Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts.

The nucleotide and amino acid sequences of 40 influenza virus hemagglutinin genes of the H3 serotype from mammalian and avian species and 9 genes of the H4 serotype were compared, and their evolutionary relationships were evaluated. From these relationships, the differences in the mutational characteristics of the viral hemagglutinin in different hosts were examined and the RNA sequence changes that occurred during the generation of the progenitor of the 1968 human pandemic strain were examined. Three major lineages were defined: one containing only equine virus isolates; one containing only avian virus isolates; and one containing avian, swine, and human virus isolates. The human pandemic strain of 1968 was derived from an avian virus most similar to those isolated from ducks in Asia, and the transfer of this virus to humans probably occurred in 1965. Since then, the human viruses have diverged from this progenitor, with the accumulation of approximately 7.9 nucleotide and 3.4 amino acid substitutions per year. Reconstruction of the sequence of the hypothetical ancestral strain at the avian-human transition indicated that only 6 amino acids in the mature hemagglutinin molecule were changed during the transition between an avian virus strain and a human pandemic strain. All of these changes are located in regions of the molecule known to affect receptor binding and antigenicity. Unlike the human H3 influenza virus strains, the equine virus isolates have no close relatives in other species and appear to have diverged from the avian viruses much earlier than did the human virus strains. Mutations were estimated to have accumulated in the equine virus lineage at approximately 3.1 nucleotides and 0.8 amino acids per year. Four swine virus isolates in the analysis each appeared to have been introduced into pigs independently, with two derived from human viruses and two from avian viruses. A comparison of the coding and noncoding mutations in the mammalian and avian lineages showed a significantly lower ratio of coding to total nucleotide changes in the avian viruses. Additionally, the avian virus lineages of both the H3 and H4 serotypes, but not the mammalian virus lineages, showed significantly greater conservation of amino acid sequence in the internal branches of the phylogenetic tree than in the terminal branches. The small number of amino acid differences between the avian viruses and the progenitor of the 1968 pandemic strain and the great phenotypic stability of the avian viruses suggest that strains similar to the progenitor strain will continue to circulate in birds and will be available for reintroduction into humans.     

Gene Expression Signature-Based Screening Identifies New Broadly Effective Influenza A Antivirals

Classical antiviral therapies target viral proteins and are consequently subject to resistance. To counteract this limitation, alternative strategies have been developed that target cellular factors. We hypothesized that such an approach could also be useful to identify broad-spectrum antivirals. The influenza A virus was used as a model for its viral diversity and because of the need to develop therapies against unpredictable viruses as recently underlined by the H1N1 pandemic. We proposed to identify a gene-expression signature associated with infection by different influenza A virus subtypes which would allow the identification of potential antiviral drugs with a broad anti-influenza spectrum of activity. We analyzed the cellular gene expression response to infection with five different human and avian influenza A virus strains and identified 300 genes as differentially expressed between infected and non-infected samples. The most 20 dysregulated genes were used to screen the connectivity map, a database of drug-associated gene expression profiles. Candidate antivirals were then identified by their inverse correlation to the query signature. We hypothesized that such molecules would induce an unfavorable cellular environment for influenza virus replication. Eight potential antivirals including ribavirin were identified and their effects were tested in vitro on five influenza A strains. Six of the molecules inhibited influenza viral growth. The new pandemic H1N1 virus, which was not used to define the gene expression signature of infection, was inhibited by five out of the eight identified molecules, demonstrating that this strategy could contribute to identifying new broad anti-influenza agents acting on cellular gene expression. The identified infection signature genes, the expression of which are modified upon infection, could encode cellular proteins involved in the viral life cycle. This is the first study showing that gene expression-based screening can be used to identify antivirals. Such an approach could accelerate drug discovery and be extended to other pathogens.     

Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus

Annual outbreaks of influenza A infection are an ongoing public health threat and novel influenza strains can periodically emerge to which humans have little immunity, resulting in devastating pandemics. The 1918 pandemic killed at least 40 million people worldwide and pandemics in 1957 and 1968 caused hundreds of thousands of deaths. The influenza A virus is capable of enormous genetic variation, both by continuous, gradual mutation and by reassortment of genome segments between viruses. Both the 1957 and 1968 pandemic strains are thought to have originated as reassortants in which one or both human-adapted viral surface proteins were replaced by proteins from avian influenza strains. Analyses of the genes of the 1918 pandemic virus, however, indicate that this strain might have had a different origin. The haemagglutinin and nucleoprotein genome segments in particular are unlikely to have come directly from an avian source that is similar to those that are currently being sequenced. Determining whether a pandemic influenza virus can emerge by different mechanisms will affect the scope and focus of surveillance and prevention efforts.     

