What is the number of influenza strains occurring at a given time?

What is the number of influenza strains occurring at a given time?

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My question was initiated by reading on mock-up influenza vaccines. I understand that the manufacturer pre-prepares a certain vaccine and tries to get it tested and ready before it is actually needed, by taking a novel virus strain that has infected a few people, and could potentially cause a pandemic.

My questions:

  • How many different influenza strains are there at any given time at one geographical spot (say, one US state? - narrow down at will). I always thought there would be just one novel strain appearing each year, explaining why people get sick time and again of the 'same' virus. However, the mock-up virus vaccine production seems to imply there are various strains occurring at once?

  • Do influenza strains vary on an annual basis, or is it a continuous process (like evolution), and is a new strain re-named after it becomes potentially dangerous (like speciation?) - i.e., what is an influenza strain? It seems, given the vaccine preps, that various strains occur at once, and strains survive for extended periods? Any hard evidence for these indirect assumptions?

And as a side -

  • Do vaccine manufacturers prepare a whole bunch of mock-up influenza vaccines on a yearly basis just awaiting to identify a dangerous strain and start manufacturing it once people get sick? Or are mock-up vaccines combined into a cocktail mix containing many strains and are these cocktails re-vamped annually? Or is a yearly shot necessary because immunity only lasts a year?
    I might have to re-post this last question at Health.SE as a dedicated question, but since related to above I prefer to leave it here for now. Grab me in chat if need be!

Antigenic drift in the coding portions of the genes that involve antibody binding sites accounts for the seasonal variability of flu strains, i.e. they mutated enough that the antigens require new antibodies, hence a new strain. The process is as you said constant, like evolution, and in an analysis of pandemic H1N1 the authors are able to note 1 - 18.1 mutations per genome per replication cycle (ref). Because of this, flu vaccines tend to have more A strains than B strains due to the greater mutability of influenza A, at approx. 30% more mutations per site per infectious cycle (ref). At current It will take me time to research the remaining questions.

Interestingly enough, however, and I've never looked at this but CDC offers weekly influenza reports that detail by region what they're finding (CDC):

Source: CDC's so-called FluView

Beyond that, I'm pretty sure they take this weekly data, see what strain is on the rise in hospitals, and manufacture the vaccine to mirror the "popular" cocktail of strains.

In April 2009, the world was hit with a swine flu pandemic. The Centers for Disease Control estimates that within that first year, 43 to 89 million people worldwide contracted the swine flu and that it contributed to 8,870 to 18,300 deaths. Some people with swine flu were spared serious complications, such as Mateo, who you read about it at the beginning of this chapter. At the time, the swine flu spread rapidly because as a newly evolved viral strain, most people had no natural immunity against it, and the existing flu vaccine could not prevent it. But by November 2009, a swine flu vaccine was developed, and now it is included in the annual flu vaccine in the U.S. By August 2010, the World Health Organization declared the H1N1 swine flu pandemic to be over. The virus is still around, but because of the vaccine and the natural immunity of those who had the virus previously, its infection rate is no longer of pandemic proportions.

The swine flu virus appears to have originated in pigs and later evolved the ability to infect humans. How could this happen? Scientists think that a process called reassortment played a critical role. In reassortment, influenza viruses can exchange genetic material with each other if they have infected the same cells. This creates new combinations of genes, somewhat similar to the genetic mixing that occurs in sexual reproduction when two parents with different genes reproduce with each other. As you know, genes help dictate the characteristics of an organism, or in this case, a virus. Therefore, the production of novel combinations of genes due to viral reassortment can lead to the evolution of new viral characteristics.

In addition to reassortment, influenza viruses have other characteristics that cause them to evolve quickly. In contrast to sexual reproduction, the replication of viruses to produce new &ldquooffspring&rdquo particles is much more rapid. As you have learned in this chapter, evolution is typically a slow process that takes place over many generations. But if these generations are produced rapidly, as in the case of viruses and bacteria, it speeds the rate of evolution. Additionally, RNA viruses have a very high rate of genetic mutation. The rapid evolution of the influenza virus is one of the reasons why the annual seasonal flu vaccine is not always effective against every strain.

But why did this flu pandemic come from pigs? Pigs are actually an ideal &ldquomixing bowl&rdquo for the evolution of influenza viruses because pigs can become infected with influenza viruses from other species, including birds and humans. Therefore, genetic reassortment can occur in pigs between viral strains that normally infect different species. This is what scientists think occurred to produce the 2009 H1N1 swine flu virus. The 2009 H1N1 has gene segments from the birds, humans, and two different pig influenza viruses, and is therefore called a &ldquoquadruple reassortant&rdquo virus. In the case of the 2009 H1N1, this resulted in a new influenza strain that could infect humans, and be passed directly from person to person.

Figure (PageIndex<1>): Different viruses that infect pig may combine the pieces of their genetic material to make a new virus

Scientists do not know exactly when and where the 2009 H1N1 evolved, but they think that the reassortment event may have occurred several years prior to the 2009 pandemic. This is based on evidence gathered from &ldquomolecular evolution&rdquo techniques, which are similar to the molecular clock technique described in this chapter. Influenza viruses are known to mutate at a relatively steady rate. The genetic sequences of the new 2009 H1N1 strain were compared to the sequences in related, older influenza viruses to count the number of new mutations, in order to give an estimate of when the new viral strain evolved.

Probably one of the final events that resulted in the generation of the 2009 H1N1 virus was contact between North American and Eurasian pigs. This is because prior to 2009, there were &ldquotriple reassortant&rdquo variants of H1N1 with gene segments from a bird, human, and North American pig influenza already in existence. The 2009 H1N1 strain additionally contained gene segments from influenza from Eurasian pigs, resulting in the &ldquoquadruple reassortant&rdquo virus. Scientists think that contact between pigs from these different regions, through international trade or other methods of contact, could have created this new strain. As you have learned in this chapter, the migration of organisms to new locations as well as contact between different organisms can influence evolution in many ways. Some examples are the migration of ancestral camels throughout the world, the coevolution of flowers and their pollinators, and the &ldquofounder effect&rdquo of small populations that move to new locations, such as the Amish.

