禽流感感染人的分子机制研究进展

禽流感病毒可以感染多种动物,包括人、猪、鸟、马、海豹、鲸和雪貂等。流感病毒在不同的宿主存在一定的屏障,但禽流感毒株能突破宿主屏障直接感染人,造成死亡。因此流感病毒的变异和病毒如何选择物种跨越物种流行的机制,对预防和控制流感的爆发是非常重要的。本文综述病毒毒力的分子生物学基础、禽流感染人的分子机制研究进展及在控制禽流感方面的研究。     

(Highly pathogenic) avian influenza as a zoonotic agent

Zoonotic agents challenging the world every year afresh are influenza A viruses. In the past, human pandemics caused by influenza A viruses had been occurring periodically. Wild aquatic birds are carriers of the full variety of influenza virus A subtypes, and thus, most probably constitute the natural reservoir of all influenza A viruses. Whereas avian influenza viruses in their natural avian reservoir are generally of low pathogenicity (LPAIV), some have gained virulence by mutation after transmission and adaptation to susceptible gallinaceous poultry. Those so-called highly pathogenic avian influenza viruses (HPAIV) then cause mass die-offs in susceptible birds and lead to tremendous economical losses when poultry is affected. Besides a number of avian influenza virus subtypes that have sporadically infected mammals, the HPAIV H5N1 Asia shows strong zoonotic characteristics and it was transmitted from birds to different mammalian species including humans. Theoretically, pandemic viruses might derive directly from avian influenza viruses or arise after genetic reassortment between viruses of avian and mammalian origin. So far, HPAIV H5N1 already meets two conditions for a pandemic virus: as a new subtype it has been hitherto unseen in the human population and it has infected at least 438 people, and caused severe illness and high lethality in 262 humans to date (August 2009). The acquisition of efficient human-to-human transmission would complete the emergence of a new pandemic virus. Therefore, fighting H5N1 at its source is the prerequisite to reduce pandemic risks posed by this virus. Other influenza viruses regarded as pandemic candidates derive from subtypes H2, H7, and H9 all of which have infected humans in the past. Here, we will give a comprehensive overview on avian influenza viruses in concern to their zoonotic potential.     

Different infection routes of avian influenza A (H5N1) virus in mice

The continuing outbreaks of avian influenza A H5N1 virus infection in Asia and Africa have caused worldwide concern because of the high mortality rates in poultry, suggesting its potential to become a pandemic influenza virus in humans. The transmission route of the virus among either the same species or different species is not yet clear. Broilers and BABL/c mice were inoculated with the H5N1 strain of influenza A virus isolated from birds. The animals were inoculated with 0.1 mL 10^6.83 TCID₅₀ of H5N1 virus oronasally, intraperitoneally and using eye drops. The viruses were examined by virological and pathological assays. In addition, to detect horizontal transmission, in each group, healthy chicks and mice were mixed with those infected. Viruses were detected in homogenates of the heart, liver, spleen, kidney and blood of the infected mice and chickens. Virus antigen was not detected in the spleen, kidney or gastrointestinal tract, but detected by Plaque Forming Unit (PFU) assay in the brain, liver and lung without degenerative change in these organs (in the group inoculated using eye drops. The detection results for mice inoculated using eye drops suggest that this virus might have a different tissue tropism from other influenza viruses mainly restricted to the respiratory tract in mice. All chicken samples tested positive for the virus, regardless of the method of inoculation. Avian influenza A H5N1 viruses are highly pathogenic to chickens, but its virulence in other animals is not yet known. To sum up, the results suggest that the virus replicates not only in different animal species but also through different routes of infection. In addition, the virus was detection not only in the respiratory tract but also in multiple extra‐respiratory tissues. This study demonstrates that H5N1 virus infection in mice can cause systemic disease and spread through potentially novel routes within and between mammalian hosts.     