Analysis of Adaptation Mutants in the Hemagglutinin of the Influenza A(H1N1)pdm09 Virus

Hemagglutinin is the major surface glycoprotein of influenza viruses. It participates in the initial steps of viral infection through receptor binding and membrane fusion events. The influenza pandemic of 2009 provided a unique scenario to study virus evolution. We performed molecular dynamics simulations with four hemagglutinin variants that appeared throughout the 2009 influenza A (H1N1) pandemic. We found that variant 1 (S143G, S185T) likely arose to avoid immune recognition. Variant 2 (A134T), and variant 3 (D222E, P297S) had an increased binding affinity for the receptor. Finally, variant 4 (E374K) altered hemagglutinin stability in the vicinity of the fusion peptide. Variants 1 and 4 have become increasingly predominant, while variants 2 and 3 declined as the pandemic progressed. Our results show some of the different strategies that the influenza virus uses to adapt to the human host and provide an example of how selective pressure drives antigenic drift in viral proteins.     

Evolution and ecology of influenza A viruses.

In this review we examine the hypothesis that aquatic birds are the primordial source of all influenza viruses in other species and study the ecological features that permit the perpetuation of influenza viruses in aquatic avian species. Phylogenetic analysis of the nucleotide sequence of influenza A virus RNA segments coding for the spike proteins (HA, NA, and M2) and the internal proteins (PB2, PB1, PA, NP, M, and NS) from a wide range of hosts, geographical regions, and influenza A virus subtypes support the following conclusions. (i) Two partly overlapping reservoirs of influenza A viruses exist in migrating waterfowl and shorebirds throughout the world. These species harbor influenza viruses of all the known HA and NA subtypes. (ii) Influenza viruses have evolved into a number of host-specific lineages that are exemplified by the NP gene and include equine Prague/56, recent equine strains, classical swine and human strains, H13 gull strains, and all other avian strains. Other genes show similar patterns, but with extensive evidence of genetic reassortment. Geographical as well as host-specific lineages are evident. (iii) All of the influenza A viruses of mammalian sources originated from the avian gene pool, and it is possible that influenza B viruses also arose from the same source. (iv) The different virus lineages are predominantly host specific, but there are periodic exchanges of influenza virus genes or whole viruses between species, giving rise to pandemics of disease in humans, lower animals, and birds. (v) The influenza viruses currently circulating in humans and pigs in North America originated by transmission of all genes from the avian reservoir prior to the 1918 Spanish influenza pandemic; some of the genes have subsequently been replaced by others from the influenza gene pool in birds. (vi) The influenza virus gene pool in aquatic birds of the world is probably perpetuated by low-level transmission within that species throughout the year. (vii) There is evidence that most new human pandemic strains and variants have originated in southern China. (viii) There is speculation that pigs may serve as the intermediate host in genetic exchange between influenza viruses in avian and humans, but experimental evidence is lacking. (ix) Once the ecological properties of influenza viruses are understood, it may be possible to interdict the introduction of new influenza viruses into humans.     

Evolutional analysis of human influenza A virus N2 neuraminidase genes based on the transition of the low-pH stability of sialidase activity

The 1957 and 1968 human pandemic influenza A virus strains as well as duck viruses possess sialidase activity under low-pH conditions, but human H3N2 strains isolated after 1968 do not possess such activity. We investigated the transition of avian (duck)-like low-pH stability of sialidase activities with the evolution of N2 neuraminidase (NA) genes in human influenza A virus strains. We found that the NA genes of H3N2 viruses isolated from 1971 to 1982 had evolved from the side branches of NA genes of H2N2 epidemic strains isolated in 1968 that were characterized by the low-pH-unstable sialidase activities, though the NA genes of the 1968 pandemic strains preserved the low-pH-stable sialidase. These findings suggest that the prototype of the H3N2 epidemic influenza strains isolated after 1968 probably acquired the NA gene from the H2N2 low-pH-unstable sialidase strain by second genetic reassortment in humans.     

