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。     

H5N1亚型禽流感病毒进化的研究进展

由H5N1流感病毒引起的高致病性禽流感,在禽类之间广泛传播。当人类接触这些禽类时,可能会被感染并产生严重的呼吸道症状,且死亡率高达60%。血凝素(hemagglutinin,HA)是H5N1病毒中和抗体的主要抗原,为了便于对病毒的HA突变进行研究,根据HA遗传基因的差异远近,所有的H5病毒株都被划分在20个分支内。对于H5N1病毒进化的研究在禽流感疫苗的研制、禽流感大流行的预防等方面均具有重要意义。现对禽流感、H5N1病毒特征、血凝素的结构功能、H5N1病毒的分支以及病毒进化的研究进行概述。     

一株H5N1亚型禽流感病毒的分离与鉴定

2004年1月湖北宜昌某鸡场暴发疫病,从该鸡场濒死鸡肺组织中分离到了一株病毒,电镜切片观察到典型的禽流感病毒粒子;采用ELISA检测禽流感抗原为阳性;RT-PCR扩增HA、NA基因并测序,经BLAST分析,HA基因与A／Goose／Guangdong／1／96(H5N1)HA基因同源性为97％;NA基因与A／Goose／Guangdong／1／96(H5N1)NA基因同源性为96％,确定该分离株为禽流感病毒H5N1亚型(A／Chicken／Yichang／Lung-1／04(H5N1))。     

H5N1亚型禽流感病毒拯救体系的建立

选择鸡胚高产的鸭源H5N1亚型禽流感病毒A/Duck/Shandong/093/2004株作为骨架病毒,在完成了全基因组序列测定基础上,设计合成的11对引物对病毒的8个基因分11段进行扩增。通过与转录载体PHW2000连接,构建A/SD/04的8个基因的拯救载体,经测序获得序列准确的拯救质粒:2412、42、243、244、245、246、247和248。A/SD/04的8质粒与PR8(H1N1)进行不同组合的拯救,获得8个均含A/SD/04 HA基因的H5重组流感病毒。鸡胚尿囊液中重组病毒的血凝效价在28~210,EID50在10-8.5~10-9之间,MDT在34~46h之间,均与野生A/SD/04(wt A/SD/04)相似。重组病毒对6周龄的SPF鸡的静脉接种指数(IVPI)与wt A/SD/04却有明显的差异,说明不同组合的内部基因影响病毒对鸡的致病力,但不影响病毒的鸡胚致死能力、对鸡胚的感染能力和病毒在鸡胚中的繁殖能力。构建的A/SD/04的8个质粒拯救系统,为H5N1的基因功能研究和新型疫苗开发奠定基础。     

一株H5N1亚型禽流感病毒的分离与鉴定

2004年1月湖北宜昌某鸡场暴发疫病,从该鸡场濒死鸡肺组织中分离到了一株病毒,电镜切片观察到典型的禽流感病毒粒子;采用ELISA检测禽流感抗原为阳性;RT-PCR扩增HA、NA基因并测序,经BLAST分析,HA基因与A/Goose/Guangdong/1/96(H5N1)HA基因同源性为97%;NA基因与A/Goose/Guangdong/1/96(H5N1)NA基因同源性为96%,确定该分离株为禽流感病毒H5N1亚型(A/Chicken/Yichang/Lung-1/04(H5N1)).     

H9N2亚型猪流感病毒感染BALB/c小鼠动物模型的建立

目的建立H9N2亚型猪流感病毒感染BALB/c小鼠动物模型,为研究病毒致病机制提供模型动物。方法通过滴鼻的方法将H9N2亚型猪流感病毒感染BALB/c小鼠,观察小鼠的症状和组织病理变化。结果 BALB/c小鼠的临床症状明显,病理变化典型。结论 H9N2亚型猪流感病毒感染BALB/c小鼠的疾病模型成功建立。     

H5N8亚型高致病性禽流感病毒的全球流行概况

H5N8亚型高致病性禽流感病毒(highly pathogenic avian influenza virus,HPAIV)随候鸟的迁徙活动及商业贸易活动现已蔓延至亚洲、欧洲、非洲、美洲等国家和地区.2014-2015和2016-2019年H5N8亚型HPAIV已引发两波全球疫情,现正经历第三波疫情,导致家禽及野生鸟类...     

