H5N1禽流感病毒感染孕鼠的研究

目的用H5N1禽流感病毒感染昆明孕鼠,检测病毒在感染孕鼠各组织脏器中的复制及分布情况,并证明病毒能否通过孕鼠的胎盘垂直传染给胎鼠。方法用虎源H5N1亚型禽流感病毒滴鼻感染妊娠10-12 d的昆明孕鼠,观察孕鼠感染后的临床症状。接种病毒后第3、4、5、6和7 d分别处死3只孕鼠,取孕鼠的肺、脑、脾、肾、子宫、胎盘及胎鼠,利用RT-PCR、Real-time PCR和病毒分离方法检测各组织中的病毒核酸和病毒滴度,并进行病理组织学与免疫组织化学检测。结果昆明孕鼠接种病毒后第3 d,即可在肺、脑、脾、肾、子宫及胎盘组织中检测出H5N1禽流感病毒核酸,并从子宫、胎盘分离出H5N1禽流感病毒;感染后第6 d,从胎鼠体内检测到病毒核酸并分离出H5N1禽流感病毒。结论H5N1亚型禽流感病毒可以感染孕鼠,在孕鼠子宫和胎盘复制,感染后期可通过胎盘屏障传给胎鼠。     

H5N1高致病性禽流感病毒研究进展

流行性感冒病毒属正粘病毒科,根据内部核蛋白（NP）和基质蛋白（MP）抗原性的不同,将流感病毒分为A型、B型和c型3型。A型流感病毒的基因组是由8个分节段的单负链RNA组成,编码11种蛋白质,表面糖蛋白为HA和NA,根据HA和NA的抗原差异分为16种HA亚型（H1一H16）和9种NA亚型（N1～N9）。     

The evolution of H5N1 influenza viruses in ducks in southern China

The pathogenicity of avian H5N1 influenza viruses to mammals has been evolving since the mid-1980s. Here, we demonstrate that H5N1 influenza viruses, isolated from apparently healthy domestic ducks in mainland China from 1999 through 2002, were becoming progressively more pathogenic for mammals, and we present a hypothesis explaining the mechanism of this evolutionary direction. Twenty-one viruses isolated from apparently healthy ducks in southern China from 1999 through 2002 were confirmed to be H5N1 subtype influenza A viruses. These isolates are antigenically similar to A/Goose/Guangdong/1/96 (H5N1) virus, which was the source of the 1997 Hong Kong "bird flu" hemagglutinin gene, and all are highly pathogenic in chickens. The viruses form four pathotypes on the basis of their replication and lethality in mice. There is a clear temporal pattern in the progressively increasing pathogenicity of these isolates in the mammalian model. Five of six H5N1 isolates tested replicated in inoculated ducks and were shed from trachea or cloaca, but none caused disease signs or death. Phylogenetic analysis of the full genome indicated that most of the viruses are reassortants containing the A/Goose/Guangdong/1/96-like hemagglutinin gene and the other genes from unknown Eurasian avian influenza viruses. This study is a characterization of the H5N1 avian influenza viruses recently circulating in ducks in mainland China. Our findings suggest that immediate action is needed to prevent the transmission of highly pathogenic avian influenza viruses from the apparently healthy ducks into chickens or mammalian hosts.     

4株野鸭源H5N1亚型禽流感病毒的全基因组鉴定

目的为了解H5N1病毒对基因进化情况,方法对4株野鸭源的H5N1禽流感病毒(A/mallard/Huadong/S/2005(S),A/mallard/Huadong/lk/2005(lk),A/mallard/Huadong/Y/2003(Y),A/mallard/Huadong/hn/2005(hn))进行全基因组测序分析,并鉴定对非免疫麻鸭的毒力。结果4株病毒全基因组序列及其氨基酸序列无明显差异,仅在HA裂解位点区,S和lk病毒的322位是Leu,329位缺失,而Y和hn在此两处分别是Gln和Lys。在遗传距离上,Y和hn比较接近,S与lk比较接近。结论根据H5亚型病毒最新分类规则,S和lk的与clade 2.3.4一致,而Y属于clade 2,hn位于clade 3。对麻鸭致病性试验表明,Y和hn是低致病性病毒,而S及lk是高致病性病毒。HA的322位的Leu和329位缺失可能是clade 2.3.4这类病毒的一个遗传进化标志。     

