Special features of the 2009 pandemic swine-origin influenza A H1N1 hemagglutinin and neuraminidase

Since the 2009 pandemic H1N1 swine-origin influenza A virus (09 S-OIV) has reminded the world about the global threat of the ever changing influenza virus,many questions regarding the detailed re-assortment of influenza viruses yet remain unanswered.Influenza A virus is the causative agent of the pandemic flu and contains 2 major antigenic glycoproteins on its surface:(i) hemagglutinin (HA);and (ii) neuraminidase (NA).The structures of the 09 S-OIV HA and NA proteins (09H1 and 09N1) have recently been resolved in our laboratory and provide some clues as to why the 09 S-OIV re-assortment virus is highly infectious with severe consequences in humans.For example,the 09H1 is highly similar to the HA of the 1918 influenza A pandemic virus in overall structure and especially in regards to its 5 defined antibody binding epitopes.For 09N1,its most distinctive feature is the lack of a 150-loop active site cavity,which was previously predicted to be present in all N1 NAs,and we hypothesize that the 150-loop may play a important role in the substrate specificity (α2,3 or α2,6 linked sialic acid receptors) and enzymatic mechanism of influenza NA.Combination of the HA and NA with special characteristics for the 09 S-OIV might contribute to its high increased transmissibility in humans.     

Influence of extreme weather and meteorological anomalies on outbreaks of influenza A (H1N1)

Biological experiments and epidemiological evidence indicate that variations in environment have important effect on the occurrence and transmission of epidemic influenza.It is therefore important to understand the characteristic patterns of transmission for prevention of disease and reduction of disease burden.Based on case records,we analyzed the environmental characteristics including climate variables in Changsha,and then constructed a meteorological anomaly susceptive-infective-removal (SIR) model on the basis of the results of influenza A (H1N1) transmission.The results showed that the outbreak of influenza A (H1N1) in Changsha showed significant correlation with meteorological conditions;the spread of influenza was sensitive to meteorological anomalies,and that the outbreak of influenza A (H1N1) in Changsha was influenced by a combination of absolute humidity anomalous weather conditions,contact rates of the influenza patients and changes in population movements.These findings will provide helpful information regarding prevention strategies under different conditions,a fresh understanding of the emergence and re-emergence of influenza outbreaks,and a new perspective on the transmission dynamics of influenza.     

野生鸟类在禽流感传播中所起的作用

野生鸟类很可能是高致病性禽流感（HPAI）在全球传播的媒介．这一论点来自于全球机构在过去数十年对野生鸟类体内禽流感病毒的调查、检测和统计结果．这一结果表明：（1）禽流感病毒能够感染所有种类的野鸟,但以雁鸭类为主的水禽是病毒的天然贮存库;（2）多数禽流感病毒亚型对宿主具有选择性,共同感染多种病毒亚型具有非随机性,这使病毒基因重组突变产生亚型的机会并不多,此结论建议防控野生鸟类传播禽流感的基本措施是：（1）隔离各类动物,避免相互接触．提高生物安全措施;（2）针对数量庞大的水禽．建立高效的检测和监测机制．     

IFITM3 restricts the morbidity and mortality associated with influenza

The 2009 H1N1 influenza pandemic showed the speed with which a novel respiratory virus can spread and the ability of a generally mild infection to induce severe morbidity and mortality in a subset of the population. Recent in vitro studies show that the interferon-inducible transmembrane (IFITM) protein family members potently restrict the replication of multiple pathogenic viruses. Both the magnitude and breadth of the IFITM proteins' in vitro effects suggest that they are critical for intrinsic resistance to such viruses, including influenza viruses. Using a knockout mouse model, we now test this hypothesis directly and find that IFITM3 is essential for defending the host against influenza A virus in vivo. Mice lacking Ifitm3 display fulminant viral pneumonia when challenged with a normally low-pathogenicity influenza virus, mirroring the destruction inflicted by the highly pathogenic 1918 'Spanish' influenza. Similar increased viral replication is seen in vitro, with protection rescued by the re-introduction of Ifitm3. To test the role of IFITM3 in human influenza virus infection, we assessed the IFITM3 alleles of individuals hospitalized with seasonal or pandemic influenza H1N1/09 viruses. We find that a statistically significant number of hospitalized subjects show enrichment for a minor IFITM3 allele (SNP rs12252-C) that alters a splice acceptor site, and functional assays show the minor CC genotype IFITM3 has reduced influenza virus restriction in vitro. Together these data reveal that the action of a single intrinsic immune effector, IFITM3, profoundly alters the course of influenza virus infection in mouse and humans.     

