Pathogenicity of a Hong Kong-origin H5N1 highly pathogenic avian influenza virus for emus, geese, ducks, and pigeons

The H5N1 type A influenza viruses that emerged in Hong Kong in 1997 are a unique lineage of type A influenza viruses with the capacity to transmit directly from chickens to humans and produce significant disease and mortality in both of these hosts. The objective of this study was to ascertain the susceptibility of emus (Dramaius novaehollandiae), domestic geese (Anser anser domesticus), domestic ducks (Anas platyrhynchos), and pigeons (Columba livia) to intranasal (i.n.) inoculation with the A/chicken/Hong Kong/220/97 (H5N1) highly pathogenic avian influenza virus. No mortality occurred within 10 days postinoculation (DPI) in the four species investigated, and clinical disease, evident as neurologic dysfunction, was observed exclusively in emus and geese. Grossly, pancreatic mottling and splenomegaly were identified in these two species. In addition, the geese had cerebral malacia and thymic and bursal atrophy. Histologically, both the emus and geese developed pancreatitis, meningoencephalitis, and mild myocarditis. Influenza viral antigen was demonstrated in areas with histologic lesions up to 10 DPI in the geese. Virus was reisolated from oropharyngeal and cloacal swabs and from the lung, brain, and kidney of the emus and geese. Moderate splenomegaly was observed grossly in the ducks. Viral infection of the ducks was pneumotropic, as evidenced by mild inflammatory lesions in the respiratory tract and virus reisolation from oropharyngeal swabs and from a lung. Pigeons were resistant to HK/220 infection, lacking gross and histologic lesions, viral antigen, and reisolation of virus. These results imply that emus and geese are susceptible to i.n. inoculation with the HK/220 virus, whereas ducks and pigeons are more resistant. These latter two species probably played a minimal epidemiologic role in the perpetuation of the H5N1 Hong Kong-origin influenza viruses.     

对麻鸭不同致病力H5N1亚型禽法流感病毒的鉴定

禽流感病毒(avain influenza virus,AIV)根据其HA和NA蛋白分为不同亚型,目前16种HA亚型和9种NA亚型均已从水禽中分离到[1].     

Hemagglutinin glycosylation modulates the pathogenicity and antigenicity of the H5N1 avian influenza virus

The location and number of glycosylation in HA proteins exhibit large variations among H5 subtype avian influenza viruses (AIVs). To investigate the effect of glycosylation in the globular head of HA on the pathogenicity and antigenicity of H5N1 AIVs, seven rescued AIVs differing in their glycosylation patterns (144N, 158N and 169N) within the HA globular head of A/Mallard/Huadong/S/2005 were generated using site directed mutagenesis. Results showed that loss of glycosylation 158N was the prerequisite for H5 AIV binding to the α2,6-linked receptor. Only in conjunction with the removal of the 158N glycosylation, the H5 AIVs harboring both 144N and 169N glycosylations obtained an optimal binding preference to the α2,6-linked receptor. Compared with the wild-type virus, growth of viruses lacking glycosylation at either 158N or 169N was significantly reduced both in MDCK and A549 cells, while replication of viruses with additional glycosylation 144N was significantly promoted. Mutant viruses with loss of 158N or 169N glycosylation sites showed increased pathogenicity, systemic spread and pulmonary inflammation in mice compared to the wild-type H5N1 virus. In addition, chicken studies demonstrated that inactivated de-glycosylation 169N mutant induced cross-reaction HI and neutralization antibody against various clades of H5N1 AIVs. Moreover, this type of glycan pattern vaccine virus provided better cross-protection in chickens compared to wild-type vaccine virus. Thus, the glycosylation alteration of HA should be considered in the global surveillance and vaccine design of H5 subtype AIVs.     

果子狸源H5N1亚型禽流感病毒的分离及生物学特性鉴定

2011年,对广西自治区进行禽流感流行病学调查时从野生哺乳动物果子狸体内分离到一株H5N1亚型禽流感病毒(AⅣ),命名为A/palm-civet/Guangxi/26/2011 (H5N1) (PC/GX/26/11).本研究对其全基因组序列进行测定及其对BALB/c鼠的致病性试验.全基因序列分析表明:该病毒属于Clade 2.3.2分支,其血凝素(HA)蛋白裂解位点存在多个连续的碱性氨基酸(-RRRR-),具有高致病性AIV (HPAIV)的典型分子特征,其HA、NA、PA、NS基因节段与A/muscovy duck/Vietnam/LBM57/2011 (MD/VN/LBM57/11) (H5N1)有较高的同源性,为99.2％～99.6％;PB2、PB1、M、NP4个基因节段与人源AIV A/Hubei/1/2010 (HuB/1/10) (H5N1)的同源性最高,为99.0％～99.5％.动物试验显示:该病毒对小鼠的半数致死量为4.9 log EID50,对小鼠具有较高的致病性.在小鼠的肺和鼻甲骨中均能够进行良好的复制.以上结果表明,该分离株未经适应便具备感染哺乳动物并有较高致死的能力.     

