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.     

Characterization of the 1918 influenza virus polymerase genes

The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Here we present sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus, and propose that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans.     

Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus

The influenza pandemic of 1918-19 was responsible for about 50 million deaths worldwide. Modern histopathological analysis of autopsy samples from human influenza cases from 1918 revealed significant damage to the lungs with acute, focal bronchitis and alveolitis associated with massive pulmonary oedema, haemorrhage and rapid destruction of the respiratory epithelium. The contribution of the host immune response leading to this severe pathology remains largely unknown. Here we show, in a comprehensive analysis of the global host response induced by the 1918 influenza virus, that mice infected with the reconstructed 1918 influenza virus displayed an increased and accelerated activation of host immune response genes associated with severe pulmonary pathology. We found that mice infected with a virus containing all eight genes from the pandemic virus showed marked activation of pro-inflammatory and cell-death pathways by 24 h after infection that remained unabated until death on day 5. This was in contrast with smaller host immune responses as measured at the genomic level, accompanied by less severe disease pathology and delays in death in mice infected with influenza viruses containing only subsets of 1918 genes. The results indicate a cooperative interaction between the 1918 influenza genes and show that study of the virulence of the 1918 influenza virus requires the use of the fully reconstructed virus. With recent concerns about the introduction of highly pathogenic avian influenza viruses into humans and their potential to cause a worldwide pandemic with disastrous health and economic consequences, a comprehensive understanding of the global host response to the 1918 virus is crucial. Moreover, understanding the contribution of host immune responses to virulent influenza virus infections is an important starting point for the identification of prognostic indicators and the development of novel antiviral therapies.     

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.     

Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus

The 1918 influenza pandemic was unusually severe, resulting in about 50 million deaths worldwide. The 1918 virus is also highly pathogenic in mice, and studies have identified a multigenic origin of this virulent phenotype in mice. However, these initial characterizations of the 1918 virus did not address the question of its pathogenic potential in primates. Here we demonstrate that the 1918 virus caused a highly pathogenic respiratory infection in a cynomolgus macaque model that culminated in acute respiratory distress and a fatal outcome. Furthermore, infected animals mounted an immune response, characterized by dysregulation of the antiviral response, that was insufficient for protection, indicating that atypical host innate immune responses may contribute to lethality. The ability of influenza viruses to modulate host immune responses, such as that demonstrated for the avian H5N1 influenza viruses, may be a feature shared by the virulent influenza viruses.     

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.     

Increased viral pathogenicity after insertion of a 28S ribosomal RNA sequence into the haemagglutinin gene of an influenza virus

The haemagglutinin glycoprotein HA of influenza viruses is responsible for the attachment of the virus to neuraminic acid-containing receptors at the cell surface and subsequent penetration by triggering fusion of the viral envelope with cellular membranes. To express full activity of the newly synthesized precursor, HA has to be modified by post-translational proteolytic cleavage into the polypeptides HA1 and HA2 by cellular enzymes. If proteases suitable for cleavage are not present in the host cell, the resulting virus particles are non-infectious. During adaptation of the apathogenic influenza virus A/turkey/Oregon/71 to chicken embryo cells, which are not permissive for HA cleavage, we obtained an infectious virus variant with increased pathogenicity. Sequence analysis revealed that during adaptation 54 nucleotides were inserted into the HA gene; their sequence corresponds to a region of the 28S ribosomal RNA. This insertion is probably responsible for increased cleavability of HA, as well as for infectivity and pathogenicity of the adapted virus.     

Molecular virology: was the 1918 pandemic caused by a bird flu?

Taubenberger et al. have sequenced the polymerase genes of the pandemic 'Spanish' influenza A virus of 1918, thereby completing the decoding of the genome of this virus. The authors conclude from these sequences that the virus jumped from birds to humans shortly before the start of the pandemic and that it was not derived from earlier viruses by gene shuffling, a process called reassortment. However, we believe that their evidence does not convincingly support these conclusions and that some of their results even indicate that, on the contrary, the virus evolved in mammals before the pandemic began and that it was a reassortant. In light of this alternative interpretation, we suggest that the current intense surveillance of influenza viruses should be broadened to include mammalian sources.     

Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus-comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus-that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures.     

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.     

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.     

Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.     

Molecular characterization of H1N1 influenza A viruses from human cases in North America

Subtypes of H1N1 influenza virus can be found in humans in North America, while they are also associated with the infection of swine. Characterization of the genotypes of viral strains in human populations is important to understand the source and distribution of viral strains. Genomic and protein sequences of 10 isolates of the 2009 outbreak of influenza A （H1N1） virus in North America were obtained from GenBank database. To characterize the genotypes of these viruses, phylogenetic trees of genes PB2, PB1, PA, HA, NP, NA, NS and M were constructed by Phylip3.67 program and N-Linked glycosylation sites of HA, NA, PB2, NS1 and M2 proteins were analyzed online by NetNGlyc1.0 program. Phylogenetic analysis indicated that these isolates are virtually identical but may be recombinant viruses because their genomic fragments come from different viruses. The isolates also contain a characteristic lowly pathogenic amino acid motif at their HA cleavage sites （IPSIQSR↓GL）, and an E residue at position 627 of the PB2 protein which shows its high affinity to humans. The homologous model of M proteins showed that the viruses had obtained the ability of anti-amantadine due to the mutation at the drug-sensitive site, while sequence analysis of NA proteins indicated that the viruses are still susceptible to the neuraminidase inhibitor drug （i.e. oseltamivir and zanamivir） because no mutations have been observed. Our results strongly suggested that the viruses responsible for the 2009 outbreaks of influenza A （H1N1） virus have the ability to cross species barriers to infect human and mammalian animals based on molecular analysis. These findings may further facilitate the therapy and prevention of possible transmission from North America to other countries.     