Urban structure and the risk of influenza A (H1N1) outbreaks in municipal districts

Changsha was one of the most affected areas during the 2009 A (H1N1) influenza pandemic in China. Here, we analyze the spatial–temporal dynamics of the 2009 pandemic across Changsha municipal districts, evaluate the relationship between case incidence and the local urban spatial structure and predict high-risk areas of influenza A (H1N1). We obtained epidemiological data on all cases of influenza A (H1N1) reported across municipal districts in Changsha during period May 2009–December 2010 and data on population density and basic geographic characteristics for 239 primary schools, 97 middle schools, 347 universities, 96 malls and markets, 674 business districts and 121 hospitals. Spatial–temporal K functions, proximity models and logistic regression were used to analyze the spatial distribution pattern of influenza A (H1N1) incidence and the association between influenza A (H1N1) cases and spatial risk factors and predict the infection risks. We found that the 2009 influenza A (H1N1) was driven by a transmission wave from the center of the study area to surrounding areas and reported cases increased significantly after September 2009. We also found that the distribution of influenza A (H1N1) cases was associated with population density and the presence of nearest public places, especially universities (OR = 10.166). The final predictive risk map based on the multivariate logistic analysis showed high-risk areas concentrated in the center areas of the study area associated with high population density. Our findings support the identification of spatial risk factors and high-risk areas to guide the prioritization of preventive and mitigation efforts against future influenza pandemics.     

Orisin and future distribution of the new A （H 1N1 ） influenza virus emerging in North America in 2009

The origin of the new A （H1N1） influenza virus recently emerging in North America is a hot controversial topic of significance in disease control and risk assessment. Some experts claimed that it was an unusually mongrelized mix of human, avian and swine influenza viruses, while some others concluded that it was totally a simple re-assortment hybrid of two lineages of swine influenza viruses. Here the phylogenetic diversity of the viral PB1, PA and PB2 gene sequences using online web servers, and the results suggest that all the 8 genetic segments of the new virus were possibly from two lineages of swine influenza viruses, and one of the lineage was a mongrelized mix of human, avian and swine influenza viruses emerging in the world approximately 10 years ago. Considering the recent epidemiological trends of the new virus, we believe it will spread more widely in the world and persist long in human populations. It also could spread among swine populations. The future wide spreading of the new virus may coincide the disappearance of a subtype of previous human influenza A virus.     

Genome evolution of novel influenza A （H1N1 ） viruses in humans

The epidemic situation of A H1N1 flu arose in North America in April 2009, which rapidly expanded to three continents of Europe, Asia and Africa, with the risk ranking up to 5. Until May 13th, the flu virus of A H1N1 had spread into 33 countries and regions, with a laboratory confirmed case number of 5728, including 61 deaths. Based on IRV and EpiFluDB database, 425 parts of A H1N1 flu virus sequence were achieved, followed by sequenced comparison and evolution analysis. The results showed that the current predominant A H1N1 flu virus was a kind of triple reassortment A flu virus： （i） HA, NA, MP, NP and NS originated from swine influenza virus; PB2 and PA originated from bird influenza virus; PB1 originated from human influenza virus. （ii） The origin of swine influenza virus could be subdivided as follows： HA, NP and NS originated from classic swine influenza virus of H1N1 subtype; NA and MP originated from bird origin swine influenza virus of H1N1 subtype. （iii） A H1N1 flu virus experienced no significant mutation during the epidemic spread, accompanied with no reassortment of the virus genome. In the paper, the region of the representative strains for sequence analysis （A/California/04/2009 （H1N1） and A/Mexico/4486/2009 （H1N1）） included USA and Mexico and was relatively wide, which suggested that the analysis results were convincing.     

鸡胚尿囊膜法评价板蓝根提取液抗H1N1流感病毒活性作用

 为探讨板蓝根药材、板蓝根颗粒提取液及板蓝根活性组分对流感病毒活性的影响作用,应用鸡胚尿囊膜法评价了板蓝根药材及板蓝根颗粒提取液对流感病毒的预防、治疗和病毒中和作用;评价了板蓝根活性组分对流感病毒的中和应用。研究结果表明,板蓝根药材、板蓝根颗粒提取液及板蓝根活性组分对流感病毒活性具有良好的抑制作用。板蓝根药材提取液对流感病毒具有预防和中和病毒的作用,板蓝根颗粒提取液和板蓝根活性组分具有中和流感病毒的作用。     

流感病毒疫苗研究现状、发展趋势和展望

 流感大流行严重威胁人类的生命健康和社会经济的发展。及时提供充足、有效的流感疫苗是目前各国应对流感大流行的首选方法。然而,目前流感疫苗还存在许多问题,如生产周期长、保护范围有限（不具备广谱性）、易产生过敏反应等。为此,各国政府和科研团体都在进行着不懈的努力以应对流感的大流行。本文介绍了目前国内外使用的流感疫苗,比较了流感灭活疫苗和减毒活疫苗的优缺点;针对当前流感病毒疫苗研制中亟待解决的问题,即疫苗的保护时效及合适的培养基问题,探讨了解决的思路以及取得的进展;结合中国在流感方面的主要研究进展,探讨了中国流感病毒疫苗相关基础研究中需要解决的关键科学问题,并对未来流感的防控进行了展望。     

