Protection from the 2009 H1N1 Pandemic Influenza by an Antibody from Combinatorial Survivor-Based Libraries

Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06) against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 “Swine” H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population.     

Unseasonal Transmission of H3N2 Influenza A Virus During the Swine-Origin H1N1 Pandemic

The initial wave of swine-origin influenza A virus (pandemic H1N1/09) in the United States during the spring and summer of 2009 also resulted in an increased vigilance and sampling of seasonal influenza viruses (H1N1 and H3N2), even though they are normally characterized by very low incidence outside of the winter months. To explore the nature of virus evolution during this influenza “off-season,” we conducted a phylogenetic analysis of H1N1 and H3N2 sequences sampled during April to June 2009 in New York State. Our analysis revealed that multiple lineages of both viruses were introduced and cocirculated during this time, as is typical of influenza virus during the winter. Strikingly, however, we also found strong evidence for the presence of a large transmission chain of H3N2 viruses centered on the south-east of New York State and which continued until at least 1 June 2009. These results suggest that the unseasonal transmission of influenza A viruses may be more widespread than is usually supposed.The recent emergence of swine-origin H1N1 influenza A virus (pandemic H1N1/09) in humans has heightened awareness of how the burden of morbidity and mortality due to influenza is associated with the appearance of new genetic variants (5) and of the genetic and epidemiological determinants of viral transmission (8). The emergence of pandemic H1N1/09 is also unprecedented in recorded history as it means that three antigenically distinct lineages of influenza A virus—pandemic H1N1/09 and the seasonal H1N1 and H3N2 viruses— currently cocirculate within human populations.Although the presence of multiple subtypes of influenza A virus may place an additional burden on public health resources, it also provides a unique opportunity to compare the patterns and dynamics of evolution in these viruses on a similar time scale. Indeed, one of the most interesting secondary effects of the current H1N1/09 pandemic has been an increased vigilance for cases of influenza-like illness and hence an intensified sampling of seasonal H1N1 and H3N2 viruses during the typical influenza “off-season” (i.e., spring-summer) in the northern hemisphere. Because the influenza season in the northern hemisphere generally runs from November through March, with a usual peak in January or February, influenza viruses sampled outside of this period are of special interest.The current model for the global spatiotemporal dynamics of influenza A virus is that the northern and southern hemispheres represent ecological “sinks” for this virus, with little ongoing viral transmission during the summer months (9). In contrast, more continual viral transmission occurs within the tropical “source” population (13) that is most likely centered on an intense transmission network in east and southeast Asia (10). However, the precise epidemiological and evolutionary reasons for this major geographic division, and for the seasonality of influenza A virus in general, remain uncertain (1, 4). Evidence for this “sink-source” ecological model is that viruses sampled from successive seasons in localities such as New York State do not usually form linked clusters on phylogenetic trees, indicating that they are not connected by direct transmission through the summer months (7). Similar conclusions can be drawn for the United States as a whole and point to multiple introductions of phylogenetically distinct lineages during the winter (6), followed by complex patterns of spatial diffusion (14). However, despite the growing epidemiological and phylogenetic data supporting this model, it is also evident that there is relatively little sequence data from seasonal influenza viruses that are sampled from April to October in the northern hemisphere. Hence, it is uncertain whether extended chains of transmission can occur during this time period, even though this may have an important bearing on our understanding of influenza seasonality.To address these issues, we examined the evolutionary behavior of seasonal H1N1 and H3N2 viruses as they cocirculated during a single time period—(late) April to June 2009—within a single locality (New York State). Not only are levels of influenza virus transmission in the northern hemisphere usually very low during this time period, but in this particular season the human host population was also experiencing the emerging epidemic of pandemic H1N1/09.     

