Isolation and characterization of avian influenza viruses, including highly pathogenic H5N1, from poultry in live bird markets in Hanoi, Vietnam, in 2001

Since 1997, outbreaks of highly pathogenic (HP) H5N1 and circulation of H9N2 viruses among domestic poultry in Asia have posed a threat to public health. To better understand the extent of transmission of avian influenza viruses (AIV) to humans in Asia, we conducted a cross-sectional virologic study in live bird markets (LBM) in Hanoi, Vietnam, in October 2001. Specimens from 189 birds and 18 environmental samples were collected at 10 LBM. Four influenza A viruses of the H4N6 (n = 1), H5N2 (n = 1), and H9N3 (n = 2) subtypes were isolated from healthy ducks for an isolation frequency of over 30% from this species. Two H5N1 viruses were isolated from healthy geese. The hemagglutinin (HA) genes of these H5N1 viruses possessed multiple basic amino acid motifs at the cleavage site, were HP for experimentally infected chickens, and were thus characterized as HP AIV. These HA genes shared high amino acid identities with genes of other H5N1 viruses isolated in Asia during this period, but they were genetically distinct from those of H5N1 viruses isolated from poultry and humans in Vietnam during the early 2004 outbreaks. These viruses were not highly virulent for experimentally infected ducks, mice, or ferrets. These results establish that HP H5N1 viruses with properties similar to viruses isolated in Hong Kong and mainland China circulated in Vietnam as early as 2001, suggest a common source for H5N1 viruses circulating in these Asian countries, and provide a framework to better understand the recent widespread emergence of HP H5N1 viruses in Asia.     

Phylogenetic analysis of H7 avian influenza viruses isolated from the live bird markets of the Northeast United States

The presence of low-pathogenic H7 avian influenza virus (AIV), which is associated with live-bird markets (LBM) in the Northeast United States, was first detected in 1994 and, despite efforts to eradicate the virus, surveillance of these markets has resulted in numerous isolations of H7 AIVs from several states from 1994 through 1998. The hemagglutinin, nonstructural, and matrix genes from representative H7 isolates from the LBM and elsewhere were sequenced, and the sequences were compared phylogenetically. The hemagglutinin gene of most LBM isolates examined appeared to have been the result of a single introduction of the hemagglutinin gene. Evidence for evolutionary changes were observed with three definable steps. The first isolate from 1994 had the amino acid threonine at the -2 position of the hemagglutinin cleavage site, which is the most commonly observed amino acid at this site for North American H7 AIVs. In January 1995 a new genotype with a proline at the -2 position was detected, and this genotype eventually became the predominant virus isolate. A third viral genotype, detected in November 1996, had an eight-amino-acid deletion within the putative receptor binding site. This viral genotype appeared to be the predominant isolate, although isolates with proline at the -2 position without the deletion were still observed in viruses from the last sampling date. Evidence for reassortment of multiple viral genes was evident. The combination of possible adaptive evolution of the virus and reassortment with different influenza virus genes makes it difficult to determine the risk of pathogenesis of this group of H7 AIVs.     

家禽市场污水来源的H5N1亚型禽流感病毒NS基因分析

对长沙市家禽市场污水来源的H5N1亚型禽流感病毒(Avian influenza viruses,AIV)的非结构蛋白(Non-structural,NS)基因进行进化和分子特征分析,探讨污水中H5N1病毒的传播风险。9份家禽市场环境污水H5N1亚型AIV标本进行NS基因TA克隆测序,测序结果利用Lasergene和Mega5软件进行氨基酸(amine acid,aa)比对和进化树分析。共得到8个阳性克隆,进化树构建显示8个H5N1的NS基因均属于A亚群,其编码的NS1和NS2蛋白与A亚群代表株(A/chicken/Hubei/w h/1999)aa同源性分别为90.1%～92.5%和91.0%～92.6%,8个H5N1的NS1和NS2aa之间的同源性分别为93.8%～100.0%和98.4%～100.0%。8个H5N1的NS1蛋白均具有缺失80～84位aa、C末端携带有ESEV的PL基序和第92位aa为E的高致病性分子特征。家禽市场污水来源的H5N1亚型AIV的NS基因具有高致病性的分子特征,这种基因特征表明污水可能传播H5N1病毒。     