Drosophila RNAi screen identifies host genes important for influenza virus replication

All viruses rely on host cell proteins and their associated mechanisms to complete the viral life cycle. Identifying the host molecules that participate in each step of virus replication could provide valuable new targets for antiviral therapy, but this goal may take several decades to achieve with conventional forward genetic screening methods and mammalian cell cultures. Here we describe a novel genome-wide RNA interference (RNAi) screen in Drosophila that can be used to identify host genes important for influenza virus replication. After modifying influenza virus to allow infection of Drosophila cells and detection of influenza virus gene expression, we tested an RNAi library against 13,071 genes (90% of the Drosophila genome), identifying over 100 for which suppression in Drosophila cells significantly inhibited or stimulated reporter gene (Renilla luciferase) expression from an influenza-virus-derived vector. The relevance of these findings to influenza virus infection of mammalian cells is illustrated for a subset of the Drosophila genes identified; that is, for three implicated Drosophila genes, the corresponding human homologues ATP6V0D1, COX6A1 and NXF1 are shown to have key functions in the replication of H5N1 and H1N1 influenza A viruses, but not vesicular stomatitis virus or vaccinia virus, in human HEK 293 cells. Thus, we have demonstrated the feasibility of using genome-wide RNAi screens in Drosophila to identify previously unrecognized host proteins that are required for influenza virus replication. This could accelerate the development of new classes of antiviral drugs for chemoprophylaxis and treatment, which are urgently needed given the obstacles to rapid development of an effective vaccine against pandemic influenza and the probable emergence of strains resistant to available drugs.     

流感病毒NS1蛋白在复制中的作用研究

流感病毒NS1基因敲除毒株(delNS1毒株)的获得可以使我们建立更有效的生物学关系,主要表现在流感病毒繁殖周期和致病的过程中,流感病毒NS1蛋白和双链RNA活性蛋白激酶(PKR)之间的生物学关系.实验结果表明,缺少功能性的PKR可以使delNS1毒株在其他非允许宿主中繁殖,这表明流感病毒NS1蛋白的主要功能是对抗或阻止PKR调解抗病毒效应.     

Subtyping of type A influenza by sequencing the variable regions of HA gene specifically amplified with RT-PCR

The outbreak of a novel influenza A （H1N1） virus across the globe poses a threat to human health. It is of paramount importance to develop a rapid, reliable and inexpensive diagnostic procedure. Based on the bioinformatic information from public database, primers specific for influenza A virus surface protein haemagglutinin （HA） of several subtypes （including H1, H2, H3, H5, H7 and H9） were designed. Primer-specific PCR products were subiected to sequencing for accurately distinguishing H1 and H3 subtypes from others. This sequencing-based detection method will not only be applied to rapid detection and simultaneous subtype identification of new influenza A virus H1N1, but also provide the strategies to monitor other new types of influenza virus with explosive potential.     

Epidemiological and risk analysis of the H7N9 subtype influenza outbreak in China at its early stage

Dozens of human cases infected with H7N9 subtype avian influenza virus (AIV) have been confirmed in China since March, 2013. Distribution data of sexes, ages, professions and regions of the cases were analyzed in this report. The results showed that the elderly cases, especially the male elderly, were significantly more than expected, which is different from human cases of H5N1 avian influenza and human cases of the pandemic H1N1 influenza. The outbreak was rated as a Grade Ⅲ (severe) outbreak, and it would evolve into a Grade IV (very severe) outbreak soon, using a method reported previously. The H7N9 AIV will probably circulate in humans, birds and pigs for years. Moreover, with the driving force of natural selection, the virus will probably evolve into highly pathogenic AIV in birds, and into a deadly pandemic influenza virus in humans. Therefore, the H7N9 outbreak has been assumed severe, and it is likely to become very or extremely severe in the future, highlighting the emergent need of forceful scientific measures to eliminate any infected animal flocks. We also described two possible mild scenarios of the future evolution of the outbreak.     

