H5N1 avian influenza in China

H5N1 highly pathogenic avian influenza virus was first detected in a goose in Guangdong Province of China in 1996. Multiple genotypes of H5N1 viruses have been identified from apparently healthy waterfowl since 1999. In the years 2004–2008, over 100 outbreaks in domestic poultry occurred in 23 provinces and caused severe economic damage to the poultry industry in China. Beginning from 2004, a culling plus vaccination strategy has been implemented for the control of epidemics. Since then, over 35420000 poultry have been depopulated, and over 55 billion doses of the different vaccines have been used to control the outbreaks. Although it is logistically impossible to vaccinate every single bird in China due to the large poultry population and the complicated rearing styles, there is no doubt that the increased vaccination coverage has resulted in decreased disease epidemic and environmental virus loading. The experience in China suggests that vaccination has played an important role in the protection of poultry from H5N1 virus infection, the reduction of virus load in the environment, and the prevention of H5N1 virus transmission from poultry to humans. Supported by the Key Animal Infectious Disease Control Program of the Ministry of Agriculture, the Chinese National S&T Plan(Grant No. 2004BA519A-57), National Key Basic Research and Development Program of China (Grant Nos: 2005CB523005, 2005CB523200).     

Highly pathogenic H5N1 avian influenza viruses exhibit few barriers to gene flow in Vietnam

Locating areas where genetic change is inhibited can illuminate underlying processes that drive evolution of pathogens. The persistence of highly pathogenic H5N1 avian influenza in Vietnam since 2003, and the continuous molecular evolution of Vietnamese avian influenza viruses, indicates that local environmental factors are supportive not only of incidence but also of viral adaptation. This article explores whether gene flow is constant across Vietnam, or whether there exist boundary areas where gene flow exhibits discontinuity. Using a dataset of 125 highly pathogenic H5N1 avian influenza viruses, principal components analysis and wombling analysis are used to indicate the location, magnitude, and statistical significance of genetic boundaries. Results show that a small number of geographically minor boundaries to gene flow in highly pathogenic H5N1 avian influenza viruses exist in Vietnam, but that overall there is little division in genetic exchange. This suggests that differences in genetic characteristics of viruses from one region to another are not the result of barriers to H5N1 viral exchange in Vietnam, and that H5N1 avian influenza is able to spread relatively unimpeded across the country.     

Pathogenesis of avian influenza A (H5N1) viruses in ferrets

Highly pathogenic avian influenza A H5N1 viruses caused outbreaks of disease in domestic poultry and humans in Hong Kong in 1997. Direct transmission of the H5N1 viruses from birds to humans resulted in 18 documented cases of respiratory illness, including six deaths. Here we evaluated two of the avian H5N1 viruses isolated from humans for their ability to replicate and cause disease in outbred ferrets. A/Hong Kong/483/97 virus was isolated from a fatal case and was highly pathogenic in the BALB/c mouse model, whereas A/Hong Kong/486/97 virus was isolated from a case with mild illness and exhibited a low-pathogenicity phenotype in mice. Ferrets infected intranasally with 10(7) 50% egg infectious doses (EID(50)) of either H5N1 virus exhibited severe lethargy, fever, weight loss, transient lymphopenia, and replication in the upper and lower respiratory tract, as well as multiple systemic organs, including the brain. Gastrointestinal symptoms were seen in some animals. In contrast, weight loss and severe lethargy were not noted in ferrets infected with 10(7) EID(50) of two recent human H3N2 viruses, although these viruses were also isolated from the brains, but not other extrapulmonary organs, of infected animals. The results demonstrate that both H5N1 viruses were highly virulent in the outbred ferret model, unlike the differential pathogenicity documented in inbred BALB/c mice. We propose the ferret as an alternative model system for the study of these highly pathogenic avian viruses.     