两株H5N1亚型禽流感病毒的基因分析及其在不同宿主中致病性的比较

为了解两株高致病性禽流感病毒(HPAIV)的分子特征及其对不同宿主的致病性,本研究对DK/HuN/4/08和CK/GX/2/09进行全基因组序列的测定,分析结果表明:两个病毒分离株HA基因的裂解位点均具有HPAIV特有的基序(341RRR(R)KR345/346),并且均属于Clade2.3.2分支,基因组同源性在97.4%～98.3%之间。致病性试验显示,两个病毒分离株均能够以106EID50感染量在3 d内引起鸡全部死亡,并且各脏器均检测到高滴度的病毒含量;两者在SPF鸭中呈现不同的致病性,CK/GX/2/09在4 d内可以使感染鸭100%死亡,而DK/HuN/4/08只引起25%的死亡率;同样在小鼠试验中,两者致病力差异与在鸭体中的反应一致,其MLD50分别为1.63 log10EID50和6.2 log10EID50。本研究表明,这两株遗传背景相似的HPAIV在鸡、鸭和小鼠中的致病性不同,为进一步利用反向遗传技术研究这两株病毒对水禽和哺乳动物致病力差异的分子机制奠定了基础。     

一株鸭源H5N2禽流感病毒全基因组序列分析及对鸡的致病性研究

2012年在我国重庆市活禽交易市场进行流行病学调查时,从鸭体内分离到1株H5N2亚型禽流感病毒(AIV),DK/CQ036/12(H5N2).为了解该株H5N2亚型AIV的生物学特性,本研究对其进行了全基因组分析及对SPF鸡和BALB/c小鼠的致病性试验.序列分析显示:HA裂解位点序列为341R-----346TRGLF350,为低致病性AIV特征.内部基因来源较复杂,与KD/CQ/036/12分离株的M基因NP基因同源性最高的病毒株均来自H4、H7等亚型分离株,呈明显的异源性.分离株的感染性试验显示,该分离株在鸡体内可以通过呼吸道和消化道向外排毒,但并不能在鸡体内有效的复制.对小鼠的感染性试验结果显示,仅在鼻甲和肺能检测到病毒存在,其他脏器病毒滴定结果为阴性,表明病毒对鸡和小鼠均呈低致病性.     

野鸟源H5N8禽流感病毒遗传变异分析与致病性评估

本试验在野鸟禽流感病毒紧急疫情检测过程中鉴定并分离到1株H5N8高致病性禽流感病毒,利用病毒全基因组测序、系统发育及关键氨基酸位点分析解析了该野鸟源H5N8禽流感病毒分离株遗传进化情况,通过体外复制动力学试验及小鼠感染试验,评价了该野鸟源H5N8禽流感病毒分离株对哺乳动物致病性。进化分析显示,该病毒株属于Clade 2.3.4.4,可以不经适应直接感染小鼠并在呼吸系统内复制,表现出有限的组织嗜性,对小鼠呈低致病性。其在体内外复制能力较低。结果表明,本试验加深了对野生鸟携带H5N8禽流感病毒的认识和理解、对野鸟源H5N8禽流感病毒生物学特性的评价,为预测野鸟源H5N8禽流感病毒遗传进化趋势及其生物安全风险评估提供借鉴和参考。     

Epidemiology of H5N1 avian influenza

High pathogenic (HP) H5N1 avian influenza (AI) infection has been reported in domestic poultry, wildlife, and human populations since 1996. Risk of infection is associated with direct contact with infected birds. The mode of H5N1 spread from Asia to Europe, Africa and the Far East is unclear; risk factors such as legal and illegal domestic poultry and exotic bird trade, and migratory bird movements have been documented. Measures used to control disease such as culling, stamping out, cleaning and disinfection, and vaccination have not been successful in eradicating H5N1 in Asia, but have been effective in Europe.     