Along with fossils, comparative anatomy and embryology, DNA analysis, and biogeography, evidence for evolution includes direct observation of it occurring. Peter and Rosemary Grant observed evolution occurring in the change in beak size of Galápagos finches. The evolution of the swine flu virus is another example of evolution in action. Evolution is not just a thing of the past &mdash it is an ongoing and important process that affects our ecosystem, species, and even our health. Like viruses, bacteria also evolve rapidly, and the evolution of antibiotic resistance in bacteria is a growing public health concern. You can see that evolution is very relevant to our lives today.


Influenza type A viruses are RNA viruses categorized into subtypes based on the type of two proteins on the surface of the viral envelope:

The hemagglutinin is central to the virus's recognizing and binding to target cells, and also to its then infecting the cell with its RNA. The neuraminidase, on the other hand, is critical for the subsequent release of the daughter virus particles created within the infected cell so they can spread to other cells.

Different influenza viruses encode for different hemagglutinin and neuraminidase proteins. For example, the H5N1 virus designates an influenza A subtype that has a type 5 hemagglutinin (H) protein and a type 1 neuraminidase (N) protein. There are 18 known types of hemagglutinin and 11 known types of neuraminidase, so, in theory, 198 different combinations of these proteins are possible. [5] [6]

Some variants are identified and named according to the isolate they resemble, thus are presumed to share lineage (example Fujian flu virus-like) according to their typical host (example human flu virus) according to their subtype (example H3N2) and according to their deadliness (example LP, low pathogenic). So a flu from a virus similar to the isolate A/Fujian/411/2002(H3N2) is called Fujian flu, human flu, and H3N2 flu.

Variants are sometimes named according to the species (host) in which the strain is endemic or to which it is adapted. The main variants named using this convention are:

Variants have also sometimes been named according to their deadliness in poultry, especially chickens:

  • Low pathogenic avian influenza (LPAI)
  • Highly pathogenic avian influenza (HPAI), also called deadly flu or death flu

Most known strains are extinct strains. For example, the annual flu subtype H3N2 no longer contains the strain that caused the Hong Kong flu.

The annual flu (also called "seasonal flu" or "human flu") in the US. "results in approximately 36,000 deaths and more than 200,000 hospitalizations each year. In addition to this human toll, influenza is annually responsible for a total cost of over $10 billion in the U.S." [10] Globally the toll of influenza virus is estimated at 290,000–645,000 deaths annually, exceeding previous estimates. [11]

The annually updated, trivalent influenza vaccine consists of hemagglutinin (HA) surface glycoprotein components from influenza H3N2, H1N1, and B influenza viruses. [12]

Measured resistance to the standard antiviral drugs amantadine and rimantadine in H3N2 has increased from 1% in 1994 to 12% in 2003 to 91% in 2005.

"Contemporary human H3N2 influenza viruses are now endemic in pigs in southern China and can reassort with avian H5N1 viruses in this intermediate host." [13]

FI6 antibody Edit

FI6, an antibody that targets the hemagglutinin protein, was discovered in 2011. FI6 is the only known antibody effective against all 16 subtypes of the influenza A virus. [14] [15] [16]

Influenza type A viruses are very similar in structure to influenza viruses types B, C, and D. [19] The virus particle (also called the virion) is 80–120 nanometers in diameter such that the smallest virions adopt an elliptical shape. [20] [18] The length of each particle varies considerably, owing to the fact that influenza is pleomorphic, and can be in excess of many tens of micrometers, producing filamentous virions. [21] Confusion about the nature of influenza virus pleomorphy stems from the observation that lab adapted strains typically lose the ability to form filaments [22] and that these lab adapted strains were the first to be visualized by electron microscopy. [23] Despite these varied shapes, the virions of all influenza type A viruses are similar in composition. They are all made up of a viral envelope containing two main types of proteins, wrapped around a central core. [24]

The two large proteins found on the outside of viral particles are hemagglutinin (HA) and neuraminidase (NA). HA is a protein that mediates binding of the virion to target cells and entry of the viral genome into the target cell. NA is involved in release from the abundant non-productive attachment sites present in mucus [25] as well as the release of progeny virions from infected cells. [26] These proteins are usually the targets for antiviral drugs. [27] Furthermore, they are also the antigen proteins to which a host's antibodies can bind and trigger an immune response. Influenza type A viruses are categorized into subtypes based on the type of these two proteins on the surface of the viral envelope. There are 16 subtypes of HA and 9 subtypes of NA known, but only H 1, 2 and 3, and N 1 and 2 are commonly found in humans. [28]

The central core of a virion contains the viral genome and other viral proteins that package and protect the genetic material. Unlike the genomes of most organisms (including humans, animals, plants, and bacteria) which are made up of double-stranded DNA, many viral genomes are made up of a different, single-stranded nucleic acid called RNA. Unusually for a virus, though, the influenza type A virus genome is not a single piece of RNA instead, it consists of segmented pieces of negative-sense RNA, each piece containing either one or two genes which code for a gene product (protein). [24] The term negative-sense RNA just implies that the RNA genome cannot be translated into protein directly it must first be transcribed to positive-sense RNA before it can be translated into protein products. The segmented nature of the genome allows for the exchange of entire genes between different viral strains. [24]

The entire Influenza A virus genome is 13,588 bases long and is contained on eight RNA segments that code for at least 10 but up to 14 proteins, depending on the strain. The relevance or presence of alternate gene products can vary: [29]