Human avian influenza A (H5N1) virus infection in China

Highly pathogenic influenza A (H5N1) virus causes a widespread poultry deaths worldwide. The first human H5N1 infected case was reported in Hong Kong Special Administrative Region of China in 1997. Since then, the virus re-emerged in 2003 and continues to infect people worldwide. Currently, over 400 human infections have been reported in more than 15 countries and mortality rate is greater than 60%. H5N1 viruses still pose a potential pandemic threat in the future because of the continuing global spread and evolution. Here, we summarize the epidemiological, clinical and virological characteristics of human H5N1 infection in China monitored and identified by our national surveillance systems. Chinese Nature Science Foundation Key Project (Grant No. 30599433), Chinese Basic Science Research Program (973)Key Project (Grant No. 2005CB523006)     

Evolution of highly pathogenic H5N1 avian influenza viruses in Vietnam between 2001 and 2007

Highly pathogenic avian influenza (HPAI) H5N1 viruses have caused dramatic economic losses to the poultry industry of Vietnam and continue to pose a serious threat to public health. As of June 2008, Vietnam had reported nearly one third of worldwide laboratory confirmed human H5N1 infections. To better understand the emergence, spread and evolution of H5N1 in Vietnam we studied over 300 H5N1 avian influenza viruses isolated from Vietnam since their first detection in 2001. Our phylogenetic analyses indicated that six genetically distinct H5N1 viruses were introduced into Vietnam during the past seven years. The H5N1 lineage that evolved following the introduction in 2003 of the A/duck/Hong Kong/821/2002-like viruses, with clade 1 hemagglutinin (HA), continued to predominate in southern Vietnam as of May 2007. A virus with a clade 2.3.4 HA newly introduced into northern Vietnam in 2007, reassorted with pre-existing clade 1 viruses, resulting in the emergence of novel genotypes with neuraminidase (NA) and/or internal gene segments from clade 1 viruses. A total of nine distinct genotypes have been present in Vietnam since 2001, including five that were circulating in 2007. At least four of these genotypes appear to have originated in Vietnam and represent novel H5N1 viruses not reported elsewhere. Geographic and temporal analyses of H5N1 infection dynamics in poultry suggest that the majority of viruses containing new genes were first detected in northern Vietnam and subsequently spread to southern Vietnam after reassorting with pre-existing local viruses in northern Vietnam. Although the routes of entry and spread of H5N1 in Vietnam remain speculative, enhanced poultry import controls and virologic surveillance efforts may help curb the entry and spread of new HPAI viral genes.     

The changing nature of avian influenza A virus (H5N1)

Highly pathogenic avian influenza A virus subtype H5N1 has been endemic in some bird species since its emergence in 1996 and its ecology, genetics and antigenic properties have continued to evolve. This has allowed diverse virus strains to emerge in endemic areas with altered receptor specificity, including a new H5 sublineage with enhanced binding affinity to the human-type receptor. The pandemic potential of H5N1 viruses is alarming and may be increasing. We review here the complex dynamics and changing nature of the H5N1 virus that may contribute to the emergence of pandemic strains.     

Receptor recognition mechanism of human influenza A H1N1 (1918), avian influenza A H5N1 (2004), and pandemic H1N1 (2009) neuraminidase

Influenza A neuraminidase (NA) is a target for anti-influenza drugs. The function of this enzyme is to cleave a glycosidic linkage of a host cell receptor that links sialic acid (Sia) to galactose (Gal), to allow the virus to leave an infected cell and propagate. The receptor is an oligosaccharide on the host cell surface. There are two types of oligosaccharide receptor; the first, which is found mainly on avian epithelial cell surfaces, links Sia with Gal by an α2,3 glycosidic linkage; in the second, found mainly on human epithelial cell surfaces, linkage is via an α2,6 linkage. Some researchers believe that NAs from different viruses show selectivity for each type of linkage, but there is limited information available to confirm this hypothesis. To see if the linkage type is more specific to any particular NA, a number of NA-receptor complexes of human influenza A H1N1 (1918), avian influenza A H5N1 (2004), and a pandemic strain of H1N1 (2009) were constructed using homology modeling and molecular dynamics simulation. The results show that the two types of receptor analogues bound to NAs use different mechanisms. Moreover, it was found that a residue unique to avian virus NA is responsible for the recognition of the Siaα2,3Gal receptor, and a residue unique to human virus NA is responsible for the recognition of Siaα2,6Gal. We believe that this finding could explain how NAs of different virus origins always possess some unique residues.     