1株H6N6亚型禽流感病毒分子遗传特性分析

本研究采用无特定病原体(specific pathogen free,SPF)鸡胚,从某活禽市场环境中分离出1株H6N6亚型禽流感病毒(A/environment/Zhenjiang/zj18/2013,en/zj18)。通过二代测序技术进行全基因组测序,通过BLASTn 进行同源性检索,并采用MEGA5.0软件构建系统发生树。基因进化树分析表明,分离株en/zj18的所有8个基因节段(PB2、PB1、PA、HA、NP、NA、M和NS)均与近年来中国华东地区流行的H6N6亚型禽流感病毒的相应基因位于同一进化分支,与参考株的核苷酸同源性达96.7%～99.6%。分离株en/zj18的HA蛋白裂解位点为PQIETR↓GL,是低致病性禽流感病毒的分子特征。HA蛋白上关键受体结合位点190和228位(按H3亚型的HA蛋白序列排序)氨基酸分别是E和G,理论上更易与α2,3-半乳糖苷唾液酸受体结合。结果提示,需加强活禽市场禽流感病毒的持续监测,从而为有效应对禽流感病毒对公共卫生的持续威胁提供科学依据。     

Comparative histopathological characteristics of highly pathogenic avian influenza (HPAI) in chickens and domestic ducks in 2008 Korea

We compared characteristic lesions occurring in chickens and domestic ducks naturally infected with H5N1 HPAI virus in April and May 2008. Infected chickens generally exhibited pale-green, watery diarrhoea, depression, neurological signs and cyanosis of wattles and combs, and infected ducks generally exhibited neurological signs and watery diarrhoea. Gross petechial or ecchymotic haemorrhage affected the heart, proventriculus, liver, muscle, fat, and pancreas in chickens, and muscle in ducks. Necrotic foci were primarily present in the pancreas of both species and in the heart of domestic ducks. Histopathologically, chickens exhibited multifocal encephalomalacia, multifocal lymphohistiocytic myocarditis, multifocal necrotic pancreatitis and haemorrhage of several organs and tissues; ducks exhibited lymphohistiocytic meningoencephalitis with multifocal haemorrhages, multifocal necrotic pancreatitis, and severe necrotic myocarditis with mineralisation. The characteristic histopathologic findings of 2008 HPAI were multifocal encephalomalacia and necrotic pancreatitis accompanied by lymphohistiocytic myocarditis, and haemorrhage in various organs and tissues in chickens, whereas in ducks, they were severe necrotic myocarditis with mineralisation and necrotic pancreatitis, accompanied with lymphohistiocytic meningoencephalitis. The high mortality of domestic ducks may be intimately associated with heart failure resulting from increased H5N1 HPAI viral cardiotropism.     

Cross-reactive, cell-mediated immunity and protection of chickens from lethal H5N1 influenza virus infection in Hong Kong poultry markets

In 1997, avian H5N1 influenza virus transmitted from chickens to humans resulted in 18 confirmed infections. Despite harboring lethal H5N1 influenza viruses, most chickens in the Hong Kong poultry markets showed no disease signs. At this time, H9N2 influenza viruses were cocirculating in the markets. We investigated the role of H9N2 influenza viruses in protecting chickens from lethal H5N1 influenza virus infections. Sera from chickens infected with an H9N2 influenza virus did not cross-react with an H5N1 influenza virus in neutralization or hemagglutination inhibition assays. Most chickens primed with an H9N2 influenza virus 3 to 70 days earlier survived the lethal challenge of an H5N1 influenza virus, but infected birds shed H5N1 influenza virus in their feces. Adoptive transfer of T lymphocytes or CD8(+) T cells from inbred chickens (B(2)/B(2)) infected with an H9N2 influenza virus to naive inbred chickens (B(2)/B(2)) protected them from lethal H5N1 influenza virus. In vitro cytotoxicity assays showed that T lymphocytes or CD8(+) T cells from chickens infected with an H9N2 influenza virus recognized target cells infected with either an H5N1 or H9N2 influenza virus in a dose-dependent manner. Our findings indicate that cross-reactive cellular immunity induced by H9N2 influenza viruses protected chickens from lethal infection with H5N1 influenza viruses in the Hong Kong markets in 1997 but permitted virus shedding in the feces. Our findings are the first to suggest that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds in live markets and create a situation for the perpetuation of H5N1 influenza viruses.     