  • Segment 1 encodes RNA polymerase subunit (PB2).
  • Segment 2 encodes RNA polymerase subunit (PB1) and the PB1-F2 protein, which induces cell death, by using different reading frames from the same RNA segment.
  • Segment 3 encodes RNA polymerase subunit (PA) and the PA-X protein, which has a role in host transcription shutoff. [30]
  • Segment 4 encodes for HA (hemagglutinin). About 500 molecules of hemagglutinin are needed to make one virion. HA determines the extent and severity of a viral infection in a host organism.
  • Segment 5 encodes NP, which is a nucleoprotein.
  • Segment 6 encodes NA (neuraminidase). About 100 molecules of neuraminidase are needed to make one virion.
  • Segment 7 encodes two matrix proteins (M1 and M2) by using different reading frames from the same RNA segment. About 3,000 matrix protein molecules are needed to make one virion.
  • Segment 8 encodes two distinct non-structural proteins (NS1 and NEP) by using different reading frames from the same RNA segment.

The RNA segments of the viral genome have complementary base sequences at the terminal ends, allowing them to bond to each other with hydrogen bonds. [26] Transcription of the viral (-) sense genome (vRNA) can only proceed after the PB2 protein binds to host capped RNAs, allowing for the PA subunit to cleave several nucleotides after the cap. This host-derived cap and accompanied nucleotides serve as the primer for viral transcription initiation. Transcription proceeds along the vRNA until a stretch of several uracil bases is reached, initiating a 'stuttering' whereby the nascent viral mRNA is poly-adenylated, producing a mature transcript for nuclear export and translation by host machinery. [31]

The RNA synthesis takes place in the cell nucleus, while the synthesis of proteins takes place in the cytoplasm. Once the viral proteins are assembled into virions, the assembled virions leave the nucleus and migrate towards the cell membrane. [32] The host cell membrane has patches of viral transmembrane proteins (HA, NA, and M2) and an underlying layer of the M1 protein which assist the assembled virions to budding through the membrane, releasing finished enveloped viruses into the extracellular fluid. [32]

The subtypes of influenza A virus are estimated to have diverged 2,000 years ago. Influenza viruses A and B are estimated to have diverged from a single ancestor around 4,000 years ago, while the ancestor of influenza viruses A and B and the ancestor of influenza virus C are estimated to have diverged from a common ancestor around 8,000 years ago. [33]

Influenza virus is able to undergo multiplicity reactivation after inactivation by UV radiation, [34] [35] or by ionizing radiation. [36] If any of the eight RNA strands that make up the genome contains damage that prevents replication or expression of an essential gene, the virus is not viable when it alone infects a cell (a single infection). However, when two or more damaged viruses infect the same cell (multiple infection), viable progeny viruses can be produced provided each of the eight genomic segments is present in at least one undamaged copy. That is, multiplicity reactivation can occur.

Upon infection, influenza virus induces a host response involving increased production of reactive oxygen species, and this can damage the virus genome. [37] If, under natural conditions, virus survival is ordinarily vulnerable to the challenge of oxidative damage, then multiplicity reactivation is likely selectively advantageous as a kind of genomic repair process. It has been suggested that multiplicity reactivation involving segmented RNA genomes may be similar to the earliest evolved form of sexual interaction in the RNA world that likely preceded the DNA world. [38] (Also see RNA world hypothesis.)

"Human influenza virus" usually refers to those subtypes that spread widely among humans. H1N1, H1N2, and H3N2 are the only known influenza A virus subtypes currently circulating among humans. [39]

Genetic factors in distinguishing between "human flu viruses" and "avian influenza viruses" include:

PB2: (RNA polymerase): Amino acid (or residue) position 627 in the PB2 protein encoded by the PB2 RNA gene. Until H5N1, all known avian influenza viruses had a Glu at position 627, while all human influenza viruses had a lysine. HA: (hemagglutinin): Avian influenza HA binds alpha 2–3 sialic acid receptors, while human influenza HA binds alpha 2–6 sialic acid receptors. Swine influenza viruses have the ability to bind both types of sialic acid receptors.

Human flu symptoms usually include fever, cough, sore throat, muscle aches, conjunctivitis and, in severe cases, breathing problems and pneumonia that may be fatal. The severity of the infection will depend in large part on the state of the infected person's immune system and if the victim has been exposed to the strain before, and is therefore partially immune. Follow-up studies on the impact of statins on influenza virus replication show that pre-treatment of cells with atorvastatin suppresses virus growth in culture. [40]

Highly pathogenic H5N1 avian influenza in a human is far worse, killing 50% of humans who catch it. In one case, a boy with H5N1 experienced diarrhea followed rapidly by a coma without developing respiratory or flu-like symptoms. [41]

The influenza A virus subtypes that have been confirmed in humans, ordered by the number of known human pandemic deaths, are:

    caused "Spanish flu" in 1918 and the 2009 swine flu pandemic caused "Asian flu" in the late 1950s caused "Hong Kong flu" in the late 1960s considered a global influenza pandemic threat through its spread in the mid-2000s is responsible for a 2013 epidemic in China [42] and considered by Dr. Michael Greger, author of How Not to Die, to have the greatest pandemic threat of the Influenza A viruses [43] has some zoonotic potential: it has rarely caused disease in humans [44][45] is currently endemic in pigs and has rarely caused disease in humans. [46] , H7N2, H7N3, H5N2, H10N7, and H10N3

A highly pathogenic strain of H5N9 caused a minor flu outbreak in 1966 in Ontario and Manitoba, Canada in turkeys. [57] H7N2

In May 2021, in Zhenjiang, China H10N3 was reported for the first time in humans. One person was infected. [63]

Evolution Edit

"All influenza A pandemics since [the Spanish flu pandemic], and indeed almost all cases of influenza A worldwide (excepting human infections from avian viruses such as H5N1 and H7N7), have been caused by descendants of the 1918 virus, including "drifted" H1N1 viruses and reassorted H2N2 and H3N2 viruses. The latter are composed of key genes from the 1918 virus, updated by subsequently incorporated avian influenza genes that code for novel surface proteins, making the 1918 virus indeed the "mother" of all pandemics." [64]