Novel Avian Influenza A (H7N9) Virus Induces Impaired Interferon Responses in Human Dendritic Cells

In March 2013 a new avian influenza A(H7N9) virus emerged in China and infected humans with a case fatality rate of over 30%. Like the highly pathogenic H5N1 virus, H7N9 virus is causing severe respiratory distress syndrome in most patients. Based on genetic analysis this avian influenza A virus shows to some extent adaptation to mammalian host. In the present study, we analyzed the activation of innate immune responses by this novel H7N9 influenza A virus and compared these responses to those induced by the avian H5N1 and seasonal H3N2 viruses in human monocyte-derived dendritic cells (moDCs). We observed that in H7N9 virus-infected cells, interferon (IFN) responses were weak although the virus replicated as well as the H5N1 and H3N2 viruses in moDCs. H7N9 virus-induced expression of pro-inflammatory cytokines remained at a significantly lower level as compared to H5N1 virus-induced “cytokine storm” seen in human moDCs. However, the H7N9 virus was extremely sensitive to the antiviral effects of IFN-α and IFN-β in pretreated cells. Our data indicates that different highly pathogenic avian viruses may show considerable differences in their ability to induce host antiviral responses in human primary cell models such as moDCs. The unexpected appearance of the novel H7N9 virus clearly emphasizes the importance of the global influenza surveillance system. It is, however, equally important to systematically characterize in normal human cells the replication capacity of the new viruses and their ability to induce and respond to natural antiviral substances such as IFNs.     

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

Inter-Seasonal Influenza is Characterized by Extended Virus Transmission and Persistence

The factors that determine the characteristic seasonality of influenza remain enigmatic. Current models predict that occurrences of influenza outside the normal surveillance season within a temperate region largely reflect the importation of viruses from the alternate hemisphere or from equatorial regions in Asia. To help reveal the drivers of seasonality we investigated the origins and evolution of influenza viruses sampled during inter-seasonal periods in Australia. To this end we conducted an expansive phylogenetic analysis of 9912, 3804, and 3941 hemagglutinnin (HA) sequences from influenza A/H1N1pdm, A/H3N2, and B, respectively, collected globally during the period 2009-2014. Of the 1475 viruses sampled from Australia, 396 (26.8% of Australian, or 2.2% of global set) were sampled outside the monitored temperate influenza surveillance season (1 May – 31 October). Notably, rather than simply reflecting short-lived importations of virus from global localities with higher influenza prevalence, we documented a variety of more complex inter-seasonal transmission patterns including “stragglers” from the preceding season and “heralds” of the forthcoming season, and which included viruses sampled from clearly temperate regions within Australia. We also provide evidence for the persistence of influenza B virus between epidemic seasons, in which transmission of a viral lineage begins in one season and continues throughout the inter-seasonal period into the following season. Strikingly, a disproportionately high number of inter-seasonal influenza transmission events occurred in tropical and subtropical regions of Australia, providing further evidence that climate plays an important role in shaping patterns of influenza seasonality.     

Predicting the Antigenic Structure of the Pandemic (H1N1) 2009 Influenza Virus Hemagglutinin

The pandemic influenza virus (2009 H1N1) was recently introduced into the human population. The hemagglutinin (HA) gene of 2009 H1N1 is derived from “classical swine H1N1” virus, which likely shares a common ancestor with the human H1N1 virus that caused the pandemic in 1918, whose descendant viruses are still circulating in the human population with highly altered antigenicity of HA. However, information on the structural basis to compare the HA antigenicity among 2009 H1N1, the 1918 pandemic, and seasonal human H1N1 viruses has been lacking. By homology modeling of the HA structure, here we show that HAs of 2009 H1N1 and the 1918 pandemic virus share a significant number of amino acid residues in known antigenic sites, suggesting the existence of common epitopes for neutralizing antibodies cross-reactive to both HAs. It was noted that the early human H1N1 viruses isolated in the 1930s–1940s still harbored some of the original epitopes that are also found in 2009 H1N1. Interestingly, while 2009 H1N1 HA lacks the multiple N-glycosylations that have been found to be associated with an antigenic change of the human H1N1 virus during the early epidemic of this virus, 2009 H1N1 HA still retains unique three-codon motifs, some of which became N-glycosylation sites via a single nucleotide mutation in the human H1N1 virus. We thus hypothesize that the 2009 H1N1 HA antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in humans. Indeed, amino acid substitutions predicted here are occurring in the recent 2009 H1N1 variants. The present study suggests that antibodies elicited by natural infection with the 1918 pandemic or its early descendant viruses play a role in specific immunity against 2009 H1N1, and provides an insight into future likely antigenic changes in the evolutionary process of 2009 H1N1 in the human population.     