Avian flu: isolation of drug-resistant H5N1 virus

The persistence of H5N1 avian influenza viruses in many Asian countries and their ability to cause fatal infections in humans have raised serious concerns about a global flu pandemic. Here we report the isolation of an H5N1 virus from a Vietnamese girl that is resistant to the drug oseltamivir, which is an inhibitor of the viral enzyme neuraminidase and is currently used for protection against and treatment of influenza. Further investigation is necessary to determine the prevalence of oseltamivir-resistant H5N1 viruses among patients treated with this drug.     

Antigenic epitope peptides of influenza H3N2 virus neuraminidase gene based on experiments

The neuraminidase (NA) in viral surface is one of the main subtype-specific antigen of influenza type A viruses.Neuraminidase is an enzyme to break the bonds between hemagglutinin (HA) and sialic acid to release newly formed viruses from infected cells.In this study,the H3N2 subtype virus NA genes were sequenced and NA proteins were screened for B-cell epitopes and assessed based on immunoinformatics.Based on this results,four peptides DR6,EY7,VG8 and RE8 (covering amino acid residues 151-156,368-374,398-405 and 428-435,respectively) of the NA protein were synthesized artificially.These peptides were used to immunize New Zealand rabbits subcutaneously to raise antisera.Experimental results showed that these four peptides were capable of eliciting antibodies against H3N2 viruses in a specific and sensitive feature,detected in vitro by enzyme-linked immunosorbent assay.Moreover,hemadsorption anti-releasing effects took place in three three-antisera-mixtures at a dilution of 1:40.Alignment using NA gene database showed that amino acid residues in these four epitope peptides were substituted at specific sites in all the NAs sequenced in this study.It was suggested that these NA epitope peptides might be used in combination with HA proteins as vaccine antigens.     

Molecular virology: was the 1918 flu avian in origin?

Taubenberger et al. claim that the 1918 influenza virus was derived from an avian source and adapted to humans shortly before the pandemic. However, we do not believe that this conclusion, which has been widely disseminated in the popular press and in scientific journals, is supported by their phylogenetic evidence.     

Isolation and characterization of H7N9 viruses from live poultry markets--Implication of the source of current H7N9 infection in humans

On March 31, 2013, the National Health and Family Planning Commission announced that human infections with a previously undescribed influenza A (H7N9) virus had occurred in Shanghai and Anhui Province, China. To investigate the possible origins of the H7N9 viruses causing these human infections, we collected 970 samples, including drinking water, soil, and cloacal and tracheal swabs of poultry from live poultry markets and poultry farms in Shanghai and Anhui Province. Twenty samples were positive for the H7N9 influenza virus. Notably, all 20 viruses were isolated from samples collected from live poultry markets in Shanghai. Phylogenetic analyses showed that the six internal genes of these novel human H7N9 viruses were derived from avian H9N2 viruses, but the ancestor of their HA and NA genes is uncertain. When we examined the phylogenetic relationship between the H7N9 isolates from live poultry markets and the viruses that caused the human infections, we found that they shared high homology across all eight gene segments. We thus identified the direct avian origin of the H7N9 influenza viruses that caused the human infections. Importantly, we observed that the H7N9 viruses isolated from humans had acquired critical mutations that made them more "human-like". It is therefore imperative to take strong measures to control the spread of H7N9 viruses in birds and humans to prevent further threats to human health.     

流感病毒致病的分子基础

自然感染，流感病毒感染的宿主范围有较强的特异性，各毒株所表现的毒力也所不同，决定流感病毒宿主特异性和毒力的因素很多，本文主要从流感病毒的分子水平上来揭示流感病毒的宿主特异性及其毒力差异。     

Recovery of immunodeficient mice from a vaccinia virus/IL-2 recombinant infection

Vaccinia virus recombinants that express cloned genes encoding antigens of unrelated infectious agents, such as hepatitis B virus and human immunodeficiency virus (HIV), provide a new approach to the development of live vaccines. Although there is evidence that genetically engineered vaccinia viruses have reduced pathogenicity a major obstacle to their use as vaccines is that severe complications can occur after vaccination, especially in immunodeficient individuals. We describe here a recombinant vaccinia virus expressing murine interleukin-2 (IL-2) and show that athymic nude mice infected with the recombinant virus resolve the virus infection rapidly whereas mice infected with control virus develop a progressive vaccinal disease. By incorporating the gene for IL-2 in live virus vaccines it may be possible to prevent the severe complications that arise in recipients with an impaired immune system.     

KMB17细胞培养流感病毒的实验研究

 探讨了流感病毒在KMB₁₇细胞上培养的可行性.将3株不同型别的流感病毒(A/Wisconsin/67/2005H3N2,A/New Caledonia/20/1999 H1N1和B/Malaysia/2506/2004)分别接种于KMB₁₇细胞上,在无血清的条件下于不同pH值和不同胰酶浓度的维持液中35℃培养72 h后收获病毒,测定血凝效价.实验结果表明,所用的3个毒株中的H3N2亚型毒株(A/Wisconsin/67/2005 H3N2)血凝效价明显高于另外的H1N1亚型(A/NewCaledonia/20/1999 H1N1)和B型(B/Malaysia/2506/2004)毒株.因此认为KMB₁₇细胞可作为培养流感病毒的备选培养基质.