Researchers from the National Institutes of Health used data from the Influenza Genome Sequencing Project and concluded that during the ten-year period examined, most of the time the hemagglutinin gene in H3N2 showed no significant excess of mutations in the antigenic regions while an increasing variety of strains accumulated. This resulted in one of the variants eventually achieving higher fitness, becoming dominant, and in a brief interval of rapid evolution, rapidly sweeping through the population and eliminating most other variants. [65]

In the short-term evolution of influenza A virus, a 2006 study found that stochastic, or random, processes are key factors. [66] Influenza A virus HA antigenic evolution appears to be characterized more by punctuated, sporadic jumps as opposed to a constant rate of antigenic change. [67] Using phylogenetic analysis of 413 complete genomes of human influenza A viruses that were collected throughout the state of New York, the authors of Nelson et al. 2006 were able to show that genetic diversity, and not antigenic drift, shaped the short-term evolution of influenza A via random migration and reassortment. The evolution of these viruses is dominated more by the random importation of genetically different viral strains from other geographic locations and less by natural selection. Within a given season, adaptive evolution is infrequent and had an overall weak effect as evidenced from the data gathered from the 413 genomes. Phylogenetic analysis revealed the different strains were derived from newly imported genetic material as opposed to isolates that had been circulating in New York in previous seasons. Therefore, the gene flow in and out of this population, and not natural selection, was more important in the short term.

Fowl act as natural asymptomatic carriers of influenza A viruses. Prior to the current [ when? ] H5N1 epizootic, strains of influenza A virus had been demonstrated to be transmitted from wildfowl to only birds, pigs, horses, seals, whales and humans and only between humans and pigs and between humans and domestic fowl and not other pathways such as domestic fowl to horse. [68]

Wild aquatic birds are the natural hosts for a large variety of influenza A viruses. Occasionally, viruses are transmitted from these birds to other species and may then cause devastating outbreaks in domestic poultry or give rise to human influenza pandemics. [3] [4]

H5N1 has been shown to be transmitted to tigers, leopards, and domestic cats that were fed uncooked domestic fowl (chickens) with the virus. H3N8 viruses from horses have crossed over and caused outbreaks in dogs. Laboratory mice have been infected successfully with a variety of avian flu genotypes. [69]

Influenza A viruses spread in the air and in manure, and survives longer in cold weather. They can also be transmitted by contaminated feed, water, equipment, and clothing however, there is no evidence the virus can survive in well-cooked meat. Symptoms in animals vary, but virulent strains can cause death within a few days. Avian influenza viruses that the World Organisation for Animal Health and others test for to control poultry disease include H5N1, H7N2, H1N7, H7N3, H13N6, H5N9, H11N6, H3N8, H9N2, H5N2, H4N8, H10N7, H2N2, H8N4, H14N5, H6N5, and H12N5.

Known outbreaks of highly pathogenic flu in poultry 1959–2003 [70]

Year Area Affected Subtype
1959 Scotland Chicken H5N1
1963 England Turkey H7N3
1966 Ontario (Canada) Turkey H5N9
1976 Victoria (Australia) Chicken H7N7
1979 Germany Chicken H7N7
1979 England Turkey H7N7
1983 Pennsylvania (US)* Chicken, turkey H5N2
1983 Ireland Turkey H5N8
1985 Victoria (Australia) Chicken H7N7
1991 England Turkey H5N1
1992 Victoria (Australia) Chicken H7N3
1994 Queensland (Australia) Chicken H7N3
1994 Mexico* Chicken H5N2
1994 Pakistan* Chicken H7N3
1997 New South Wales (Australia) Chicken H7N4
1997 Hong Kong (China)* Chicken H5N1
1997 Italy Chicken H5N2
1999 Italy* Turkey H7N1
2002 Hong Kong (China) Chicken H5N1
2002 Chile Chicken H7N3
2003 Netherlands* Chicken H7N7

*Outbreaks with significant spread to numerous farms, resulting in great economic losses. Most other outbreaks involved little or no spread from the initially infected farms.

More than 400 harbor seal deaths were recorded in New England between December 1979 and October 1980, from acute pneumonia caused by the influenza virus, A/Seal/Mass/1/180 (H7N7). [71]

What is the number of influenza strains occurring at a given time? - Biology

Current vaccine strategies against influenza focus on generating robust antibody responses. Because of the high degree of antigenic drift among circulating influenza strains over the course of a year, vaccine strains must be reformulated specifically for each influenza season. The time delay from isolating the pandemic strain to large-scale vaccine production would be detrimental in a pandemic situation. A vaccine approach based on cell-mediated immunity that avoids some of these drawbacks is discussed here. Specifically, cell-mediated responses typically focus on peptides from internal influenza proteins, which are far less susceptible to antigenic variation. We review the literature on the role of CD4+ and CD8+ T cell–mediated immunity in influenza infection and the available data on the role of these responses in protection from highly pathogenic influenza infection. We discuss the advantages of developing a vaccine based on cell-mediated immune responses toward highly pathogenic influenza virus and potential problems arising from immune pressure.

Vaccine approaches against respiratory virus infections such as influenza have relied on inducing antibodies that protect against viral infection by neutralizing virions or blocking the virus's entry into cells. These humoral immune responses target external viral coat proteins that are conserved for a given strain. Antibody-mediated protection is therefore effective against homologous viral strains but inadequate against heterologous strains with serologically distinct coat proteins. This distinction is of consequence since many viruses rapidly mutate their coat proteins an effective humoral response&ndashbased vaccine against a form of the virus may be ineffective against next season's variant. In contrast, T cells, which mediate cellular immune responses, can target internal proteins common to heterologous viral strains. This property gives vaccines that induce protective cellular immune responses the potential to protect against heterologous viral strains.