虎源H5N1亚型禽流感病毒感染小鼠模型的建立

为研究H5N1亚型禽流感病毒的病原特性、致病机理及对其疫苗与救治药物效果评价提供平台,利用本室分离鉴定的虎源A/Tiger/Harbin/01/2002株(简称HAB/01)H5N1亚型禽流感病毒进行连续10倍稀释后,对4～6周龄雄性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定其MLD50,检测小鼠感染、致病的多项指标,观察期为14d。结果感染小鼠呈现出规律的以肺炎为主的临床症状、病理变化及病死率;测得该病毒对小鼠的MLD50为10-7.1/0.05mL。成功建立了虎源H5N1亚型禽流感病毒感染BALB/c小鼠的实验模型。     

禽流感病毒H5N1亚型HA基因表达及其产物的应用

根据GenBank公布的禽流感病毒H5N1亚型血凝素基因 (HA) (GenBank DQ023145) 序列设计一对引物P1、P2,以重组质粒pUC-HA为模板扩增去除信号肽的HA成熟蛋白. PCR 产物克隆入pMD18-T载体,经测序发现在967位A突变为T,形成一个终止密码子TAA. 在突变位点附近设计两条有21个碱基配对的突变引物P3、P4,采用重叠延伸剪切法 (SOE) 用A定点替换T碱基,然后将正确的基因片段定向插入到表达载体pET-32a (+) 中,诱导表达获得正确的表达产物.Western-blot分析表明,表达的重组蛋白能与经BL21(DE3)大肠杆菌菌体裂解液处理的H5亚型禽流感病毒阳性抗血清发生特异性反应.利用纯化的重组HA蛋白初步建立了检测H5亚型禽流感病毒抗体的间接ELISA方法,该方法可以代替传统的血凝与血凝抑制方法用于区分禽流感病毒的血清亚型.本研究为禽流感病毒亚单位疫苗及新型诊断试剂盒的研究奠定了基础.     

禽流感病毒H5N1亚型HA基因表达及其产物的应用

根据GenBank公布的禽流感病毒H5N1亚型血凝素基因(HA)(GenBank:DQ023145)序列设计一对引物P1、P2,以重组质粒pUC-HA为模板扩增去除信号肽的HA成熟蛋白。PCR产物克隆入pMD18-T载体,经测序发现在967位A突变为T,形成一个终止密码子TAA。在突变位点附近设计两条有21个碱基配对的突变引物P3、P4,采用重叠延伸剪切法(SOE)用A定点替换T碱基,然后将正确的基因片段定向插入到表达载体pET-32a( )中,诱导表达获得正确的表达产物。Western-blot分析表明,表达的重组蛋白能与经BL21(DE3)大肠杆菌菌体裂解液处理的H5亚型禽流感病毒阳性抗血清发生特异性反应。利用纯化的重组HA蛋白初步建立了检测H5亚型禽流感病毒抗体的间接ELISA方法,该方法可以代替传统的血凝与血凝抑制方法用于区分禽流感病毒的血清亚型。本研究为禽流感病毒亚单位疫苗及新型诊断试剂盒的研究奠定了基础。     

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。     

禽流感H5N1病毒感染BALB/c小鼠的细胞免疫动态变化

[目的]测定H5N1病毒感染BALB/c的小鼠模型的细胞免疫动态变化,探讨病毒对机体免疫系统的影响。[方法]通过流式细胞仪测定CD3+T、CD4+T、CD8+T等细胞免疫变化。[结果]感染H5N1病毒的小鼠血液中CD3+T、CD4+T、CD8+T细胞数量下降(P<0.05),脾脏中T细胞数量下降的趋势与血液相同,CD4+T/CD8+T的比例上升,只是两者的时间有所差别。[结论]说明病毒对细胞免疫T细胞数量影响较大,而且CD8+T受到的影响更为明显,反应了机体特异性细胞免疫功能受抑制,并且彼此之间的平衡受到破坏。     

Proinflammatory Cytokine Response and Viral Replication in Mouse Bone Marrow Derived Macrophages Infected with Influenza H1N1 and H5N1 Viruses

