致病性禽流感病毒对水禽致病性差异的分子致病机理.[方法]我们对从野鸭分离到的H5N1亚型禽流感病毒的生物学特性进行鉴定,其中A/mallard/Huadong/Y/2003(Y)是对麻鸭无致病性病毒,而 A/mallard/Huadong/S/2005(S)是对麻鸭高致病性病毒.利用反向遗传技术构建一系列单个和多个基因组合替换基因重排病毒,并验证重排病毒在麻鸭上的致病力.[结果]研究表明,PB2, PB1, PA(3P), HA单基因以及3P基因组合替换的使S病毒对麻鸭的毒力完全致弱,但相应的基因替换后仅使Y病毒对麻鸭的毒力略有上升.两病毒的其它基因对毒力影响较小.[结论]H5N1亚型禽流感病毒对麻鸭的致病力受多基因调控,且这种调控作用在不同病毒骨架上的影响不一致,强毒受影响程度远比弱毒的大.     

Immunization of Chickens with Newcastle Disease Virus Expressing H5 Hemagglutinin Protects against Highly Pathogenic H5N1 Avian Influenza Viruses

Background

Highly-pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are the two most important poultry viruses in the world. Natural low-virulence NDV strains have been used as vaccines over the past 70 years with proven track records. We have previously developed a reverse genetics system to produce low-virulent NDV vaccine strain LaSota from cloned cDNA. This system allows us to use NDV as a vaccine vector for other avian pathogens.

Methodology/Principal Finding

Here, we constructed two recombinant NDVs (rNDVs) each of which expresses the hemagglutinin (HA) gene of HPAIV H5N1strain A/Vietnam/1203/2004 from an added gene. In one, rNDV (rNDV-HA), the open reading frame (ORF) of HA gene was expressed without modification. In the second, rNDV (rNDV-HAF), the ORF was modified so that the transmembrane and cytoplasmic domains of the encoded HA gene were replaced with those of the NDV F protein. The insertion of either version of the HA ORF did not increase the virulence of the rNDV vector. The HA protein was found to be incorporated into the envelopes of both rNDV-HA and rNDV-HAF. However, there was an enhanced incorporation of the HA protein in rNDV-HAF. Chickens immunized with a single dose of either rNDV-HA or rNDV-HAF induced a high titer of HPAIV H5-specific antibodies and were completely protected against challenge with NDV as well as lethal challenges of both homologous and heterologous HPAIV H5N1.

Conclusion and Significance

Our results suggest that these chimeric viruses have potential as safe and effective bivalent vaccines against NDV and. HPAIV. These vaccines will be convenient and affordable, which will be highly beneficial to the poultry industry. Furthermore, immunization with these vaccines will permit serological differentiation of vaccinated and avian influenza field virus infected animals.     

Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.     

禽流感的哺乳类动物模型研究进展

N1禽流感为新的人畜共患性疾病,建立良好的哺乳动物模型是进一步深入研究人禽流感防治的重要基础。本文报道了禽流感哺乳动物模型研究进展,对病原株、造模方法、模型的临床特征、病理变化、病毒复制等方面进行归纳,比较了小鼠模型、雪貂模型和非人灵长类动物模型特点。各种禽流感动物模型有各自的优点和缺点,应根据不同的实验目的和实验条件选择合适的病毒株和实验动物进行造模。     

禽流感H5N1亚型病毒对五个不同品系小鼠致病性的比较(英文)

目的比较了不同遗传背景小鼠对禽流感H5N1亚型病毒的致病敏感性,为H5N1禽流感模型制作和机理研究提供依据。方法近交系BALB/c、C57BL/6和封闭群ICR、NIHSwiss和KMSwiss共五个不同品系小鼠。每个品系实验动物30只,分接毒组20只,空白对照组10只,每组雌雄各半。病毒株为A/Goose/Guangdong/NH/2003(H5N1),经测定TCID50为10-4.875/mL。接毒组通过鼻腔接种0.1mL病毒液,对照组接种正常鸡胚尿囊液。小鼠接毒后连续观察14d,观察记录临床症状、体温、体重变化,对在实验期间死亡和实验14d结束后仍然存活的小鼠均进行组织器官病理取材,进行RT-PCR病毒分离检测、HE染色及H5N1抗原特异性免疫组化染色。结果①临床症状:H5N1禽流感病毒能感染五个品系的小鼠,引起呼吸急促等症状和一过性体重、体温下降。②死亡情况:小鼠在接毒后第1天即出现死亡,死亡的高峰期集中在接毒后第3～6天。五个品系小鼠死亡率存在差异,BALB/c为70%,ICR为50%,NIHSwiss为40%,C57BL/6为25%,KMSwiss为10%;③病毒分离:各组接毒小鼠在死亡后均进行了病毒分离,死亡小鼠的肺脏均分离到病毒,其他脏器未分离到病毒。④病理变化:实验期间五个品系死亡小鼠肺脏病理改变相近。大体观:死亡小鼠肺部淤血,呈暗红色,体积增大,局部肺组织实变。镜下观:死亡小鼠的共同病理改变为间质性肺炎,具体表现为肺泡腔及间质出血、炎性细胞浸润;间质增生,肺泡隔增宽;肺泡腔中见纤维素性渗出,透明膜形成。⑤免疫组化结果 :在死亡小鼠的气管上皮细胞和肺巨噬细胞可观察到H5N1禽流感病毒阳性表达。结论小鼠作为H5N1禽流感病毒模型具有普适性,不同品系小鼠感染鹅源H5N1禽流感病毒的临床症状、病程和病理变化与人禽流感病例相似。不同品系小鼠的死亡比例有明显差别,可以根据不同的实验目的 ,选择不同品系的小鼠制作H5N1禽流感动物模型。不同品系的遗传特性对禽流感易感性产生明显的影响,遗传背景可能与H5N1禽流感病毒感染应答机理存在联系:BALB/c和C57BL/6均为近交系,其中BALB/c小鼠的品系特征之一表现为干扰素产量低,接种H5N1病毒后表现为高死亡率(70%),而C57BL/6小鼠的干扰素产量高,接种H5N1病毒后表现为低死亡率(25%),提示不同遗传背景小鼠的干扰素水平与H5N1感染致死具相关性。为进一步研究H5N1禽流感病毒易感性相关基因以及其与宿主免疫反应的关系提供了一个研究基础。