Antigen-specific ligation of T-cell receptors induces effector mechanisms that either directly or indirectly promote lysis of infected cells. Functionally distinct T-cell subsets are broadly identified according to their differential expression of CD4 and CD8 coreceptors. The CD4+ T helper cells are primarily responsible for helping other immune cells through direct cell-cell interactions or by secreting cytokines after recognizing viral peptides bound to major histocompatibility complex (MHC) class II molecules. The cytotoxic T lymphocytes (CTLs) typically express CD8 and induce apoptosis of cells on which they recognize foreign antigens presented by MHC class I molecules, providing a defense against intracellular pathogens such as viruses. This association of phenotype and function is not absolute, since CD4+ cells may exhibit lytic activity, while CD8+ cells secrete antiviral cytokines, notably interferon-&gamma (IFN-&gamma) and tumor necrosis factor. Greater understanding of how each subset contributes to protective immunity and how T-cell memory is maintained and recalled in a secondary infection would contribute to development of effective vaccines that use these basic features of the immune response.

Immune Models of Influenza

Influenza is a contagious, acute respiratory disease caused by infection of the host respiratory tract mucosa by an influenza virus (1). The influenza A viruses infect host epithelial cells by attaching to a cellular receptor (sialic acid) by the viral surface protein hemagglutinin (HA). The virus is subsequently released because of the action of another surface glycoprotein, the enzyme neuraminidase (NA), several hours after infection.

Mouse models of influenza A virus pneumonia provide a well-developed experimental system to analyze T cell&ndashmediated immunity. In particular, the T-cell immune response to influenza infection has been well characterized in C57BL/6 (B6,H2 b ) mice. While influenza infection of mice does not precisely replicate the natural infection in human, avian, or other vertebrate species, the availability of reagents and genetically modified mouse models has enabled extensive analysis of the cellular immune response. Emerging evidence indicates that findings from mouse studies are pertinent to immunopathology in human disease. In the BL/6 model, virus is cleared 10 days after infection, with no indication of persistent antigen or viral RNA (2). Recovery or prevention of influenza relies on targeting both innate and adaptive responses to the respiratory tract mucosa.

CD8+ T-cell Response to Influenza

Much of the current knowledge on murine CD8+ T-cell responses to influenza has come from analyzing the response to challenge with the HKx31 (H3N2) and PR/8 (H1N1) influenza viruses. A role for CD8+ T cells in protective immunity has been discerned from studies citing delayed influenza virus clearance in CD8+ T cell&ndashdeficient mice (3,4). Furthermore, CD8+ T cells can promote recovery from otherwise lethal secondary viral infections in mice that lack mature B cells or antibodies (5,6), and cloned influenza-specific CTLs can passively transfer protection (7). Despite a seemingly protective role for CD8+ T cells, vaccination with dominant influenza determinants in either a vector or in a recombinant form is only mildly protective (8&ndash10). Moreover, in a T cell&ndashreceptor transgenic mouse model, devoid of antibodies, influenza-specific CTL can either contribute to survival or exacerbate lethal influenza pneumonia (11). This study highlights the need to understand the many facets of the immune response to influenza.

The influenza A virus&ndashspecific CD8+ T-cell response has been characterized by using intracellular cytokine staining and MHC class I tetramer labeling. These techniques have enabled each phase of the response to be tracked. After intranasal infection, priming, activation, and expansion of naive influenza-specific CD8+ T cells occur in the draining mediastinal lymph node 3&ndash4 days after infection (12,13). The antiviral capacity of these virus-specific CD8+ cells is strongly dependent on their ability to migrate and localize to the lungs and infected airway epithelium (14), where they appear 5&ndash7 days after infection (15). Because viral replication is confined to cells in the respiratory epithelium (16,17), CD8+ T cells exert their effector functions at this site, producing antiviral cytokines and lysing target cells presenting viral determinants for which they bear a specific T-cell receptor. Lysis of infected epithelial cells is mediated by exocytosis granules containing perforin and granzymes (18,19). The release of perforin and granzymes from influenza-specific CTLs is tightly regulated, occurring shortly after activation at or near the contact point between CTLs and the infected target cell (18).

Influenza-specific CD8+ T cells recognize multiple viral epitopes on target cells and antigen-presenting cells. The HKx31 and PR8 strains share 6 internal genes derived from PR8 that are processed to generate peptides recognized by influenza-specific CD8+ T cells. The primary response to either strain is dominated by CD8+ T cells' recognition of 2 determinants, the nucleoprotein (NP366-374, H2D b ) (20) and the acid polymerase (PA224-233, H2D b ) (21). A similarly low proportion of CD8+ T cells recognizes 4 other determinants: the basic polymerase subunit 1 (PB1703-711, H2K b ), nonstructural protein 2 (NS2114-121, H2K d ), matrix protein 1 (M1128-135, H2K b ), and a protein derived from an alternative open reading frame within the PB1 gene (PB1-F262-70, H2D b ) (22). The subsequent memory populations appears to be stable D b NP366-374 and D b PA224-233 CD8+ memory cells are still detectable >570 days after initial infection (K. Kedzierska and J. Stambas, unpub. data).

Secondary influenza-specific CTL responses arise &asymp2 days faster than the primary response, with a greatly increased level of activity. Depletion of CD8+ T cells reduces the capacity of primed mice to respond to influenza infection, which indicates a role for CD8+ T cells in the protective secondary response. Prime and challenge experiments can be conducted with HKx31 and PR/8 as all of the recognized epitopes are derived from internal proteins. Furthermore, cross-reactive neutralizing antibodies are avoided because HKx31 and PR/8 express different surface HA and NA or proteins. Despite a similar magnitude to D b PA224-233 in the primary response, D b NP366-374-specific CD8+ T cells dominate the secondary response to HKx31&rarrPR/8 challenge, accounting for up to 80% of the influenza-specific CD8+ T cells. This dominance is maintained in the memory population the numbers of NP-specific CD8+ T cells exceed all other quantified influenza-specific CD8+ T-cell populations (23). Despite the NP dominance, CD8+ T cells specific for the other 5 determinants can still be isolated after secondary challenge, albeit at low frequency.