Transmissibility of 1918 pandemic influenza

The 1918 influenza pandemic killed 20-40 million people worldwide, and is seen as a worst-case scenario for pandemic planning. Like other pandemic influenza strains, the 1918 A/H1N1 strain spread extremely rapidly. A measure of transmissibility and of the stringency of control measures required to stop an epidemic is the reproductive number, which is the number of secondary cases produced by each primary case. Here we obtained an estimate of the reproductive number for 1918 influenza by fitting a deterministic SEIR (susceptible-exposed-infectious-recovered) model to pneumonia and influenza death epidemic curves from 45 US cities: the median value is less than three. The estimated proportion of the population with A/H1N1 immunity before September 1918 implies a median basic reproductive number of less than four. These results strongly suggest that the reproductive number for 1918 pandemic influenza is not large relative to many other infectious diseases. In theory, a similar novel influenza subtype could be controlled. But because influenza is frequently transmitted before a specific diagnosis is possible and there is a dearth of global antiviral and vaccine stores, aggressive transmission reducing measures will probably be required.     

Molecular characterization of H3N2 and H4N6 subtypes avian influenza viruses isolated from mallards in Poyang Lake, China in 2010

Poyang Lake is the largest inland freshwater lake in China and contains many species of wild birds and waterfowls.We conducted a survey of avian influenza viruses in nine semi-artificial waterfowl farms in Poyang Lake during January to March of 2010.Out of 1036 cloacal swabs collected,three H3N2 and one H4N6 influenza viruses were isolated from healthy mallards.All the isolates were genetically and phylogenetically characterized.The analysis of putative HA cleavage sites showed that all the four isolates possessed the molecular characteristics(QTRGL for H3N2 viruses,PEKASR for H4N6 virus) of lowly pathogenic avian influenza(LPAI) virus.The phylogenetic analysis of the viral genomes showed that all four virus isolates clustered in the Eurasian clade of influenza viruses.The M gene of the viruses possessed the highest homology with highly pathogenic H5N1 influenza viruses.In addition,co-infection of H3N2 and H4N6 in the same farm was observed.And interestingly,we isolated two subtypes viruses(H3N2 and H4N6) and their progeny virus(H3N2) with evidence of genome reassortment from the same farm,in which the PB1 and PB2 gene segments of H4N6 replaced those of the H3N2 strain.The results of animal infection experiments showed that all the four isolated viruses were lowly pathogenic to chickens and not pathogenic to mice,which was consistent with the results of genetic analysis.     

Long-term evolution and transmission dynamics of swine influenza A virus

Swine influenza A viruses (SwIV) cause significant economic losses in animal husbandry as well as instances of human disease and occasionally give rise to human pandemics, including that caused by the H1N1/2009 virus. The lack of systematic and longitudinal influenza surveillance in pigs has hampered attempts to reconstruct the origins of this pandemic. Most existing swine data were derived from opportunistic samples collected from diseased pigs in disparate geographical regions, not from prospective studies in defined locations, hence the evolutionary and transmission dynamics of SwIV are poorly understood. Here we quantify the epidemiological, genetic and antigenic dynamics of SwIV in Hong Kong using a data set of more than 650 SwIV isolates and more than 800 swine sera from 12?years of systematic surveillance in this region, supplemented with data stretching back 34?years. Intercontinental virus movement has led to reassortment and lineage replacement, creating an antigenically and genetically diverse virus population whose dynamics are quantitatively different from those previously observed for human influenza viruses. Our findings indicate that increased antigenic drift is associated with reassortment events and offer insights into the emergence of influenza viruses with epidemic potential in swine and humans.     

板蓝根粗提液抑制流感病毒的实验研究

对板蓝根凝聚粗提液的抗流感病毒作用进行了研究。用丙酮脱脂提取板蓝根生药的凝集素，并分别测定各样品的血凝活性，并用45.3mg/mL的样品对流感病毒（A1／京防／97－53H1N1，A1／京防／262／95）进行了体外抑制试验。结果表明：板蓝根凝集素对流感病毒具有显著的直接杀灭作用和预防作用及较好的治疗作用，而且得出抑制流感病毒的效果与板蓝根凝集素血凝活性的高低有关。     

Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors

Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era. Recent work has recovered the gene sequences of this unusual strain, so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes. However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 haemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. Here we show that of the 32 individuals tested that were born in or before 1915, each showed seroreactivity with the 1918 virus, nearly 90 years after the pandemic. Seven of the eight donor samples tested had circulating B cells that secreted antibodies that bound the 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that showed potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but did not cross-react with HAs of more contemporary human influenza viruses. The antibody genes had an unusually high degree of somatic mutation. The antibodies bound to the 1918 HA protein with high affinity, had exceptional virus-neutralizing potency and protected mice from lethal infection. Isolation of viruses that escaped inhibition suggested that the antibodies recognize classical antigenic sites on the HA surface. Thus, these studies demonstrate that survivors of the 1918 influenza pandemic possess highly functional, virus-neutralizing antibodies to this uniquely virulent virus, and that humans can sustain circulating B memory cells to viruses for many decades after exposure-well into the tenth decade of life.