Evolution of avian influenza viruses

Although influenza viruses can infect a wide variety of birds and mammals, the natural host of the virus is wild waterfowl, shorebirds, and gulls. When other species of animals, including chickens, turkeys, swine, horses, and humans, are infected with influenza viruses, they are considered aberrant hosts. The distinction between the normal and aberrant host is important when describing virus evolution in the different host groups. The evolutionary rate of influenza virus in the natural host reservoirs is believed to be slow, while in mammals the rate is much higher. The higher rate of evolution in mammals is thought to be a result of selective pressure on the virus to adapt to an aberrant host species. Chickens and turkey influenza virus isolates have previously and incorrectly been lumped together with wild waterfowl, gull, and shorebird influenza viruses when determining rates of evolutionary change. To determine mutational and evolutionary rates of a virus in any host species, two primary assumptions must be met: first, all isolates included in the analysis must have descended from a single introduction of the virus, and second, the outbreak must continue long enough to determine a trend. For poultry, three recent outbreaks of avian influenza meet these criteria, and the sequences of the hemagglutinin and nonstructural genes were compared. Sequences from all three outbreaks were compared to an avian influenza virus consensus sequence, which at the amino acid level is highly conserved for all the internal viral proteins. The consensus sequence also provides a common point of origin to compare all influenza viruses. The evolutionary rates determined for all three outbreaks were similar to what is observed in mammals, providing strong evidence of adaptation of influenza to the new host species, chickens and turkeys.     

Influenza pandemic planning

In this article the most important properties of influenza A viruses are described to understand influenza pandemics. There are at least three possibilities: (1) By reassortment between an avian and the prevailing human influenza A virus viruses with a new surface are created, against which no neutralizing antibodies are present in the human population. Such a virus can spread immediately worldwide. (2) Viruses, which have been present in the human population some time ago, reappear and infect the new generation, which has not been in contact with this virus before. (3) An avian influenza virus crosses the species barrier to humans and forms there a new stable lineage. In relation to pandemic planning, the first possibility can be more or less excluded, since the now-a-days human influenza A viruses have evolved so far away from their original source, the avian influenza viruses, that the formation of a well-growing and well-spreading reassortant is practically not possible anymore. Point two is a dangerous possibility, in that, e.g., a human H2N2 virus could reappear, which had disappeared in 1968 from the human population. The third possibility is at the moment the most dangerous situation, if, e.g., a highly neurotropic H5N1 virus from Southeast Asia crosses the species barrier to humans. An infection with such a pandemic virus presumably cannot be treated efficiently by antivirals. In such a situation only a rapid vaccination would be successful. In this respect in the last year important results have been obtained by using the reverse genetics. Meanwhile in about 50 countries there have been drawn up pandemic-preparedness plans.     

Novel human H7N9 influenza virus in China

Outbreaks of H7N9 avian influenza in humans in 5 provinces and 2 municipalities of China have reawakened concern that avian influenza viruses may again cross species barriers to infect the human population and thereby initiate a new influenza pandemic. Evolutionary analysis shows that human H7N9 influenza viruses originated from the H9N2, H7N3 and H11N9 avian viruses, and that it is as a novel reassortment influenza virus. This article reviews current knowledge on 11 subtypes of influenza A virus from human which can cause human infections.     

H9N2亚型禽流感病毒的研究现状

H9N2亚型禽流感病毒已在世界范围内的禽类中分离确认,并被证实可以传播到人类和低等哺乳类动物。对于它存在的潜在危害已经越来越多地受到关注,相关的研究也相继开展。许多遗传进化的分析为禽或猪流感可以直接感染人提供了证据,通过在人体的适应或与人流感病毒基因重组,可以形成新的病毒株,引起人类流感疫情暴发。文章提示应当密切监控H9N2亚型禽流感病毒,防止人类流感大流行。     

Avian influenza virus

Avian influenza viruses do not typically replicate efficiently in humans, indicating direct transmission of avian influenza virus to humans is unlikely. However, since 1997, several cases of human infections with different subtypes (H5N1, H7N7, and H9N2) of avian influenza viruses have been identified and raised the pandemic potential of avian influenza virus in humans. Although circumstantial evidence of human to human transmission exists, the novel avian-origin influenza viruses isolated from humans lack the ability to transmit efficiently from person-to-person. However, the on-going human infection with avian-origin H5N1 viruses increases the likelihood of the generation of human-adapted avian influenza virus with pandemic potential. Thus, a better understanding of the biological and genetic basis of host restriction of influenza viruses is a critical factor in determining whether the introduction of a novel influenza virus into the human population will result in a pandemic. In this article, we review current knowledge of type A influenza virus in which all avian influenza viruses are categorized.     