Conservation of these 6 internal genes and persistence of the corresponding antigen-specific CD8+ T cells makes these genes an attractive target for vaccine therapies. However, although cell-mediated immunity can promote viral clearance, it does not provide sterile resistance because, unlike humoral immunity, it cannot prevent infection of the host cells. In humans, the level of influenza-specific CTLs correlates with the rate of viral clearance but not with susceptibility to infection or subsequent illness (24). Despite this limitation, vaccines that promote cell-mediated immunity may be a favorable option to fight potentially lethal, highly pathogenic influenza strains.

CD4+ T cell&ndashspecific Responses to Viruses

In contrast to the body of literature that has characterized the role of CD8+ T cells specifically in models of influenza infection, relatively little is known about the role of CD4+ T cells as direct mediators of effector function. That CD4+ T-cell help is central to adaptive immunity is well established, but few antigen-specific systems have been developed to dissect the role of CD4+ T cells in a viral infection. While knowledge of CD8+ T-cell antigen-specific responses has increased substantially in the past several years as a result of tetramer technology, these reagents have been more difficult to develop for the CD4+ subset. Further, identification of CD4+ T cell&ndashspecific epitopes has been less successful for a variety of pathogens. For instance, in influenza, the CD8+ restricted epitopes have all been largely identified for some time, particularly in the BL/6 model system in contrast, only very recently have confirmed CD4 epitopes been found, and they are much more poorly characterized (25).

Still, a substantial amount of work has been done with various knockout, depletion, and cell-transfer models to investigate the role of CD4+ T cells in primary, secondary, and memory responses to influenza infection in the mouse model (26,27). Controversy still exists in the field, and an antigen-specific system would help address it, but certain findings appear to be consistent across different experimental systems (28).

In the primary response, CD4+ T cells are not required for expansion or development of functional CD8+ CTL (27,29), which may in part result from the ability of influenza virus to directly activate dendritic cells, aiding in the development of CD8+ responses that substitute for functional CD4+ T cells (30). Similarly, in the case of a murine &gamma-herpesvirus, the lack of CD4+ T cells can be compensated for by the addition of anti-CD40 stimulation (31). In mice in which both the CD4+ T-cell and B-cell compartments were defective, the primary CD8+ T-cell response to influenza appeared to be stunted in terms of recruitment and expansion (vs. mice in which B cells alone were knocked out) the remaining CD8+ T cells had a robust level of functionality as assayed by IFN-&gamma intracellular cytokine production (27). The defect in the CD8+ T-cell primary response was less obvious in mice with intact B cells, though viral clearance was delayed. Still, not until the secondary and memory responses are examined can the dramatic effect of CD4+ T-cell deletion be observed.

In multiple systems, a defect of CD8+ T-cell secondary and memory responses have been observed when the primary response lacks CD4+ T cells (26,32,33). In influenza, a dramatic drop was observed in the size and magnitude of the recall response to secondary infection. The rate of viral clearance was also slowed considerably, beyond the degree seen in the primary response. Similarly, in the Listeria monocytogenes model system, the primary response was largely intact, while the long-term memory response was defective (34). In mice that lacked CD4+ T cells during the primary response, the memory pool of CD8+ T cells was initially similar in size and functionality to that seen in wild-type mice but began to decline after longer intervals, leading eventually to the recrudescence of the infection. Secondary challenge also demonstrated a reduced antigen-specific CD8+ T-cell compartment.

In the influenza model, although the draining lymph node and spleen CD8+ responses were defective in secondary infection of CD4+ T cell&ndashdeficient mice, the CD8+ T-cell responses in bronchoalveolar lavage were equivalent to those seen in wild-type mice (29). This finding implies that the high levels of activation and inflammation, in large part mediated by innate immune effectors at the site of infection, were capable of providing the right maturation milieu to expand the response to wild-type levels this finding suggests CD4+ T cell&ndashspecific help is not required at the site of the pathologic changes, at least when the infection induces a high level of other immune stimulation, though it is essential in the lymphoid organs in the generation and maintenance of memory.

A role for CD4+ T cells as effectors has been found in a number of other systems, including the mouse &gamma-herpesvirus model (35) and in HIV-infected humans (36,37). In these studies, CD4+ T cells contribute to infection control by supplementing their helper role with cytotoxicity. In the case of the &gamma-herpesvirus, the effector CD4+ population was important only in immunoglobulin &ndash/&ndash &muMT mice, while the HIV studies were conducted in infected (and presumably immune-irregular) patients. However, effector CD4+ T cells have been found in multiple stages of the disease and in long-term patients whose disease is not progressing because viral replication is controlled. Finally, a recent report demonstrated a similar cytotoxic CD4+ T-cell effector population in protozoan-infected cattle (38).

Relatively few established mouse models are available for studying the CD4+ response to influenza virus. On the IA d BALB/c background, an HA epitope was discovered, and a transgenic mouse was developed to analyze specific responses (the HNT model) (39). This model has been extremely useful for studying several aspects of CD4+ biology in influenza infection, particularly in regards to aging and the development of primary responses leading to acute memory (39). Several investigators have introduced external epitopes in influenza to follow CD4+ T-cell responses in defined systems. These include the hen egg lysozyme p46&ndash63 sequence (40) and the ovalbumin 323&ndash339 (OT-II) epitope inserted into the NA stalk of WSN influenza virus (41). We have inserted the OT-II epitope into the HA of the PR8 H1N1 virus and the X31 H3N2 virus. In contrast to the robust responses achieved with CD8+ T-cell epitopes and transgenics, the CD4+ T-cell responses seem relatively weak (unpub. data). Other naturally occurring epitopes have similarly low frequencies after infection (25). The antigen-specific CD4+ response may not develop the dramatic immunodominance hierarchies that are well-known for CD8+ T cells and may be directed at many epitopes, more than are seen in the more-delimited CD8+ T-cell response. Much work needs to be done before this conclusion is certain, and examples of respiratory infections in mice produce robust and dominant responses toward individual class II epitopes (42).