Baculovirus Displaying Hemagglutinin Elicits Broad Cross-Protection against Influenza in Mice

The widespread influenza virus infection further emphasizes the need for novel vaccine strategies that effectively reduce the impact of epidemic as well as pandemic influenza. Conventional influenza vaccines generally induce virus neutralizing antibody responses which are specific for a few antigenically related strains within the same subtype. However, antibodies directed against the conserved stalk domain of HA could neutralize multiple subtypes of influenza virus and thus provide broad-spectrum protection. In this study, we designed and constructed a recombinant baculovirus-based vaccine, rBac-HA virus, that expresses full-length HA of pandemic H1N1 influenza virus (A/California/04/09) on the viral envelope. We demonstrated that repeated intranasal immunizations with rBac-HA virus induced HA stalk-specific antibody responses and protective immunity against homologous as well as heterosubtypic virus challenge. The adoptive transfer experiment shows that the cross-protection is conferred by the immune sera which contain HA stalk-specific antibodies. These results warrant further development of rBac-HA virus as a broad-protective vaccine against influenza. The vaccine induced protection against infection with the same subtype as well as different subtype, promising a potential universal vaccine for broad protection against different subtypes to control influenza outbreaks including pandemic.     

Manifestations of influenza-virus variability during its cultivation in the system of passively immunized chick embryos]

A method of immunological actions on the influenza virus in the system of chick embryos passively immunized with isologous sera was elaborated. With the aid of the suggested method it was possible to induce in strains (with different hemagglutinin type and different degree of attenuation) the transformation of signs characterizing the virus variability in the epidemic process. Variants of the vaccine strains of the influenza virus with increased immunogenic activity and signs of antigenic "outrunning" were obtained. Experimental vaccines from these variants were nonreactogenic for humans and caused a more frequent seroconversion than the initial strain. The diagnostic agents from the variants revealed antibodies in human sera and gamma-globulin, similarly to the "new" viruses isolated from man. The theoretical significance and the aspects of practical utilization of the data obtained are discussed.     

Influenza Nucleoprotein Delivered with Aluminium Salts Protects Mice from an Influenza A Virus That Expresses an Altered Nucleoprotein Sequence

Influenza virus poses a difficult challenge for protective immunity. This virus is adept at altering its surface proteins, the proteins that are the targets of neutralizing antibody. Consequently, each year a new vaccine must be developed to combat the current recirculating strains. A universal influenza vaccine that primes specific memory cells that recognise conserved parts of the virus could prove to be effective against both annual influenza variants and newly emergent potentially pandemic strains. Such a vaccine will have to contain a safe and effective adjuvant that can be used in individuals of all ages. We examine protection from viral challenge in mice vaccinated with the nucleoprotein from the PR8 strain of influenza A, a protein that is highly conserved across viral subtypes. Vaccination with nucleoprotein delivered with a universally used and safe adjuvant, composed of insoluble aluminium salts, provides protection against viruses that either express the same or an altered version of nucleoprotein. This protection correlated with the presence of nucleoprotein specific CD8 T cells in the lungs of infected animals at early time points after infection. In contrast, immunization with NP delivered with alum and the detoxified LPS adjuvant, monophosphoryl lipid A, provided some protection to the homologous viral strain but no protection against infection by influenza expressing a variant nucleoprotein. Together, these data point towards a vaccine solution for all influenza A subtypes.     

Influenza activity in Czechoslovakia and the USSR, 1980-1985

Between 1980 and 1985, Czechoslovakia had experienced 4 and the USSR 3 major influenza outbreaks. Of the 3 epidemic outbreaks in the USSR, 2 were associated with influenza B virus (in the 1980/81 and 1983/84 seasons) and 1 with influenza A virus of the H3N2 subtype. In the USSR, influenza A (H1N1) virus never predominated as a cause of epidemic during the 5 years period. In Czechoslovakia, 2 epidemics (in the 1980/81 and 1983/84 seasons) were due to influenza A (H1N1) virus. The epidemic in the 1981/82 season had two waves of unequal heights and a mixed type B and subtype A (H3N2) etiology; a two-wave epidemic associated with isolates of influenza A (H1N1) and influenza B viruses was also recorded in the 1983/84 season. The influenza A (H3N2) epidemic in 1983 was of explosive character. All influenza viruses circulating in the two countries between 1980 and 1985 were of the same antigenic profile, but were isolated from the epidemics that occurred in different influenza seasons. The virological surveillance revealed strains of virus closely related to drift variants detected from outbreaks in 1977-1979 and the new variants A/Chile 1/83, A/Philippines 2/82, A/Caen 1/84 and B/USSR 100/83.     