Variant Influenza Associated with Live Animal Markets,Minnesota

Variant influenza viruses are swine‐origin influenza A viruses that cause illness in humans. Surveillance for variant influenza A viruses, including characterization of exposure settings, is important because of the potential emergence of novel influenza viruses with pandemic potential. In Minnesota, we have documented variant influenza A virus infections associated with swine exposure at live animal markets.     

The epidemiology and evolution of influenza viruses in pigs

Pigs serve as major reservoirs of H1N1 and H3N2 influenza viruses which are endemic in pig populations world-wide and are responsible for one of the most prevalent respiratory diseases in pigs. The maintenance of these viruses in pigs and the frequent exchange of viruses between pigs and other species is facilitated directly by swine husbandry practices, which provide for a continual supply of susceptible pigs and regular contact with other species, particularly humans. The pig has been a contender for the role of intermediate host for reassortment of influenza A viruses of avian and human origin since it is the only domesticated mammalian species which is reared in abundance and is susceptible to, and allows productive replication, of avian and human influenza viruses. This can lead to the generation of new strains of influenza, some of which may be transmitted to other species including humans. This concept is supported by the detection of human-avian reassortant viruses in European pigs with some evidence for subsequent transmission to the human population. Following interspecies transmission to pigs, some influenza viruses may be extremely unstable genetically, giving rise to variants which could be conducive to the species barrier being breached a second time. Eventually, a stable lineage derived from the dominant variant may become established in pigs. Genetic drift occurs particularly in the genes encoding the external glycoproteins, but does not usually result in the same antigenic variability that occurs in the prevailing strains in the human population. Adaptation of a 'newly' transmitted influenza virus to pigs can take many years. Both human H3N2 and avian H1N1 were detected in pigs many years before they acquired the ability to spread rapidly and become associated with disease epidemics in pigs.     

Isolation and phylogenetic analysis of pandemic H1N1/09 influenza virus from swine in Jiangsu province of China

To investigate whether the 2009 pandemic H1N1 influenza A virus was still being transmitted in swine, a total of 1029 nasal swab samples from healthy swine were collected from January to May 2010 in Jiangsu province of China. Eight H1N1 influenza viruses were isolated and identified, and their full length genomes were sequenced. We found that all eight of the H1N1 viruses shared higher than 98.0% sequence identity with the 2009 pandemic virus A/Jiangsu/1/2009 (JS1). In addition, some of these viruses had D225G (3/8) mutations in the receptor binding sites of the hemagglutinin (HA) protein, indicating enhancement of their binding affinity to the sialic α2, 3Gal receptor. In conclusion, the 2009 pandemic H1N1 influenza A virus has retro-infected swine from humans in mainland China, and significant viral evolution is still ongoing in this species.     

Anticipating the species jump: surveillance for emerging viral threats

Zoonotic disease surveillance is typically triggered after animal pathogens have already infected humans. Are there ways to identify high‐risk viruses before they emerge in humans? If so, then how and where can identifications be made and by what methods? These were the fundamental questions driving a workshop to examine the future of predictive surveillance for viruses that might jump from animals to infect humans. Virologists, ecologists and computational biologists from academia, federal government and non‐governmental organizations discussed opportunities as well as obstacles to the prediction of species jumps using genetic and ecological data from viruses and their hosts, vectors and reservoirs. This workshop marked an important first step towards envisioning both scientific and organizational frameworks for this future capability. Canine parvoviruses as well as seasonal H3N2 and pandemic H1N1 influenza viruses are discussed as exemplars that suggest what to look for in anticipating species jumps. To answer the question of where to look, prospects for discovering emerging viruses among wildlife, bats, rodents, arthropod vectors and occupationally exposed humans are discussed. Finally, opportunities and obstacles are identified and accompanied by suggestions for how to look for species jumps. Taken together, these findings constitute the beginnings of a conceptual framework for achieving a virus surveillance capability that could predict future species jumps.     