Complete Genome Sequence of an H3N2 Avian Influenza Virus Isolated from a Live Poultry Market in Eastern China

Previous studies indicate that the H3 influenza virus has the ability to establish infection upon interspecies transmission and poses a threat to mammals. Therefore, it is important to enhance the surveillance of H3 avian influenza viruses (AIVs). In this study, A/duck/Shanghai/C84/2009(H3N2) (C84) was isolated from a live poultry market in Shanghai, China. Using PCR and sequencing analyses, we obtained the whole-genome sequence of this virus. The H3N2 virus proved to be a novel multiple-gene reassortant AIV whose genes were derived from H3N2, H4N6, H6N2, and H9N2. Knowledge regarding the complete genome sequence of the C84 virus will be useful for epidemiological surveillance.     

长沙A(H1N1)亚型季节性流感暴发疫情的实验室诊断及其HA基因特性分析

对2009 年长沙麓山国际学校流感暴发疫情进行实验室诊断, 并探索新分离的A(H1N1)亚型流感病毒血凝素(HA)的基因特性。对流感暴发疫情的25 份鼻/咽拭子标本进行RT-PCR检测和流感病毒分离, 然后利用CEQ?8000 Genetic Analysis System对病毒分离株(A/Yuelu/314/2009)进行测序, 测序结果提交至GenBank(登录号: FJ912843)并用ClustalX和Mega4.1软件进行序列分析。结果显示, 分离出A(H1N1)亚型流感毒株18株, 检出21份A(H1N1)亚型流感病毒核酸阳性; A/Yuelu/314/2009(H1N1) HA基因序列与2008~2009 年疫苗株(A/Brisbane/59/2007)比较显示: 核苷酸和氨基酸同源性均为99%, 有6个位点的氨基酸发生了变异(V148A、S158N、G202A、I203D、A206T、W435R), 其中一个S158N氨基酸变异位于B抗原表位, HA基因序列上共有潜在糖基化位点9 个(27、28、40、71、151、176、303、497、536), 与A/Brisbane/59/2007相同且氨基酸序列保守。本实验诊断出此次流感暴发疫情的病原体为A(H1N1)型季节性流感病毒, 研究还发现A/Yuelu/314/2009(H1N1)长沙分离株与A/Brisbane/59/2007 疫苗株基因序列比较显示并未形成一个新的变种, 推测是由于分离株与疫苗株之间基因特性的改变和人群对A(H1N1)亚型流感病毒免疫力降低导致了此次长沙麓山国际学校A(H1N1)亚型流感的暴发。     

Mutations in H5N1 Influenza Virus Hemagglutinin that Confer Binding to Human Tracheal Airway Epithelium

The emergence in 2009 of a swine-origin H1N1 influenza virus as the first pandemic of the 21st Century is a timely reminder of the international public health impact of influenza viruses, even those associated with mild disease. The widespread distribution of highly pathogenic H5N1 influenza virus in the avian population has spawned concern that it may give rise to a human influenza pandemic. The mortality rate associated with occasional human infection by H5N1 virus approximates 60%, suggesting that an H5N1 pandemic would be devastating to global health and economy. To date, the H5N1 virus has not acquired the propensity to transmit efficiently between humans. The reasons behind this are unclear, especially given the high mutation rate associated with influenza virus replication. Here we used a panel of recombinant H5 hemagglutinin (HA) variants to demonstrate the potential for H5 HA to bind human airway epithelium, the predominant target tissue for influenza virus infection and spread. While parental H5 HA exhibited limited binding to human tracheal epithelium, introduction of selected mutations converted the binding profile to that of a current human influenza strain HA. Strikingly, these amino-acid changes required multiple simultaneous mutations in the genomes of naturally occurring H5 isolates. Moreover, H5 HAs bearing intermediate sequences failed to bind airway tissues and likely represent mutations that are an evolutionary “dead end.” We conclude that, although genetic changes that adapt H5 to human airways can be demonstrated, they may not readily arise during natural virus replication. This genetic barrier limits the likelihood that current H5 viruses will originate a human pandemic.     

Close relationship between the 2009 H1N1 virus and South Dakota AIV strains

Although previous publications suggest the 2009 pandemic influenza A (H1N1) virus was reassorted from swine viruses of North America and Eurasia, the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the 2009 H1N1 influenza, great deal of interest has been drawn to influenza, consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus, which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the 2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances, it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1 virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.     