Single vaccination provides limited protection to ducks and geese against H5N1 high pathogenicity avian influenza virus

Since 2002, high pathogenicity avian influenza (HPAI) has spread from Asia to Europe and into Africa, causing the largest epizootic of HPAI of the last 50 yr, including infecting domestic and wild waterfowl. Our study was conducted to investigate whether a single vaccination of 7-day-old domestic ducks and geese with inactivated oil emulsion vaccines resulted in protection against HPAI virus challenge at 30 days of age. In ducks, some but not all vaccines decreased oropharyngeal and cloacal viral shedding for different periods postchallenge when compared with the sham group. In geese, decreased morbidity signs and mortality were noted but limited to some vaccines. Best protection was seen with a vaccine homologous to HPAI challenge virus. Limited decreases in oropharyngeal and cloacal viral shedding and mixed results were attained when looking at seroconversion. Our results indicate a single dose of oil-emulsified vaccine optimized for chickens did not provide adequate protection for ducks and geese against HPAI virus, and, at a minimum, additional research is needed to formulate waterfowl-specific vaccines.     

Characterization of an H5N1 avian influenza virus from Taiwan

In 2003, an avian influenza (AI) virus of H5N1 subtype (A/Duck/China/E319-2/03; Dk/CHN/E319-2/03) was isolated from a smuggled duck in Kinmen Island of Taiwan. Phylogenetic analysis and pairwise comparison of nucleotide and amino acid sequences revealed that the virus displayed high similarity to the H5N1 viruses circulating in Asia during 2004 and 2005. The hemagglutinin (HA) protein of the virus contained multiple basic amino acid residues (-RERRRKR-) adjacent to the cleavage site between the HA1 and HA2 domains, showing the highly pathogenic (HP) characteristics. The HP phenotype was confirmed by experimental infection of chickens, which led up to 100% mortality within 24-72h postinfection. The virus replicated equally well in the majority of organs of the infected chickens with titers ranging from 10(7.5) to 10(4.7) 50% embryo lethal dose (ELD50) per gram of tissue. In a mouse model the virus exhibits low pathogenic characteristics with a lethal infection observed only after applying high inoculating dose (>or=10(7.6) ELD50) of the virus. The infectious virus particles were recovered only from the pulmonary system including trachea and lungs. Our study suggests that ducks infected with H5N1 AIV of HPAI pathotype showing no disease signs can carry the virus silently and that bird smuggling represent a serious risk for H5N1 HPAI transmission.     

H5N1亚型禽流感病毒低剂量感染小鼠发病模型的建立

为模拟哺乳动物感染H5亚型高致病性禽流感病毒(HPAIV)的发病进程,本研究采用对哺乳动物高度致病的H5N1亚型HPAIV株A/bar-headed goose/Qinghai/3/05 (BHG/3/05),以低剂量鼻腔接种小鼠,观察发病、存活、病毒复制及组织病理损伤情况.结果显示,100.4 EID50即能够100％感染小鼠,但发病表现缓慢,死亡延迟至8d以后,存活达60％;体内病毒复制可持续10 d以上,感染后前3d病毒的增殖限于呼吸道,随后扩散至脑、脾、肾等其他器官;组织病理学观察肺脏早期表现出渗出性炎症,第10d发展为典型的间质性肺炎.本研究结果为探讨人禽流感的病理发生机制提供了具有价值的模型.     

两株鸭源H5N1亚型禽流感病毒的序列分析及致病性研究

本研究对2010年在湖北活禽市场监测分离到的两株鸭源H5N1亚型禽流感病毒(AIV) (HuB/495/10和HuB/513/10)进行了序列分析和致病性试验研究,以了解湖北地区H5N1亚型AIV的生物学特性差异.序列分析显示:2株病毒全基因组核苷酸同源性在97.3 ％～98.6％,2株病毒的8个节段基因均与青海和香港分离的野鸟源病毒A/great crested-grebe/Qinghai/1/2009 (H5N1)和A/black-crowned night heron/Hong Kong/659/2008 (H5N1)的核苷酸高度同源,HA蛋白裂解位点序列基序为341RRRKR345,呈现典型的高致病力分子特征.以105 EID50/100 μL病毒剂量感染4周龄SPF鸭发现:HuB/495/10和HuB/513/10对鸭的致死率分别为100％和20％,病毒在鸭体内呈全身性复制并可通过呼吸道和消化道向外排毒;不同滴度的病毒感染6周龄BALB/c小鼠,HuB/495/10和HuB/513/10的MLD50分别为1.38 log10 EID50和1.68 log10 EID50,对小鼠表现为高致病力,均在肺脏中高拷贝复制.     