Conventional and future diagnostics for avian influenza

The significant and continued transboundary spread of Asian avian influenza H5N1 since 2003, paired with documented transmission from avian species to humans and other mammals, has focused global attention on avian influenza virus detection and diagnostic strategies. While the historic and conventional laboratory methods used for isolation and identification of the virus and for detection of specific antibodies continued to be widely applied, new and emerging technologies are rapidly being adapted to support avian influenza virus surveillance and diagnosis worldwide. Molecular tools in particular are advancing toward lab-on-chip and fully integrated technologies that are capable of same day detection, pathotyping, and phylogenetic characterization of influenza A viruses obtained from clinical specimens. The future of avian influenza diagnostics, rather than moving toward a single approach, is wisely adopting a strategy that takes advantage of the range of conventional and advancing technologies to be used in "fit-for-purpose" testing.     

Avian and swine influenza viruses: our current understanding of the zoonotic risk

The introduction of swine or avian influenza (AI) viruses in the human population can set the stage for a pandemic, and many fear that the Asian H5N1 AI virus will become the next pandemic virus. This article first compares the pathogenesis of avian, swine and human influenza viruses in their natural hosts. The major aim was to evaluate the zoonotic potential of swine and avian viruses, and the possible role of pigs in the transmission of AI viruses to humans. Cross-species transfers of swine and avian influenza to humans have been documented on several occasions, but all these viruses lacked the critical capacity to spread from human-to-human. The extreme virulence of H5N1 in humans has been associated with excessive virus replication in the lungs and a prolonged overproduction of cytokines by the host, but there remain many questions about the exact viral cell and tissue tropism. Though pigs are susceptible to several AI subtypes, including H5N1, there is clearly a serious barrier to infection of pigs with such viruses. AI viruses frequently undergo reassortment in pigs, but there is no proof for a role of pigs in the generation of the 1957 or 1968 pandemic reassortants, or in the transmission of H5N1 or other wholly avian viruses to humans. The major conclusion is that cross-species transmission of influenza viruses per se is insufficient to start a human influenza pandemic and that animal influenza viruses must undergo dramatic but largely unknown genetic changes to become established in the human population.     

Influenza virus infections in mammals

The natural reservoir of all known subtypes of influenza A viruses are aquatic birds, mainly of the orders Anseriformes and Charadriiformes in which the infection is asymptomatic and the viruses stay at an evolutionary equilibrium. However, mammals may occasionally contract influenza A virus infections from this pool. This article summarizes: (i) natural infections in mammals including pigs, horses, marine mammals, ferrets, minks; (ii) results from experimental infections in several animal models including mice, ferrets, primates, rats, minks, hamsters and (iii) evidence for the increased pathogenicity of the current influenza A H5N1/Asia viruses for mammals. Several reports have shown that a number of mammalian species, including pigs, cats, ferrets, minks, whales, seals and finally also man are susceptible to natural infection with influenza A viruses of purely avian genetic make up. Among the mammalian species naturally susceptible to avian influenza virus the pig and the cat might exert the greatest potential public health impact. Despite numerous studies in animal and cell culture models, the basis of the extended host spectrum and the unusual pathogenicity of the influenza A H5N1 viruses for mammals is only beginning to be unraveled. Recently, also the transmission of equine influenza A virus to greyhound racing dogs has been documented.     