The pathogenesis of human influenza H5N1 virus infection remains poorly understood and controversial. Cytokine dysregulation in human infection has been hypothesized to contribute to disease severity. We developed in vitro cultures of mouse bone marrow derived macrophages (BMDMΦ) from C57BL/6N mouse to compare influenza A (H5N1 and H1N1) virus replication and pro-inflammatory cytokine and chemokine responses. While both H1N1 and H5N1 viruses infected the mouse bone marrow derived macrophages, only the H1N1 virus had showed evidence of productive viral replication from the infected cells. In comparison with human seasonal influenza H1N1 (A/HK/54/98) and mouse adapted influenza H1N1 (A/WSN/33) viruses, the highly pathogenic influenza H5N1 virus (A/HK/483/97) was a more potent inducer of the chemokine, CXCL 10 (IP-10), while there was not a clear differential TNF-α protein expression pattern. Although human influenza viruses rarely cause infection in mice without prior adaption, the use of in vitro cell cultures of primary mouse cells is of interest, especially given the availability of gene-defective (knock-out) mice for specific genes.     

恒河猴二次接种H5N1亚型禽流感病毒

对恒河猴进行二次接种H5N1亚型禽流感病毒试验,并对二次接毒的结果进行观察,评估初次接毒对恒河猴二次接毒效果的影响.首次接种试验中,3、4、5号恒河猴用环甲膜穿刺注射方法接种含有H5N1亚型禽流感病毒的尿囊液,6号猴接种不含病毒的尿囊液.90d后,再次用环甲膜穿刺注射方法二次接种,4、5、6号猴接种7mlTCID50浓度为104.875病毒的尿囊液,3号猴接种7ml不含病毒的尿囊液.进行抗体等检测,并分别于72h无痛处死3、4、6号猴,第7d无痛处死5号猴,进行肺的病毒检测及病理观察.结果显示,3、4、5号猴至试验结束时体内依然有较高的抗H5N1亚型禽流感病毒抗体水平,6号猴没有抗体;通过RT-PCR以及免疫组化染色进行病毒检测,均只在6号猴肺部检出病毒,且6号猴肺部病理损伤最为严重.由此可以得出初步结论:在初次感染H5N1亚型禽流感病毒后90d,临床症状已基本恢复正常的恒河猴体内仍然有较高水平的抗体,此时,恒河猴抵抗H5N1亚型禽流感病毒二次感染的能力显著提高.     

Viremia Associated with Fatal Outcomes in Ferrets Infected with Avian H5N1 Influenza Virus

Avian H5N1 influenza viruses cause severe disease and high mortality in infected humans. However, tissue tropism and underlying pathogenesis of H5N1 virus infection in humans needs further investigation. The objective of this work was to study viremia, tissue tropism and disease pathogenesis of H5N1 virus infection in the susceptible ferret animal model. To evaluate the relationship of morbidity and mortality with virus loads, we performed studies in ferrets infected with the H5N1 strain A/VN/1203/04 to assess clinical signs after infection and virus load in lung, brain, ileum, nasal turbinate, nasal wash, and blood. We observed that H5N1 infection in ferrets is characterized by high virus load in the brain and and low levels in the ileum using real-time PCR. In addition, viral RNA was frequently detected in blood one or two days before death and associated with symptoms of diarrhea. Our observations further substantiate pathogenicity of H5N1 and further indicate that viremia may be a bio-marker for fatal outcomes in H5N1 infection.     

Phylogenetic and Pathogenic Analyses of Avian Influenza A H5N1 Viruses Isolated from Poultry in Vietnam

Despite great efforts to control the infection of poultry with H5N1 viruses, these pathogens continue to evolve and spread in nature, threatening public health. Elucidating the characteristics of H5N1 avian influenza virus will benefit disease control and pandemic preparation. Here, we sequenced the genomes of 15 H5N1 avian influenza viruses isolated in Vietnam in 2006 and 2007 and performed phylogenetic analyses to compare these sequences with those of other viruses available in the public databases. Molecular characterization of the H5N1 viruses revealed that seven genetically distinct clades of H5N1 viruses have appeared in Vietnam. Clade 2.3.4 viruses existed in Vietnam as early as 2005. Fifteen viruses isolated during 2006 and 2007 belonged to clade 1 and clade 2.3.4, and were divided into five genotypes. Reassortants between the clade 1 and clade 2.3.4 viruses were detected in both North and South Vietnam. We also assessed the replication and pathogenicity of these viruses in mice and found that these isolates replicated efficiently and exhibited distinct virulence in mice. Our results provide important information regarding the diversity of H5N1 viruses in nature.     