Homo- and heterosubtypic low pathogenic avian influenza exposure on H5N1 highly pathogenic avian influenza virus infection in wood ducks (Aix sponsa)

Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus has been shown to modulate the outcome of a highly pathogenic avian influenza (HPAI) virus infection in multiple domestic avian species, but few studies have addressed this effect in wild birds. In this study, the effect of pre-exposure to homosubtypic (homologous hemagglutinin) and heterosubtypic (heterologous hemagglutinin) low pathogenic avian influenza (LPAI) viruses on the outcome of a H5N1 HPAI virus infection in wood ducks (Aix sponsa) was evaluated. Pre-exposure of wood ducks to different LPAI viruses did not prevent infection with H5N1 HPAI virus, but did increase survival associated with H5N1 HPAI virus infection. The magnitude of this effect on the outcome of the H5N1 HPAI virus infection varied between different LPAI viruses, and was associated both with efficiency of LPAI viral replication in wood ducks and the development of a detectable humoral immune response. These observations suggest that in naturally occurring outbreaks of H5N1 HPAI, birds with pre-existing immunity to homologous hemagglutinin or neuraminidase subtypes of AI virus may either survive H5N1 HPAI virus infection or live longer than naïve birds and, consequently, could pose a greater risk for contributing to viral transmission and dissemination. The mechanisms responsible for this protection and/or the duration of this immunity remain unknown. The results of this study are important for surveillance efforts and help clarify epidemiological data from outbreaks of H5N1 HPAI virus in wild bird populations.     

Monitoring exposure to avian influenza viruses in wild mammals

• 1 Avian influenza (AI) viruses primarily circulate in wild waterfowl populations and are occasionally transmitted to domestic poultry flocks. However, the possible roles of other wildlife species, such as wild mammals, in AI virus ecology have not been adequately addressed.
• 2 Due to their habitat and behaviour, many wild mammals may be capable of transmitting pathogens among wild and domestic populations. Exposure to AI viruses has been reported in an array of wild and domestic animals. The presence of wild mammals on farms has been identified as a risk factor for at least one poultry AI outbreak in North America. These reports suggest the need for seroprevalence studies examining the exposure of wild mammals to AI viruses.
• 3 Serological tests are routinely used to assess domestic poultry, domestic swine and human exposure to influenza A viruses, but these tests have not been validated for use in wild mammals. As such, some of these protocols may require adjustments or may be inappropriate for use in serology testing of wild mammals. Herein, we review these serological techniques and evaluate their potential usefulness in AI surveillance of wild mammals. We call for care to be taken when applying serological tests outside their original area of validation, and for continued assay verification for multiple species and virus strains.

     

CASCIRE surveillance network and work on avian influenza viruses

正Studies on influenza virus by Chinese Academy of Sciences(CAS)could be traced back as early as 2005 by the CAS Key Laboratory of Pathogenic Microbiology and Immunology(CASPMI),who discovered that Qinghai-like Clade 2.2H5N1 subtype highly pathogenic avian influenza virus(HPAIV)first caused severe outbreak in wild birds in Qinghai Lake(Liu et al.,2005).     

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.     

Detection of avian influenza viruses from shorebirds: evaluation of surveillance and testing approaches

Although influenza A viruses have been isolated from numerous shorebird species (Family: Scolopacidae) worldwide, our understanding of natural history of these viruses in this diverse group is incomplete. Gaining this information can be complicated by sampling difficulties related to live capture, the need for large sample sizes related to a potentially low prevalence of infection, and the need to maintain flexibility in diagnostic approaches related to varied capabilities and resources. To provide information relevant to improving sampling and testing of shorebirds for influenza A viruses, we retrospectively evaluated a combined data set from Delaware Bay, USA, collected from 2000 to 2009. Our results indicate that prevalence trends and subtype diversity can be effectively determined by either direct sampling of birds or indirect sampling of feces; however, the extent of detected subtype diversity is a function of the number of viruses recovered during that year. Even in cases where a large number of viruses are identified, an underestimate of true subtype diversity is likely. Influenza A virus isolation from Ruddy Turnstones can be enhanced by testing both cloacal and tracheal samples, and matrix real-time PCR can be used as an effective screening tool. Serologic testing to target species of interest also has application to shorebird surveillance. Overall, all of the sampling and diagnostic approaches have utility as applied to shorebird surveillance, but all are associated with inherent biases that need to be considered when comparing results from independent studies.     