A Novel Nonhuman Primate Model for Influenza Transmission

Studies of influenza transmission are necessary to predict the pandemic potential of emerging influenza viruses. Currently, both ferrets and guinea pigs are used in such studies, but these species are distantly related to humans. Nonhuman primates (NHP) share a close phylogenetic relationship with humans and may provide an enhanced means to model the virological and immunological events in influenza virus transmission. Here, for the first time, it was demonstrated that a human influenza virus isolate can productively infect and be transmitted between common marmosets (Callithrix jacchus), a New World monkey species. We inoculated four marmosets with the 2009 pandemic virus A/California/07/2009 (H1N1pdm) and housed each together with a naïve cage mate. We collected bronchoalveolar lavage and nasal wash samples from all animals at regular intervals for three weeks post-inoculation to track virus replication and sequence evolution. The unadapted 2009 H1N1pdm virus replicated to high titers in all four index animals by 1 day post-infection. Infected animals seroconverted and presented human-like symptoms including sneezing, nasal discharge, labored breathing, and lung damage. Transmission occurred in one cohabitating pair. Deep sequencing detected relatively few genetic changes in H1N1pdm viruses replicating in any infected animal. Together our data suggest that human H1N1pdm viruses require little adaptation to replicate and cause disease in marmosets, and that these viruses can be transmitted between animals. Marmosets may therefore be a viable model for studying influenza virus transmission.     

Pre-existing heterosubtypic immunity provides a barrier to airborne transmission of influenza viruses

Human-to-human transmission of influenza viruses is a serious public health threat, yet the precise role of immunity from previous infections on the susceptibility to airborne infection is still unknown. Using the ferret model, we examined the roles of exposure duration and heterosubtypic immunity on influenza transmission. We demonstrate that a 48 hour exposure is sufficient for efficient transmission of H1N1 and H3N2 viruses. To test pre-existing immunity, a gap of 8–12 weeks between primary and secondary infections was imposed to reduce innate responses and ensure robust infection of donor animals with heterosubtypic viruses. We found that pre-existing H3N2 immunity did not significantly block transmission of the 2009 H1N1pandemic (H1N1pdm09) virus to immune animals. Surprisingly, airborne transmission of seasonal H3N2 influenza strains was abrogated in recipient animals with H1N1pdm09 pre-existing immunity. This protection from natural infection with H3N2 virus was independent of neutralizing antibodies. Pre-existing immunity with influenza B virus did not block H3N2 virus transmission, indicating that the protection was likely driven by the adaptive immune response. We demonstrate that pre-existing immunity can impact susceptibility to heterologous influenza virus strains, and implicate a novel correlate of protection that can limit the spread of respiratory pathogens through the air.     

Reassortant between Human-Like H3N2 and Avian H5 Subtype Influenza A Viruses in Pigs: A Potential Public Health Risk

Background

Human-like H3N2 influenza viruses have repeatedly been transmitted to domestic pigs in different regions of the world, but it is still uncertain whether any of these variants could become established in pig populations. The fact that different subtypes of influenza viruses have been detected in pigs makes them an ideal candidate for the genesis of a possible reassortant virus with both human and avian origins. However, the determination of whether pigs can act as a “mixing vessel” for a possible future pandemic virus is still pending an answer. This prompted us to gather the epidemiological information and investigate the genetic evolution of swine influenza viruses in Jilin, China.

Methods

Nasopharyngeal swabs were collected from pigs with respiratory illness in Jilin province, China from July 2007 to October 2008. All samples were screened for influenza A viruses. Three H3N2 swine influenza virus isolates were analyzed genetically and phylogenetically.

Results

Influenza surveillance of pigs in Jilin province, China revealed that H3N2 influenza viruses were regularly detected from domestic pigs during 2007 to 2008. Phylogenetic analysis revealed that two distinguishable groups of H3N2 influenza viruses were present in pigs: the wholly contemporary human-like H3N2 viruses (represented by the Moscow/10/99-like sublineage) and double-reassortant viruses containing genes from contemporary human H3N2 viruses and avian H5 viruses, both co-circulating in pig populations.

Conclusions

The present study reports for the first time the coexistence of wholly human-like H3N2 viruses and double-reassortant viruses that have emerged in pigs in Jilin, China. It provides updated information on the role of pigs in interspecies transmission and genetic reassortment of influenza viruses.     

Swine-origin influenza-virus-induced acute lung injury: Novel or classical pathogenesis?