Cell-mediated Protection against Highly Pathogenic Influenza

Highly pathogenic H5N1 influenza emerged in 1997, followed by several waves of infection from 2002 until now (43). The viruses have been remarkably virulent in multiple animal models, including mice, but little work has been done to characterize the protective immune responses toward H5N1 viruses. A series of reports has shown strong protection toward other highly pathogenic viruses mediated by cellular responses, in the absence of neutralizing antibody. Antibody-deficient mice infected with a mild, passaged strain of an H3N2 virus were more likely to survive than naive controls when challenged with a highly pathogenic H3N8 duck virus compared to naive controls (44). A double-priming protocol provided increased protection from a lethal H7N7 challenge, which correlated with an increased pool of cross-reactive antigen-specific CD8+ T cells (45). In both these cases, the initial phase of infection and viral growth seemed similar to that occurring in immunologically naive mice, but a rapid decrease in viral titers occurs after a few days of infection.

Since the emergence of the H5N1 viruses, concern has arisen that the biological activity of these viruses, including their diverse tissue tropism in a number of animal models, may influence the ability of immune responses to control infection. Furthermore, some pathology associated with these viruses has been attributed to extremely high levels of inflammatory cytokines produced in response to infection, which suggests a negative role for immune responses. However, the few studies that have been performed have shown promising results for the potential of cell-mediated responses to contribute to the control of infections. A prime-challenge protocol using an H9N2 isolate with 98% homology to the internal genes of the A/HongKong/156/97 H5N1 protected against the otherwise lethal challenge (46) with a virus with a highly cleavable H5, a characteristic of all the pathogenic H5 viruses. The priming protocol generated significant CTL activity directed at the NP and PB2 proteins.

Figure. Apparent cell-mediated protection against highly pathogenic H5N1 influenza virus. Mice (10 in each group) were immunized by intraperitoneal injection of PR8, followed by intraperitoneal injection 4 weeks later of X31. Four.

Our own work has indicated a similar ability of cell-mediated immunity to protect against virulent H5N1 challenge. In a preliminary experiment, we primed mice with the H1N1 PR8 strain and the H3N2 X31 strain followed by a challenge with A/Vietnam/1203/2004, one of the most lethal H5N1 viruses, which causes severe pathologic changes, even in ducks. While 9 of 10 naive mice died, 9 of 10 primed mice survived past day 10 of infectious challenge and recovered substantial weight (Figure). The fact that both groups lost weight indicated protection was occurring by delayed cell-mediated responses, rather than by the "immediate" cross-protective antibody response.

Cell-mediated Vaccine for Highly Pathogenic Influenza?

Despite the systems currently in place for manufacturing and distributing an influenza vaccine, pandemic influenza will require a substantially different approach. The standard influenza vaccine given during the infectious season is made from a reassortant seed strain containing the HA and NA of the circulating virus with the internal genes of a vaccine strain, usually PR8. The seed strain is grown in eggs and is formaldehyde inactivated. This strategy does not prime strong CD8 CTL responses, but it is effective in providing antibody-mediated protection to closely homologous strains (47).

One drawback to this approach is the length of time required to develop a seed strain, amplify it, and manufacture it into distributable vaccine. In the case of a potential influenza pandemic, the delivery of vaccine on this schedule would not prevent the spread of the epidemic in many countries. Furthermore, antigenic drift can occur between the original selection of the seed strain and circulating viruses before the vaccine is ready for distribution (48). This problem was faced recently in a nonpandemic situation in 2003 and 2004 when the circulating Fujian strain of H3N2 influenza had drifted from the vaccine strain (49). While the Fujian strain was predicted to be circulating at the time of vaccine delivery, a recombined seed strain could not be isolated in time for vaccine production. Although the ensuing influenza season was not as severe as initially feared, the situation highlighted a problem with the current vaccine strategy. Evidence of antigenic drift is already evident in the most recent outbreaks of H5N1 (48).

Several groups have developed reverse genetics&ndashbased methods that could speed the production of seed viruses as well as proposals for growing viruses in cell culture rather than in embyronated chicken eggs, which would allow for a much faster scale up in response to an epidemic (50). These technologies have not been approved yet for human use, though trials are underway.

Even if the development of recombinant seed strains by reverse genetics becomes standard over the next few years, questions remain about how effective the current formaldehyde-inactivated seed strain strategies would be against pandemic strains, particularly the currently circulating H5N1 strains. Assuming that seed strains could be produced rapidly, several weeks would be required to manufacture a relevant number of doses of vaccine. To address this concern, several governments have been stockpiling vaccines based on H5N1 viruses that have been circulating over the last few years. While these vaccines may provide some protection, substantial evolution and antigenic drift seem to be occurring, rendering the stockpiled strains less and less useful (48).

An approach based on conserved cellular epitopes within the internal genes has the advantage of subverting all of these issues. While cellular immunity is not sterilizing, it prevents illness and death in animal models (3). Common and immunodominant epitopes among circulating nonavian strains have been identified, and many of the same models and algorithms can be used to make predictions against the pathogenic strains (51). Mouse models are now available that have human leukocyte antigen (HLA) alleles, and they appear to recapitulate human epitope use. As described earlier, protection against death from highly pathogenic viruses has been shown in multiple systems. Cross-protective cell-mediated immunity has been found in birds for circulating chicken H5N1 and H9N2, both of which have been suggested as potential human pandemic strains (52). The notion of a "universal" vaccine for highly pathogenic strains is attractive.