不同H9N2亚型禽流感病毒株致病力的比较试验

参照NDV评价毒力的方法对1998—2008年间收集的25株H9N2AIV的致病性进行了比较研究。结果显示,25株H9N2AIV不同毒株间致病性有较大差异,大致分为3类：致病性偏强、致病性中等和致病性较弱。从中选出致病力有差异的8个毒株进行了1日龄雏鸡脑内接种指数（ICPI）、6周龄鸡静脉接种指数（IVPI）以及8周龄SPF鸡人工感染排毒试验,结果证明大部分毒株ICPI和IVPI基本上为0,排毒散毒的高峰期在攻毒后第5天到第6天。在25个毒株中,3#和12#表现出了比较高的致病性,不仅其EID50值最高（分别为10-8.8/0.2mL和10-8.8/0.2mL）、ELD50值最高（分别为10-7.9/0.2mL和10-8.7/0.2mL）,而且鸡胚平均死亡时间也最短（分别为66.5,69h）。3#、12#还出现了1日龄雏鸡脑内接种指数（分别为0.238,0.437）和6周鸡静脉内接种指数（分别为0.34,0.51）。在8周龄SPF鸡人工感染排毒试验中3#和12#毒株的排毒量大,排毒时间也明显长于其他毒株（第2～9天）。以上试验结果表明我国H9N2AIV不同毒株致病性有明显差异,呈多态性,致病性偏强的毒株造成鸡群较高的死亡率。     

H5N1亚型禽流感病毒NS1基因的克隆及表达

利用RT-PCR扩增了2株H5N1亚型禽流感病毒NS1基因,并将其克隆到pMD 18-T载体上,进行序列分析.结果显示,这2株禽流感病毒NS1基因核苷酸序列的同源性为70.2%,分别属于NS等位基因群A和等位基因群B.再将克隆的NS1基因插入到pET-28a质粒中构建原核表达载体,将其转化到DH5α大肠埃希氏菌感受态细胞中,经双酶切鉴定及序列分析,表明获得了重组质粒pET-52NS1和pET-174NS1.经SDS-PAGE分析,重组质粒转化BL21（DE3）（pLysS）感受态细胞后,经IPTG诱导,获得了分子质量约为30 ku的NS1融合蛋白.用AIV多克隆血清做Western-blotting分析,发现来自2个等位基因群的NS1蛋白都具有较好的抗原活性.     

Highly pathogenic avian influenza H5N1 virus in cats and other carnivores

The Asian lineage highly pathogenic avian influenza (HPAI) H5N1 virus is a known pathogen of birds. Only recently, the virus has been reported to cause sporadic fatal disease in carnivores, and its zoonotic potential has been dominating the popular media. Attention to felids was drawn by two outbreaks with high mortality in tigers, leopards and other exotic felids in Thailand. Subsequently, domestic cats were found naturally infected and experimentally susceptible to H5N1 virus. A high susceptibility of the dog to H3N8 equine influenza A virus had been reported earlier, and recently also HPAI H5N1 virus has been identified as a canine pathogen. The ferret, hamster and mouse are suitable as experimental animals; importantly, these species are also kept as pets. Experimental intratracheal and oral infection of cats with an HPAI H5N1 virus isolate from a human case resulted in lethal disease; furthermore, cats have been infected by the feeding of infected chickens. Spread of the infection from experimentally infected to in-contact cats has been reported. The epidemiological role of the cat and other pet animal species in transmitting HPAI H5N1 virus to humans needs continuous consideration and attention.     

Neurotropism of the 1997 Hong Kong H5N1 influenza virus in mice

The direct transmission of H5N1 influenza A viruses from chickens to humans in Hong Kong in 1997 emphasized the need to have information on the pathogenesis of avian influenza virus infection in mammals. H5N1 influenza viruses isolated from patients during the incident killed experimentally infected mice. The principal lesions of the mice were broncho-interstitial pneumonia and nonsuppurative encephalitis. Infectious viruses and/or viral antigens were detected in the brain as well as in the trigeminal and vagal ganglia but not in the blood of the mice. These findings suggest that the virus reached the brain through the vagus and/or trigeminal nerves following replication in the respiratory mucosa. The results imply that neurotropism of the H5N1 virus in mice is a novel characteristic in the pathogenesis of infection by human influenza virus isolates.