Influenza viruses are common respiratory pathogens in humans and can cause serious infection that leads to the development of pneumonia. Due to their host-range diversity, genetic and antigenic diversity, and potential to reassort genetically in vivo, influenza A viruses are continual sources of novel influenza strains that lead to the emergence of periodic epidemics and outbreaks in humans. Thus, newly emerging viral diseases are always major threats to public health. In March 2009, a novel influenza virus suddenly emerged and caused a worldwide pandemic. The novel pandemic influenza virus was genetically and antigenically distinct from previous seasonal human influenza A/H1N1 viruses; it was identified to have originated from pigs, and further genetic analysis revealed it as a subtype of A/H1N1, thus later called a swine-origin influenza virus A/H1N1. Since the novel virus emerged, epidemiological surveys and research on experimental animal models have been conducted, and characteristics of the novel influenza virus have been determined but the exact mechanisms of pulmonary pathogenesis remain to be elucidated. In this editorial, we summarize and discuss the recent pandemic caused by the novel swine-origin influenza virus A/H1N1 with a focus on the mechanism of pathogenesis to obtain an insight into potential therapeutic strategies.     

Continued Evolution of H5N1 Influenza Viruses in Wild Birds,Domestic Poultry,and Humans in China from 2004 to 2009

Despite substantial efforts to control H5N1 avian influenza viruses (AIVs), the viruses have continued to evolve and cause disease outbreaks in poultry and infections in humans. In this report, we analyzed 51 representative H5N1 AIVs isolated from domestic poultry, wild birds, and humans in China during 2004 to 2009, and 21 genotypes were detected based on whole-genome sequences. Twelve genotypes of AIVs in southern China bear similar H5 hemagglutinin (HA) genes (clade 2.3). These AIVs did not display antigenic drift and could be completely protected against by the A/goose/Guangdong/1/96 (GS/GD/1/96)-based oil-adjuvanted killed vaccine and recombinant Newcastle disease virus vaccine, which have been used in China. In addition, antigenically drifted H5N1 viruses, represented by A/chicken/Shanxi/2/06 (CK/SX/2/06), were detected in chickens from several provinces in northern China. The CK/SX/2/06-like viruses are reassortants with newly emerged HA, NA, and PB1 genes that could not be protected against by the GS/GD/1/96-based vaccines. These viruses also reacted poorly with antisera generated from clade 2.2 and 2.3 viruses. The majority of the viruses isolated from southern China were lethal in mice and ducks, while the CK/SX/2/06-like viruses caused mild disease in mice and could not replicate in ducks. Our results demonstrate that the H5N1 AIVs circulating in nature have complex biological characteristics and pose a continued challenge for disease control and pandemic preparedness.The highly pathogenic H5N1 influenza viruses that emerged over a decade ago in southern China have evolved into over 10 distinct phylogenetic clades based on their hemagglutinin (HA) genes. The viruses have spread to over 63 countries and to multiple mammalian species, including humans, resulting in 498 cases of infection and 294 deaths by 6 May 2010 according to the World Health Organization (WHO) (http://www.who.int). To date, none of the different H5N1 clades has acquired the ability to consistently transmit among mammalian species. The currently circulating H5N1 viruses are unique in that they continue to circulate in avian species. All previous highly pathogenic H5 and H7 viruses have naturally “burned out” or were stamped out because of their high pathogenicity in domestic poultry. While there is growing complacency about the potential of H5N1 “bird flu” to attain consistent transmissibility in humans and develop pandemicity, it is worth remembering that we have no knowledge of the time that it took the 1918 Spanish, the 1957 Asian, the 1968 Hong Kong, and the 2009 North American pandemics to develop their pandemic potentials. We may therefore currently be witnessing in real time the evolution of an H5N1 pandemic influenza virus.H5N1 avian influenza viruses (AIVs) were first detected in sick geese in Guangdong province in 1996, and both nonpathogenic and highly pathogenic (HP) H5N1 viruses were described (18). In 1997, H5N1 reassortant viruses that derived the HA gene from A/goose/Guangdong/1/96 (GS/GD/1/96)-like viruses and the other genes from H6N1 and/or H9N2 viruses caused lethal outbreaks in poultry and humans in Hong Kong (6, 7). Since then, long-term active surveillance of influenza viruses in domestic poultry has been performed, and multiple subtypes of influenza viruses have been detected in chickens and ducks in China (16, 19, 37). H5N1 influenza viruses have been repeatedly detected in apparently healthy ducks in southern China since 1999 (4, 13) and were also detected in pigs in Fujian province in 2001 and 2003 (39).Since the beginning of 2004, there have been significant outbreaks of H5N1 avian influenza virus infection involving multiple poultry farm flocks in more than 20 provinces in China (2). H5N1 viruses resulted in the deaths of millions of domestic poultry, including chickens, ducks, and geese, as the result of infection or of culling and the deaths of thousands of wild birds (5, 20). Thirty-eight human cases of HP H5N1 infection with 25 fatalities have been associated with direct exposure to infected poultry (WHO; http://www.who.int). Since 2004, the vaccination of domestic poultry has been used for the control of HP H5N1 influenza virus in China. While this strategy has been effective at reducing the incidence of HP H5N1 in poultry and at markedly reducing the number of human cases, it is impossible to vaccinate every single bird due to the enormous poultry population. Outbreaks of H5N1 influenza virus still continue to occur in poultry although at a reduced frequency.A previous study by Smith et al. reported that a “Fujian-like” H5N1 influenza virus emerged in late 2005 and predominated in poultry in southern China (26). Those authors suggested that vaccination may have facilitated the selection of the “Fujian-like” sublineage. Here, we analyzed 51 representative H5N1 viruses that were isolated from wild birds, domestic poultry, and humans from 2004 to 2009 in China and described their genetic evolution and antigenicity profiles. Our results indicate that H5N1 influenza viruses in southern China, including the “Fujian-like” viruses, are complicated reassortants, which could be well protected against by GS/GD/1/96 virus-based vaccines. We documented the emergence of the latest variant of H5N1 (A/chicken/Shanxi/2/06 [CK/SX/2/06]) that broke through existing poultry vaccines. We show that this variant is less pathogenic in mice and ducks than the earlier strains and propose that the variant was not selected by the use of vaccines.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