Possible circulation of H5N1 avian influenza viruses in healthy ducks on farms in northern Vietnam

To estimate the prevalence of influenza A subtype H5N1 viruses among domestic ducks in the period between October and November 2006 when H5N1 outbreaks had been absent, 1106 healthy ducks raised in northern Vietnam were collected. Inoculation of all throat and cloacae samples into embryonated eggs resulted in the isolation of subtype H3N8 in 13 ducks, but not H5N1 viruses. Serological analyses demonstrated that five ducks (0.45%) solely developed H5N1 subtype-specific hemagglutinin-inhibiting and neuraminidase-inhibiting antibodies together with anti-non-structural protein 1 antibodies. The results suggested that the ducks were naturally infected with H5N1 viruses when obvious H5N1 outbreaks were absent.     

Proposed lead molecules against Hemagglutinin of avian influenza virus (H5N1)

Human infection with avian influenza H5N1 is an emerging infectious disease characterized by respiratory symptoms and a high fatality rate. Hemagglutinin and neuraminidase are the two surface proteins responsible for infection by influenza virus. Till date, neuraminidase has been the major target for antiviral drugs. In the present study we chose hemagglutinin protein as it mediates the binding of the virus to target cells through sialic acid residues on the host cell-surface. Hemagglutinin of H5 avian influenza (PDB ID: 1JSN) was used as the receptor protein. Ligands were generated by structure-based de novo approach and virtual screening of ZINC database. A total of 11,104 conformers were generated and docked into the receptor binding site using 'High Throughput Virtual Screening'. We proposed potential lead molecules against the receptor binding site of hemagglutinin based on the results obtained from in silico docking and hydrogen bond interaction between the ligand and the 1JSN protein molecule. We found sialic acid derivative 1 to be the lead molecules amongst the ligands generated by structure based de novo approach. However the molecules obtained from ZINC database were showing better docking scores as well as conserved hydrogen bond interactions. Thus we proposed ZINC00487720 and ZINC00046810 as potential lead molecules that could be used as an inhibitor to the receptor binding site of hemagglutinin. They could now be studied in vivo to validate the in silico results.     

Phylodynamics of H5N1 avian influenza virus in Indonesia

Understanding how pathogens invade and become established in novel host populations is central to the ecology and evolution of infectious disease. Influenza viruses provide unique opportunities to study these processes in nature because of their rapid evolution, extensive surveillance, large data sets and propensity to jump species boundaries. H5N1 highly pathogenic avian influenza virus (HPAIV) is a major animal pathogen and public health threat. The virus is of particular importance in Indonesia, causing severe outbreaks among poultry and sporadic human infections since 2003. However, little is known about how H5N1 HPAIV emerged and established in Indonesia. To address these questions, we analysed Indonesian H5N1 HPAIV gene sequences isolated during 2003-2007. We find that the virus originated from a single introduction into East Java between November 2002 and October 2003. This invasion was characterized by an initially rapid burst of viral genetic diversity followed by a steady rate of lineage replacement and the maintenance of genetic diversity. Several antigenic sites in the haemagglutinin gene were subject to positive selection during the early phase, suggesting that host-immune-driven selection played a role in host adaptation and expansion. Phylogeographic analyses show that after the initial invasion of H5N1, genetic variants moved both eastwards and westwards across Java, possibly involving long-distance transportation by humans. The phylodynamics we uncover share similarities with other recently studied viral invasions, thereby shedding light on the ecological and evolutionary processes that determine disease emergence in a new geographical region.     