Predicting the Emergence of H3N2 Influenza Viruses Reveals Contrasted Modes of Evolution of HA and NA Antigens

Vaccine design for rapidly changing viruses is based on empirical surveillance of strains circulating in a given season to assess those that will most likely spread during the next season. The choice of which strains to include in the vaccine is critical, as an erroneous decision can lead to a nonimmunized human population that will then be at risk in the face of an epidemic or, worse, a pandemic. Here, we present the first steps toward a very general phylogenetic approach to predict the emergence of novel viruses. Our genomic model builds upon natural features of viral evolution such as selection and recombination / reassortment, and incorporates episodic bursts of evolution and or of recombination. As a proof-of-concept, we assess the performance of this model in a retrospective study, focusing: (i) on the emergence of an unexpected H3N2 influenza strain in 2007, and (ii) on a longitudinal design. Based on the analysis of hemagglutinin (HA) and neuraminidase (NA) genes, our results show a lack of predictive power in both experimental designs, but shed light on the mode of evolution of these two antigens: (i) supporting the lack of significance of recombination in the evolution of this influenza virus, and (ii) showing that HA evolves episodically while NA changes gradually.     

Flushuffle and Fluresort: new algorithms to identify reassorted strains of the influenza virus by mass spectrometry

ABSTRACT: BACKGROUND: Influenza is one of the oldest and deadliest infectious diseases known to man. Reassorted strains of the virus pose the greatest risk to both human and animal health and have been associated with all pandemics of the past century, with the possible exception of the 1918 pandemic, resulting in tens of millions of deaths. We have developed and tested new computer algorithms, FluShuffle and FluResort, which enable reassorted viruses to be identified by the most rapid and direct means possible. These algorithms enable reassorted influenza, and other, viruses to be rapidly identified to allow prevention strategies and treatments to be more efficiently implemented. RESULTS: The FluShuffle and FluResort algorithms were tested with both experimental and simulated mass spectra of whole virus digests. Flu Shuffle considers different combinations of viral protein identities that match the mass spectral data using a Gibbs sampling algorithm employing a mixed protein Markov chain Monte Carlo (MCMC) method. Flu Resort utilizes those identities to calculate the weighted distance of each across two or more different phylogenetic trees constructed through viral protein sequence alignments. Each weighted mean distance value is normalized by conversion to a Z-score to establish a reassorted strain. CONCLUSIONS: The new Flu Shuffle and Flu Resort algorithms can correctly identify the origins of influenza viral proteins and the number of reassortment events required to produce the strains from the high resolution mass spectral data of whole virus proteolytic digestions. This has been demonstrated in the case of constructed vaccine strains as well as common human seasonal strains of the virus. The algorithms significantly improve the capability of the proteotyping approach to identify reassorted viruses that pose the greatest pandemic risk.     

Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

The influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.     

Distinct Host Tropism Protein Signatures to Identify Possible Zoonotic Influenza A Viruses

Zoonotic influenza A viruses constantly pose a health threat to humans as novel strains occasionally emerge from the avian population to cause human infections. Many past epidemic as well as pandemic strains have originated from avian species. While most viruses are restricted to their primary hosts, zoonotic strains can sometimes arise from mutations or reassortment, leading them to acquire the capability to escape host species barrier and successfully infect a new host. Phylogenetic analyses and genetic markers are useful in tracing the origins of zoonotic infections, but there are still no effective means to identify high risk strains prior to an outbreak. Here we show that distinct host tropism protein signatures can be used to identify possible zoonotic strains in avian species which have the potential to cause human infections. We have discovered that influenza A viruses can now be classified into avian, human, or zoonotic strains based on their host tropism protein signatures. Analysis of all influenza A viruses with complete proteome using the host tropism prediction system, based on machine learning classifications of avian and human viral proteins has uncovered distinct signatures of zoonotic strains as mosaics of avian and human viral proteins. This is in contrast with typical avian or human strains where they show mostly avian or human viral proteins in their signatures respectively. Moreover, we have found that zoonotic strains from the same influenza outbreaks carry similar host tropism protein signatures characteristic of a common ancestry. Our results demonstrate that the distinct host tropism protein signature in zoonotic strains may prove useful in influenza surveillance to rapidly identify potential high risk strains circulating in avian species, which may grant us the foresight in anticipating an impending influenza outbreak.     

Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.     