Recombinant viruses of poultry as vector vaccines against fowl plague

To help in the control of fowl plague caused by highly pathogenic avian influenza A viruses of hemagglutinin (HA) subtypes H5 and H7 several vaccines have been developed. A prophylactic immunization of poultry with inactivated influenza viruses in non-endemic situations is questionable, however, due to the impairment of serological identification of field virus-infected animals which hinders elimination of the infectious agent from the population. This problem might be overcome by the use of genetically engineered marker vaccines which contain only the protective influenza virus hemagglutinin. Infected animals could then be unambiguously identified by their serum antibodies against other influenza virus proteins, e.g. neuraminidase or nucleoprotein. For such a use, purified HA or HA-expressing DNA vaccines are conceivable. Economically advantageous and easier to apply are modified live virus vaccines in use against other poultry diseases, which have been modified to express influenza virus HA. So far, recombinant HA-expressing fowlpox virus (FPV) as well as infectious laryngotracheitis and Newcastle disease viruses have been asssessed in animal experiments. An H5-expressing FPV recombinant is already in use in Central America and Southeast Asia but without accompanying marker diagnostics. Advantages and disadvantages of the different viral vectors are discussed.     

中国流行的2009甲型H1N1猪源流感病毒HA和NA基因的分子和遗传特征

目前流行的甲型H1N1流感病毒是一个复杂的基因重配病毒。对病毒的分子生物学研究,尤其是病毒囊膜蛋白血凝素（haemagglutini,HA）基因和神经氨酸酶（neuraminidase,NA）基因的研究,为控制和预防H1N1流感病毒具有重要的意义。本研究对中国流行的2009甲型H1N1猪源流感病毒的HA和NA基因与疫苗株A/California/07/2009（H1N1）,以及不同国家和地区的病毒株进行核苷酸和氨基酸序列分析。从NCBI的GenBank数据库下载所需要毒株的序列,采用Lasergene 6.0软件包中的EditSeq和MegAlign进行序列分析,进化树分析采用MEGA4.1软件。进化分析表明,中国流行的2009 H1N1流感病毒与疫苗株的核苷酸同源率分别在98.8%~99.7%和98.6%~99.6%之间;裂解位点处为I/VPSIQSR↓G,不具备高致病性流感病毒的特征;有1株NA抗性病毒。尽管与疫苗株相比,中国流行株2009甲型H1N1猪源流感病毒的HA和NA基因有部分突变,但这些突变并不是重要的。本研究首次详细分析了中国流行的2009甲型H1N1猪源流感病毒株与疫苗株的HA和NA基因的分子特征,对实时监测流感病毒HA和NA基因的变化具有重要意义。     

A Review of Simulation Modelling Approaches Used for the Spread of Zoonotic Influenza Viruses in Animal and Human Populations

Increasing incidences of emerging and re‐emerging diseases that are mostly zoonotic (e.g. severe acute respiratory syndrome, avian influenza H5N1, pandemic influenza) has led to the need for a multidisciplinary approach to tackling these threats to public and animal health. Accordingly, a global movement of ‘One‐Health/One‐Medicine’ has been launched to foster collaborative efforts amongst animal and human health officials and researchers to address these problems. Historical evidence points to the fact that pandemics caused by influenza A viruses remain a major zoonotic threat to mankind. Recently, a range of mathematical and computer simulation modelling methods and tools have increasingly been applied to improve our understanding of disease transmission dynamics, contingency planning and to support policy decisions on disease outbreak management. This review provides an overview of methods, approaches and software used for modelling the spread of zoonotic influenza viruses in animals and humans, particularly those related to the animal‐human interface. Modelling parameters used in these studies are summarized to provide references for future work. This review highlights the limited application of modelling research to influenza in animals and at the animal‐human interface, in marked contrast to the large volume of its research in human populations. Although swine are widely recognized as a potential host for generating novel influenza viruses, and that some of these viruses, including pandemic influenza A/H1N1 2009, have been shown to be readily transmissible between humans and swine, only one study was found related to the modelling of influenza spread at the swine‐human interface. Significant gaps in the knowledge of frequency of novel viral strains evolution in pigs, farm‐level natural history of influenza infection, incidences of influenza transmission between farms and between swine and humans are clearly evident. Therefore, there is a need to direct additional research to the study of influenza transmission dynamics in animals and at the animal‐human interface.