Live Attenuated Influenza Viruses Containing NS1 Truncations as Vaccine Candidates against H5N1 Highly Pathogenic Avian Influenza

Due to the high mortality associated with recent, widely circulating strains of H5N1 influenza virus in poultry, the recurring introduction of H5N1 viruses from birds to humans, and the difficulties in H5N1 eradication by elimination of affected flocks, an effective vaccine against HPAI (highly pathogenic avian influenza) is highly desirable. Using reverse genetics, a set of experimental live attenuated vaccine strains based on recombinant H5N1 influenza virus A/Viet Nam/1203/04 was generated. Each virus was attenuated through expression of a hemagglutinin protein in which the polybasic cleavage site had been removed. Viruses were generated which possessed a full-length NS1 or a C-terminally truncated NS1 protein of 73, 99, or 126 amino acids. Viruses with each NS genotype were combined with a PB2 polymerase gene which carried either a lysine or a glutamic acid at position 627. We predicted that glutamic acid at position 627 of PB2 would attenuate the virus in mammalian hosts, thus increasing the safety of the vaccine. All recombinant viruses grew to high titers in 10-day-old embryonated chicken eggs but were attenuated in mammalian cell culture. Induction of high levels of beta interferon by all viruses possessing truncations in the NS1 protein was demonstrated by interferon bioassay. The viruses were each found to be highly attenuated in a mouse model. Vaccination with a single dose of any virus conferred complete protection from death upon challenge with a mouse lethal virus expressing H5N1 hemagglutinin and neuraminidase proteins. In a chicken model, vaccination with a single dose of a selected virus encoding the NS1 1-99 protein completely protected chickens from lethal challenge with homologous HPAI virus A/Viet Nam/1203/04 (H5N1) and provided a high level of protection from a heterologous virus, A/egret/Egypt/01/06 (H5N1). Thus, recombinant influenza A/Viet Nam/1203/04 viruses attenuated through the introduction of mutations in the hemagglutinin, NS1, and PB2 coding regions display characteristics desirable for live attenuated vaccines and hold potential as vaccine candidates in poultry as well as in mammalian hosts.     

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.     

Systems-Level Comparison of Host-Responses Elicited by Avian H5N1 and Seasonal H1N1 Influenza Viruses in Primary Human Macrophages

Human disease caused by highly pathogenic avian influenza (HPAI) H5N1 can lead to a rapidly progressive viral pneumonia leading to acute respiratory distress syndrome. There is increasing evidence from clinical, animal models and in vitro data, which suggests a role for virus-induced cytokine dysregulation in contributing to the pathogenesis of human H5N1 disease. The key target cells for the virus in the lung are the alveolar epithelium and alveolar macrophages, and we have shown that, compared to seasonal human influenza viruses, equivalent infecting doses of H5N1 viruses markedly up-regulate pro-inflammatory cytokines in both primary cell types in vitro. Whether this H5N1-induced dysregulation of host responses is driven by qualitative (i.e activation of unique host pathways in response to H5N1) or quantitative differences between seasonal influenza viruses is unclear. Here we used microarrays to analyze and compare the gene expression profiles in primary human macrophages at 1, 3, and 6 h after infection with H5N1 virus or low-pathogenic seasonal influenza A (H1N1) virus. We found that host responses to both viruses are qualitatively similar with the activation of nearly identical biological processes and pathways. However, in comparison to seasonal H1N1 virus, H5N1 infection elicits a quantitatively stronger host inflammatory response including type I interferon (IFN) and tumor necrosis factor (TNF)-α genes. A network-based analysis suggests that the synergy between IFN-β and TNF-α results in an enhanced and sustained IFN and pro-inflammatory cytokine response at the early stage of viral infection that may contribute to the viral pathogenesis and this is of relevance to the design of novel therapeutic strategies for H5N1 induced respiratory disease.