一株人感染高致病性禽流感(H5N1)病毒基因组分子特征分析

【背景】H5N1禽流感病毒可以感染人类导致重症呼吸道感染,致死率高。【目的】研究我中心确认的一例人感染高致病性禽流感H5N1病毒A/Nanjing/1/2015的可能起源及基因组分子特征。【方法】对病人痰液样本中的H5N1病毒进行全基因组测序,使用CLC Genomics Workbench 9.0对序列进行拼接,使用BLAST和MEGA 5.22软件进行同源性比对和各片段分子特征分析。【结果】该株禽流感病毒属于H5亚型的2.3.2.1c家系,其8个片段均与江浙地区禽类中分离的病毒高度同源,未发现有明显的重配。分子特征显示,该病毒血凝素(Hemagglutinin,HA)蛋白裂解位点为PQRERRRR/G,受体结合位点呈现禽类受体特点,但出现D94N、S133A和T188I氨基酸置换增强了病毒对人类受体的亲和性。神经氨酸酶(Neuraminidase,NA)蛋白颈部在49-68位缺失20个氨基酸,非结构蛋白1 (Non-structure protein,NS1)存在P42S置换和80-84位氨基酸的缺失。其他蛋白中也存在多个增强病毒致病力和对人类细胞亲和力的氨基酸突变。对耐药位点分析发现存在对奥司他韦的耐药突变H_274Y,病毒对金刚烷胺仍旧敏感。【结论】人感染高致病性禽流感H5N1病毒A/Nanjing/1/2015属于2.3.2.1c家系,禽类来源,关键位点较保守,但仍出现了多个氨基酸的进化与变异使其更利于感染人类。H5N1禽流感病毒进化活跃,持续动态监测不能放松。     

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。     

Ecological determinants of highly pathogenic avian influenza (H5N1) outbreaks in Bangladesh

Background

The agro-ecology and poultry husbandry of the south Asian and south-east Asian countries share common features, however, with noticeable differences. Hence, the ecological determinants associated with risk of highly pathogenic avian influenza (HPAI-H5N1) outbreaks are expected to differ between Bangladesh and e.g., Thailand and Vietnam. The primary aim of the current study was to establish ecological determinants associated with the risk of HPAI-H5N1 outbreaks at subdistrict level in Bangladesh. The secondary aim was to explore the performance of two different statistical modeling approaches for unmeasured spatially correlated variation.

Methodology/Principal Findings

An ecological study at subdistrict level in Bangladesh was performed with 138 subdistricts with HPAI-H5N1 outbreaks during 2007–2008, and 326 subdistricts with no outbreaks. The association between ecological determinants and HPAI-H5N1 outbreaks was examined using a generalized linear mixed model. Spatial clustering of the ecological data was modeled using 1) an intrinsic conditional autoregressive (ICAR) model at subdistrict level considering their first order neighbors, and 2) a multilevel (ML) model with subdistricts nested within districts. Ecological determinants significantly associated with risk of HPAI-H5N1 outbreaks at subdistrict level were migratory birds'' staging areas, river network, household density, literacy rate, poultry density, live bird markets, and highway network. Predictive risk maps were derived based on the resulting models. The resulting models indicate that the ML model absorbed some of the covariate effect of the ICAR model because of the neighbor structure implied in the two different models.

Conclusions/Significance

The study identified a new set of ecological determinants related to river networks, migratory birds'' staging areas and literacy rate in addition to already known risk factors, and clarified that the generalized concept of free grazing duck and duck-rice cultivation interacted ecology are not significant determinants for Bangladesh. These findings will refine current understanding of the HPAI-H5N1 epidemiology in Bangladesh.     