Indications that live poultry markets are a major source of human H5N1 influenza virus infection in China

Human infections of H5N1 highly pathogenic avian influenza virus have continued to occur in China without corresponding outbreaks in poultry, and there is little conclusive evidence of the source of these infections. Seeking to identify the source of the human infections, we sequenced 31 H5N1 viruses isolated from humans in China (2005 to 2010). We found a number of viral genotypes, not all of which have similar known avian virus counterparts. Guided by patient questionnaire data, we also obtained environmental samples from live poultry markets and dwellings frequented by six individuals prior to disease onset (2008 and 2009). H5N1 viruses were isolated from 4 of the 6 live poultry markets sampled. In each case, the genetic sequences of the environmental and corresponding human isolates were highly similar, demonstrating a link between human infection and live poultry markets. Therefore, infection control measures in live poultry markets are likely to reduce human H5N1 infection in China.     

苏州活禽市场禽流感病毒(H7N9)季节变化及HA、NA、PB2基因系统进化分析

自2013年3月中国首次发现新型禽流感病毒H7N9以来,其于2013-2014年期间发生流行,2015年也有散发性感染。该病毒的流行不仅危及家禽养殖业,还对公共卫生安全造成严重威胁。为调查活禽市场中H7N9的进化史和季节性变化,本研究于2013年7-12月在H7N9主要流行地区之一江苏省苏州市活禽市场采集2 655份鸡、鸭咽拭子样本,对样本中流感病毒核酸进行检测。结果显示,冬季样本中H7N9阳性率显著高于夏季样本,同时发现样本中存在H5、H7和H9亚型毒株之间的混合感染。进一步对H7N9阳性样本的HA、NA和PB2基因序列进行分析,结果表明阳性样本中HA、NA和PB2基因序列与新型H7N9病毒的相应基因序列同源,其在家禽体内传代时也在继续进化。特别是一些样品中PB2基因序列与H5N1病毒PB2基因序列的同源性较高。结果提示,苏州存在一种新型H7N9病毒基因重排的可能性,建议在活禽市场对所有禽流感病毒亚型进行持续监控,从而有助于流感病毒的及时防控。     

Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses

H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.     

A duck enteritis virus-vectored bivalent live vaccine provides fast and complete protection against H5N1 avian influenza virus infection in ducks

Ducks play an important role in the maintenance of highly pathogenic H5N1 avian influenza viruses (AIVs) in nature, and the successful control of AIVs in ducks has important implications for the eradication of the disease in poultry and its prevention in humans. The inactivated influenza vaccine is expensive, labor-intensive, and usually needs 2 to 3 weeks to induce protective immunity in ducks. Live attenuated duck enteritis virus (DEV; a herpesvirus) vaccine is used routinely to control lethal DEV infections in many duck-producing areas. Here, we first established a system to generate the DEV vaccine strain by using the transfection of overlapping fosmid DNAs. Using this system, we constructed two recombinant viruses, rDEV-ul41HA and rDEV-us78HA, in which the hemagglutinin (HA) gene of the H5N1 virus A/duck/Anhui/1/06 was inserted and stably maintained within the ul41 gene or between the us7 and us8 genes of the DEV genome. Duck studies indicated that rDEV-us78HA had protective efficacy similar to that of the live DEV vaccine against lethal DEV challenge; importantly, a single dose of 10(6) PFU of rDEV-us78HA induced complete protection against a lethal H5N1 virus challenge in as little as 3 days postvaccination. The protective efficacy against both lethal DEV and H5N1 challenge provided by rDEV-ul41HA inoculation in ducks was slightly weaker than that provided by rDEV-us78HA. These results demonstrate, for the first time, that recombinant DEV is suitable for use as a bivalent live attenuated vaccine, providing rapid protection against both DEV and H5N1 virus infection in ducks.     

Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization

The recent emergence of highly pathogenic avian influenza virus (HPAI) strains in poultry and their subsequent transmission to humans in Southeast Asia have raised concerns about the potential pandemic spread of lethal disease. In this paper we describe the development and testing of an adenovirus-based influenza A virus vaccine directed against the hemagglutinin (HA) protein of the A/Vietnam/1203/2004 (H5N1) (VN/1203/04) strain isolated during the lethal human outbreak in Vietnam from 2003 to 2005. We expressed different portions of HA from a recombinant replication-incompetent adenoviral vector, achieving vaccine production within 36 days of acquiring the virus sequence. BALB/c mice were immunized with a prime-boost vaccine and exposed to a lethal intranasal dose of VN/1203/04 H5N1 virus 70 days later. Vaccination induced both HA-specific antibodies and cellular immunity likely to provide heterotypic immunity. Mice vaccinated with full-length HA were fully protected from challenge with VN/1203/04. We next evaluated the efficacy of adenovirus-based vaccination in domestic chickens, given the critical role of fowl species in the spread of HPAI worldwide. A single subcutaneous immunization completely protected chickens from an intranasal challenge 21 days later with VN/1203/04, which proved lethal to all control-vaccinated chickens within 2 days. These data indicate that the rapid production and subsequent administration of recombinant adenovirus-based vaccines to both birds and high-risk individuals in the face of an outbreak may serve to control the pandemic spread of lethal avian influenza.     

Identifying the genetic basis for resistance to avian influenza in commercial egg layer chickens

Two highly pathogenic avian influenza (HPAI) outbreaks have affected commercial egg production flocks in the American continent in recent years; a H7N3 outbreak in Mexico in 2012 that caused 70% to 85% mortality and a H5N2 outbreak in the United States in 2015 with over 99% mortality. Blood samples were obtained from survivors of each outbreak and from age and genetics matched non-affected controls. A total of 485 individuals (survivors and controls) were genotyped with a 600 k single nucleotide polymorphism (SNP) array to detect genomic regions that influenced the outcome of highly pathogenic influenza infection in the two outbreaks. A total of 420458 high quality, segregating SNPs were identified across all samples. Genetic differences between survivors and controls were analyzed using a logistic model, mixed models and a Bayesian variable selection approach. Several genomic regions potentially associated with resistance to HPAI were identified, after performing multidimensional scaling and adjustment for multiple testing. Analysis conducted within each outbreak identified different genomic regions for resistance to the two virus strains. The strongest signals for the Iowa H5N2 survivor samples were detected on chromosomes 1, 7, 9 and 15. Positional candidate genes were mainly coding for plasma membrane proteins with receptor activity and were also involved in immune response. Three regions with the strongest signal for the Mexico H7N3 samples were located on chromosomes 1 and 5. Neuronal cell surface, signal transduction and immune response proteins coding genes were located in the close proximity of these regions.     

Risk maps for the spread of highly pathogenic avian influenza in poultry

Devastating epidemics of highly contagious animal diseases such as avian influenza, classical swine fever, and foot-and-mouth disease underline the need for improved understanding of the factors promoting the spread of these pathogens. Here the authors present a spatial analysis of the between-farm transmission of a highly pathogenic H7N7 avian influenza virus that caused a large epidemic in The Netherlands in 2003. The authors developed a method to estimate key parameters determining the spread of highly transmissible animal diseases between farms based on outbreak data. The method allows for the identification of high-risk areas for propagating spread in an epidemiologically underpinned manner. A central concept is the transmission kernel, which determines the probability of pathogen transmission from infected to uninfected farms as a function of interfarm distance. The authors show how an estimate of the transmission kernel naturally provides estimates of the critical farm density and local reproduction numbers, which allows one to evaluate the effectiveness of control strategies. For avian influenza, the analyses show that there are two poultry-dense areas in The Netherlands where epidemic spread is possible, and in which local control measures are unlikely to be able to halt an unfolding epidemic. In these regions an epidemic can only be brought to an end by the depletion of susceptible farms by infection or massive culling. The analyses provide an estimate of the spatial range over which highly pathogenic avian influenza viruses spread between farms, and emphasize that control measures aimed at controlling such outbreaks need to take into account the local density of farms.