Characterization of an artificial swine-origin influenza virus with the same gene combination as H1N1/2009 virus: a genesis clue of pandemic strain

Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus.     

T cell-mediated protection against lethal 2009 pandemic H1N1 influenza virus infection in a mouse model

Genetic mutation and reassortment of influenza virus gene segments, in particular those of hemagglutinin (HA) and neuraminidase (NA), that lead to antigenic drift and shift are the major strategies for influenza virus to escape preexisting immunity. The most recent example of such phenomena is the first pandemic of H1N1 influenza of the 21st century, which started in 2009. Cross-reactive antibodies raised against H1N1 viruses circulating before 1930 show protective activity against the 2009 pandemic virus. Cross-reactive T-cell responses can also contribute to protection, but in vivo support of this view is lacking. To explore the protection mechanisms in vivo, we primed mice with H1 and H3 influenza virus isolates and rechallenged them with a virus derived from the 2009 H1N1 A/CA/04/09 virus, named CA/E3/09. We found that priming with influenza viruses of both H1 and H3 homo- and heterosubtypes protected against lethal CA/E3/09 virus challenge. Convalescent-phase sera from these primed mice conferred no neutralization activity in vitro and no protection in vivo. However, T-cell depletion studies suggested that both CD4 and CD8 T cells contributed to the protection. Taken together, these results indicate that cross-reactive T cells established after initial priming with distally related viruses can be a vital component for prevention of disease and control of pandemic H1N1 influenza virus infection. Our results highlight the importance of establishing cross-reactive T-cell responses for protecting against existing or newly emerging pandemic influenza viruses.     

Identification of hemagglutinin structural domain and polymorphisms which may modulate swine H1N1 interactions with human receptor

Background  

The novel A/H1N1 influenza virus, which recently emerged in North America is most closely related to North American H1N1/N2 swine viruses. Until the beginning of 2009, North American swine H1N1/N2 viruses have only sporadically infected humans as dead-end hosts. In 2009 the A/H1N1 virus acquired the capacity to spread efficiently by human to human transmission. The novel A/H1N1 influenza virus has struck thousands of people in more than 70 countries and killed more than 140, representing a public health emergency of international concern. Here we have studied properties of hemagglutinin of A/H1N1 which may modulate virus/receptor interaction.