An avian influenza H5N1 virus that binds to a human-type receptor

Avian influenza viruses preferentially recognize sialosugar chains terminating in sialic acid-alpha2,3-galactose (SAalpha2,3Gal), whereas human influenza viruses preferentially recognize SAalpha2,6Gal. A conversion to SAalpha2,6Gal specificity is believed to be one of the changes required for the introduction of new hemagglutinin (HA) subtypes to the human population, which can lead to pandemics. Avian influenza H5N1 virus is a major threat for the emergence of a pandemic virus. As of 12 June 2007, the virus has been reported in 45 countries, and 312 human cases with 190 deaths have been confirmed. We describe here substitutions at position 129 and 134 identified in a virus isolated from a fatal human case that could change the receptor-binding preference of HA of H5N1 virus from SAalpha2,3Gal to both SAalpha2,3Gal and SAalpha2,6Gal. Molecular modeling demonstrated that the mutation may stabilize SAalpha2,6Gal in its optimal cis conformation in the binding pocket. The mutation was found in approximately half of the viral sequences directly amplified from a respiratory specimen of the patient. Our data confirm the presence of H5N1 virus with the ability to bind to a human-type receptor in this patient and suggest the selection and expansion of the mutant with human-type receptor specificity in the human host environment.     

High accumulation in tobacco seeds of hemagglutinin antigen from avian (H5N1) influenza

Tobacco seeds can be used as a cost effective system for production of recombinant vaccines. Avian influenza is an important respiratory pathogen that causes a high degree of mortality and becomes a serious threat for the poultry industry. A safe vaccine against avian flu produced at low cost could help to prevent future outbreaks. We have genetically engineered tobacco plants to express extracellular domain of hemagglutinin protein from H5N1 avian influenza virus as an inexpensive alternative for production purposes. Two regulatory sequences of seed storage protein genes from Phaseolus vulgaris L. were used to direct the expression, yielding 3.0 mg of the viral antigen per g of seeds. The production and stability of seed-produced recombinant HA protein was characterized by different molecular techniques. The aqueous extract of tobacco seed proteins was used for subcutaneous immunization of chickens, which developed antibodies that inhibited the agglutination of erythrocytes after the second application of the antigen. The feasibility of using tobacco seeds as a vaccine carrier is discussed.     

Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract

Poor human-to-human transmission of influenza A H5N1 virus has been attributed to the paucity of putative sialic acid alpha2-3 virus receptors in the epithelium of the human upper respiratory tract, and thus to the presumed inability of the virus to replicate efficiently at this site. We now demonstrate that ex vivo cultures of human nasopharyngeal, adenoid and tonsillar tissues can be infected with H5N1 viruses in spite of an apparent lack of these receptors.     

Susceptibility of wood ducks to H5N1 highly pathogenic avian influenza virus

Since 2002, H5N1 highly pathogenic avian influenza (HPAI) viruses have caused mortality in numerous species of wild birds; this is atypical for avian influenza virus (AIV) infections in these avian species, especially for species within the order Anseriformes. Although these infections document the susceptibility of wild birds to H5N1 HPAI viruses and the spillover of these viruses from infected domestic birds to wild birds, it is unknown whether H5N1 HPAI viruses can persist in free-living avian populations. In a previous study, we established that wood ducks (Aix sponsa) are highly susceptible to infection with H5N1 HPAI viruses. To quantify this susceptibility and further evaluate the likelihood of H5N1 HPAI viral maintenance in a wild bird population, we determined the concentration of virus required to produce infection in wood ducks. To accomplish this, 25 wood ducks were inoculated intranasally at 12-16 wk of age with decreasing concentrations of a H5N1 HPAI virus (A/Whooper Swan/Mongolia/244/05 [H5N1]). The median infectious dose and the lethal dose of H5N1 HPAI virus in wood ducks were very low (10(0.95) and 10(1.71) median embryo infectious dose [EID(50)]/ml, respectively) and less than that of chickens (10(2.80) and 10(2.80) EID(50)/ml). These results confirm that wood ducks are highly susceptible to infection with H5N1 HPAI virus. The data from this study, combined with what is known experimentally about H5N1 HPAI virus infection in wood ducks and viral persistence in aquatic environments, suggest that the wood duck would represent a sensitive indicator species for H5N1 HPAI. Results also suggest that the potential for decreased transmission efficiency associated with reduced viral shedding (especially from the cloaca) and a loss of environmental fitness (in water), may be offset by the ability of this virus to be transmitted through a very low infectious dose.