Emergence of multiple genotypes of H5N1 avian influenza viruses in Hong Kong SAR

Although A/Hong Kong/156/97 (H5N1/97)-like viruses associated with the "bird flu" incident in Hong Kong SAR have not been detected since the slaughter of poultry in 1997, its putative precursors continue to persist in the region. One of these, Goose/Guangdong/1/96 (H5N1 Gs/Gd)-like viruses, reassorted with other avian viruses to generate multiple genotypes of H5N1 viruses that crossed to chickens and other terrestrial poultry from its reservoir in geese. Whereas none of these recent reassortants had acquired the gene constellation of H5N1/97, these events provide insight into how such a virus may have been generated. The recent H5N1 reassortants readily infect and kill chicken and quail after experimental infection, and some were associated with significant mortality of chickens within the poultry retail markets in Hong Kong. Some genotypes are lethal for mice after intra-nasal inoculation and spread to the brain. On this occasion, the early detection of H5N1 viruses in the retail, live poultry markets led to preemptive intervention before the occurrence of human disease, but these newly emerging, highly pathogenic H5N1 viruses provide cause for pandemic concern.     

人感染H6N1禽流感病毒HA基因的分子特征及进化分析

目的 检测人感染H6N1禽流感病毒HA基因的分子特征、致病机制、来源及进化。方法 从Flu和GISAID两大数据库中获得人感染H6N1及近缘禽流感病毒的HA序列,通过ML、MP、BI法构建系统发育树,推测进化速率和最近共同祖先(TMRCA)时间,并分析相关变异位点、糖基化位点及裂解位点。结果 人感染与台湾3株禽感染H6N1病毒聚成一支,最近共同祖先时间为2002年,目前正在流行,且进化速度很快。其氨基酸序列含有5个N-糖基化位点和2个O-糖基化位点,裂解位点上游仅1个碱性氨基酸,拥有两个特异的变异位点P186L和A287T。结论 人感染H6N1禽流感病毒目前仍是一种低致病性禽流感病毒,其HA基因由家鸡的序列进化而来。由于其高的进化速率及病毒重配的可能性,应引起高度重视。     

Predicting the global spread of H5N1 avian influenza

The spread of highly pathogenic H5N1 avian influenza into Asia, Europe, and Africa has resulted in enormous impacts on the poultry industry and presents an important threat to human health. The pathways by which the virus has and will spread between countries have been debated extensively, but have yet to be analyzed comprehensively and quantitatively. We integrated data on phylogenetic relationships of virus isolates, migratory bird movements, and trade in poultry and wild birds to determine the pathway for 52 individual introduction events into countries and predict future spread. We show that 9 of 21 of H5N1 introductions to countries in Asia were most likely through poultry, and 3 of 21 were most likely through migrating birds. In contrast, spread to most (20/23) countries in Europe was most likely through migratory birds. Spread in Africa was likely partly by poultry (2/8 introductions) and partly by migrating birds (3/8). Our analyses predict that H5N1 is more likely to be introduced into the Western Hemisphere through infected poultry and into the mainland United States by subsequent movement of migrating birds from neighboring countries, rather than from eastern Siberia. These results highlight the potential synergism between trade and wild animal movement in the emergence and pandemic spread of pathogens and demonstrate the value of predictive models for disease control.     

The radiologic manifestations of H5N1 avian influenza

Avian influenza is caused by the H5N1 subtype of the influenza A virus. Human transmission is either directly through close contact with infected birds usually poultry or their secretions. To date 178 people throughout South East Asia have been infected with 85 deaths. Patients usually present with a rapidly progressive pneumonia that can result in respiratory failure and acute respiratory distress syndrome. The chest radiograph therefore remains the most convenient and accessible imaging modality. Studies have shown that most radiographs are abnormal at the time of presentation with multifocal consolidation the commonest radiographic finding. During the course of disease, pleural effusions and cavitation can also develop. Consolidation that involves > or = 4 zones on presentation or at day 7 after the onset of symptoms and subsequent development of acute respiratory distress syndrome are generally associated with an adverse outcome. Chest CT examinations performed during the convalescent period have demonstrated persistent ground glass attenuation and segmental consolidation. Additional features included pseudocavitation, pneumatocoele formation, lymphadenopathy, and centrilobular nodules. Overall the appearances are suggestive of mild fibrosis.     

Revised and updated nomenclature for highly pathogenic avian influenza A (H5N1) viruses

The divergence of the hemagglutinin gene of A/goose/Guangdong/1/1996‐lineage H5N1 viruses during 2011 and 2012 (807 new sequences collected through December 31, 2012) was analyzed by phylogenetic and p‐distance methods to define new clades using the pre‐established nomenclature system. Eight new clade designations were recommended based on division of clade 1·1 (Mekong River Delta), 2·1·3·2 (Indonesia), 2·2·2 (India/Bangladesh), 2·2·1·1 (Egypt/Israel), and 2·3·2·1 (Asia). A simplification to the previously defined criteria, which adds a letter rather than number to the right‐most digit of fifth‐order clades, was proposed to facilitate this and future updates.     