Evolution of human influenza A viruses in nature: sequential mutations in the genomes of new H1N1

The genetic variation of the new pandemic H1N1 influenza A viruses isolated in 1977 was analyzed by two-dimensional oligonucleotide fingerprinting and RNA sequencing. Differences were observed in the fingerprints of the RNAs of these viruses, and analysis of the changes suggested that sequential mutations occurred in their genomes. Based on these data, a scheme is presented which proposes divergent evolution of strains from a common ancestry. Furthermore, it was found that mutations were not restricted to the genes coding for the hemagglutinin and the neuraminidase, but were scattered throughout the genome, suggesting that selective antibody pressure is not solely responsible for the emergence of genetic variants. Our data also strengthen the hypothesis that the new H1N1 influenza virus strains are derived from strains circulating in 1950.     

Exploring the Molecular Epidemiology and Evolutionary Dynamics of Influenza A Virus in Taiwan

The evolution and population dynamics of human influenza in Taiwan is a microcosm of the viruses circulating worldwide, which has not yet been studied in detail. We collected 343 representative full genome sequences of human influenza A viruses isolated in Taiwan between 1979 and 2009. Phylogenetic and antigenic data analysis revealed that H1N1 and H3N2 viruses consistently co-circulated in Taiwan, although they were characterized by different temporal dynamics and degrees of genetic diversity. Moreover, influenza A viruses of both subtypes underwent internal gene reassortment involving all eight segments of the viral genome, some of which also occurred during non-epidemic periods. The patterns of gene reassortment were different in the two subtypes. The internal genes of H1N1 viruses moved as a unit, separately from the co-evolving HA and NA genes. On the other hand, the HA and NA genes of H3N2 viruses tended to segregate consistently with different sets of internal gene segments. In particular, as reassortment occurred, H3HA always segregated as a group with the PB1, PA and M genes, while N2NA consistently segregated with PB2 and NP. Finally, the analysis showed that new phylogenetic lineages and antigenic variants emerging in summer were likely to be the progenitors of the epidemic strains in the following season. The synchronized seasonal patterns and high genetic diversity of influenza A viruses observed in Taiwan make possible to capture the evolutionary dynamic and epidemiological rules governing antigenic drift and reassortment and may serve as a “warning” system that recapitulates the global epidemic.     

Population interactions in biological system: influenza virus A--wild and domestic animals--human; reasons and consequences of introduction high pathogenic influenza virus A/H5N1 on Russian territory

Own results and literature data under the analysis of variability of a influenza virus A and population interactions in biological system: influenza virus A--wild and domestic animals--human are presented. Opportunities of virus reassortants appearance between viruses of animals and viruses of human and spreading the new pandemic variants are proved, in particular, at simultaneous infection of pigs with virus H5 and viruses of human influenza H1 and H3, circulating worldwide. Blocks of preparation to a flu pandemic, including on vaccine prophylactic are stated. It is discussed, what vaccines it is necessary to prepare. Inadmissibility the use of alive influenza vaccines is emphasized.     

Impact of host cell line adaptation on quasispecies composition and glycosylation of influenza A virus hemagglutinin

The genome of influenza A viruses is constantly changing (genetic drift) resulting in small, gradual changes in viral proteins. Alterations within antibody recognition sites of the viral membrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) result in an antigenetic drift, which requires the seasonal update of human influenza virus vaccines. Generally, virus adaptation is necessary to obtain sufficiently high virus yields in cell culture-derived vaccine manufacturing. In this study detailed HA N-glycosylation pattern analysis was combined with in-depth pyrosequencing analysis of the virus genomic RNA. Forward and backward adaptation from Madin-Darby Canine Kidney (MDCK) cells to African green monkey kidney (Vero) cells was investigated for two closely related influenza A virus PR/8/34 (H1N1) strains: from the National Institute for Biological Standards and Control (NIBSC) or the Robert Koch Institute (RKI). Furthermore, stability of HA N-glycosylation patterns over ten consecutive passages and different harvest time points is demonstrated. Adaptation to Vero cells finally allowed efficient influenza A virus replication in Vero cells. In contrast, during back-adaptation the virus replicated well from the very beginning. HA N-glycosylation patterns were cell line dependent and stabilized fast within one (NIBSC-derived virus) or two (RKI-derived virus) successive passages during adaptation processes. However, during adaptation new virus variants were detected. These variants carried "rescue" mutations on the genomic level within the HA stem region, which result in amino acid substitutions. These substitutions finally allowed sufficient virus replication in the new host system. According to adaptation pressure the composition of the virus populations varied. In Vero cells a selection for "rescue" variants was characteristic. After back-adaptation to MDCK cells some variants persisted at indifferent frequencies, others slowly diminished and even dropped below the detection limit.