H5N1亚型禽流感病毒NS1基因在大肠杆菌和昆虫细胞中的表达

目的构建大肠杆菌表达载体pET-NS1和昆虫杆状病毒转移载体pFast-NS1,将H5N1亚型禽流感病毒NS1基因分别在大肠杆菌和昆虫细胞中进行表达,表达产物用Western blot进行检测分析。方法酶切含有NS1基因的质粒pUC-NS1,分别克隆进大肠杆菌表达载体pET-28a(+)和杆状病毒转移载体pFastBac HT A中,分别获得表达载体pET-NS1和pFast-NS1。将pET-NS1转化大肠杆菌BL21,以异丙基硫代半乳糖苷(IPTG)进行诱导表达;将pFast-NS1转化DH10Bac感受态细胞,提取重组Bac-NS1DNA,以M13为通用引物作PCR鉴定,阳性Bac-NS1用脂质体转染sf9细胞,72h后收集感染细胞。大肠杆菌表达产物和细胞表达产物分别裂解后作SDS-PAGE和Western blot分析。结果成功构建了大肠杆菌表达载体pET-NS1和昆虫杆状病毒转移载体pFast-NS1,大肠杆菌和昆虫细胞中表达的融合蛋白Western blot都能检测到特异性条带。结论NS1基因在大肠杆菌和昆虫细胞中得到成功表达,为获得大量NS1蛋白进行功能研究及抗体制备奠定了基础。     

感染人类的禽流感病毒A（H5N1）研究进展

禽流感病毒A（H5N1）[avian influenza（H5N1） viruses]之前一直存在于鸟类，但却能导致人类疾病，并且具有高致死性和广泛流行的威胁。本文在综合了第二届世界卫生组织（World Health Organization，WHO）感染人类禽流感病毒A（H5N1）临床诊断咨询会议公布的研究信息基础上，对2005年的报告进行了更新。     

DG01株H5N1亚型禽流感病毒全基因组序列分析

根据国内外已公布的AIV H5N1亚型全基因组序列设计了8对引物,对H5N1亚型高致病力禽流感病毒毒株A/Duck/Guangdong/DG01/05(简称DG01)毒株进行了全基因组RT-PCR、测序及序列分析。DG01株基因组由PB22 341bp,PB1 2 341bp,PA2 233bp,HA1 779bp,NP 1 565bp,NA1 398 bp,M1 027bp,NS 875 bp 8个节段组成,与往年及同年一些流行株比较分析结果显示,DG01株具有高致病性禽流感病毒基因特征,NA基因及NS1基因均有部分缺失,从基因特点分析属于2005年流行代表株。     

Global epidemiology of human infections with highly pathogenic avian influenza A (H5N1) viruses

Abstract:   From 1997 through 2007, human infections with highly pathogenic avian influenza A (H5N1) viruses resulted in rare, sporadic, severe and fatal cases among persons in 14 countries in Asia, the Middle East, Eastern Europe and Africa. Of 369 reported human H5N1 cases that occurred from 1997 through 2007, overall mortality was 60%. Ten antigenically and genetically distinct clades of H5N1 viruses have been identified to date, and strains from four clades have infected humans. Surveillance has focused upon hospitalized cases of febrile acute lower respiratory tract disease among persons with exposure to sick or dead poultry, or to a human H5N1 case. Detection of H5N1 virus infection is based primarily upon collection of respiratory tract specimens from suspected cases for RT-PCR testing. Most human H5N1 cases were previously healthy children or young adults who developed severe acute pulmonary or multi-organ disease following direct or close contact with sick or dead H5N1 virus–infected poultry. Occasional clusters of H5N1 cases have occurred, predominantly among blood-related family members. Limited human-to-human H5N1 virus transmission has been reported or could not be excluded in some clusters. The frequency of asymptomatic or clinically mild H5N1 virus infection is unknown, but limited investigations suggest that such infections have been rare since 2003. There is no evidence of sustained human-to-human H5N1 virus spread. However, H5N1 viruses continue to circulate and evolve among poultry in many countries, and there are many unanswered questions about human infection with H5N1 viruses. Thus, the pandemic influenza threat presented by H5N1 viruses persists.