Antigenic drift due to immunologic pressure is also a concern with a CD8- or CD4-based vaccine approach. Reports have suggested that CD8+ epitopes under pressure will mutate to escape protective immunity (11). The mutation of an NP epitope that binds HLA-B35 present in strains of virus from the 1930s through the present indicates that even in nonpandemic years, immunologic pressure from cross-protective CD8+ T cells is enough to drive the evolution of the virus (53). In contrast, though, other dominant epitopes do not appear to be under the same pressure (54).

Several human peptide epitopes that have been described and characterized show evidence of remarkably little mutation over many generations of viral evolution. In the most recent outbreaks of H5N1 virus, some of these peptides are conserved in viruses isolated from human patients (Table). The conservation of so many peptides from such distantly related viruses suggests that they may be less susceptible to antigenic drift than the HA and NA glycoproteins. Vaccines that promote strong memory CTL activity toward these peptides and MHC, in combination with the antibody-based approaches already underway, could help prevent pandemic influenza. This approach could potentiate immunologic pressure on the vaccine-targeted epitopes, but an immunization strategy that targets a large number of epitopes along with the natural restriction on epitope structure due to viral function should mitigate this effect. Some evidence shows that highly conserved CTL epitopes are restricted from mutation by viral structural requirements. Given the large number of influenza viruses sequenced over time, we should be able to make reasonable assumptions about the identity of these epitopes in MHC-diverse populations and focus on how to facilitate the development of strong immune responses toward them.

Appendix Bibliography

Further literature support for the material discussed in this article is available.

1. Arnold PY, Vignali KM, Miller TB, La Gruta NL, Cauley LS, Haynes L, et al. Reliable generation and use of MHC class II:gamma2aFc multimers for the identification of antigen-specific CD4(+) T cells. J Immunol Methods. 2002271:137&ndash51. PubMed

2. Belz GT, Xie W, Doherty PC. Diversity of epitope and cytokine profiles for primary and secondary influenza a virus-specific CD8+ T cell responses. J Immunol. 2001166:4627&ndash33. PubMed

3. Boon AC, de Mutsert G, Fouchier RA, Sintnicolaas K, Osterhaus AD, Rimmelzwaan GF. Preferential HLA usage in the influenza virus-specific CTL response. J Immunol. 2004172:4435&ndash43. PubMed

4. Cerwenka A, Morgan TM, Harmsen AG, Dutton RW. Migration kinetics and final destination of type 1 and type 2 CD8 effector cells predict protection against pulmonary virus infection. J Exp Med. 1999189:423&ndash34. PubMed

5. Chen W, Bennink JR, Morton PA, Yewdell JW. Mice deficient in perforin, CD4+ T cells, or CD28-mediated signaling maintain the typical immunodominance hierarchies of CD8+ T-cell responses to influenza virus. J Virol. 200276:10332&ndash7. PubMed

6. Doherty PC, Topham DJ, Tripp RA, Cardin RD, Brooks JW, Stevenson PG. Effector CD4+ and CD8+ T-cell mechanisms in the control of respiratory virus infections. Immunol Rev. 1997159:105&ndash17. PubMed

7. Falk K, Rotzschke O, Deres K, Metzger J, Jung G, Rammensee HG. Identification of naturally processed viral nonapeptides allows their quantification in infected cells and suggests an allele-specific T cell epitope forecast. J Exp Med. 1991174:425&ndash34. PubMed

8. Flynn KJ, Belz GT, Altman JD, Ahmed R, Woodland DL, Doherty PC. Virus-specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity. 19988:683&ndash91. PubMed

9. Flynn KJ, Riberdy JM, Christensen JP, Altman JD, Doherty PC. In vivo proliferation of naive and memory influenza-specific CD8(+) T cells. Proc Natl Acad Sci U S A. 199996:8597&ndash602. PubMed

10. Hou S, Hyland L, Ryan KW, Portner A, Doherty PC. Virus-specific CD8+ T-cell memory determined by clonal burst size. Nature. 1994369:652&ndash4. PubMed

11. Hu N, D'Souza C, Cheung H, Lang H, Cheuk E, Chamberlain JW. Highly conserved pattern of recognition of influenza A wild-type and variant CD8(+) CTL epitopes in HLA-A2(+) humans and transgenic HLA-A2(+)/H2 class I-deficient mice. Vaccine. 200523:5231&ndash44. PubMed

12. Kilbourne ED. Future influenza vaccines and the use of genetic recombinants. Bull World Health Organ. 196941:643&ndash5. PubMed

13. Lamb RA, Krug RM. Orthomyxoviridae: the viruses and their replication. In: Fields BN, Knipe DM, Howley PM, Chanock RM, Melnick JL, Monath TP, et al., editors. Fields virology. 3rd ed. Philadelphia: Lippincott-Raven Publishers 1996. 1353&ndash95.

14. Lawson CM, Bennink JR, Restifo NP, Yewdell JW, Murphy BR. Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge. J Virol. 199468:3505&ndash11 . PubMed

15. Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci U S A. 2004101:5598&ndash603. PubMed

16. Rott R, Klenk HD, Nagai Y, Tashiro M. Influenza viruses, cell enzymes, and pathogenicity. Am J Respir Crit Care Med. 1995152:S16&ndash9 . PubMed

17. Stephenson I, Nicholson KG, Wood JM, Zambon MC, Katz JM. Confronting the avian influenza threat: vaccine development for a potential pandemic. Lancet Infect Dis. 20044:499&ndash509 . PubMed

18. Topham DJ, Doherty PC. Clearance of an influenza A virus by CD4+ T cells is inefficient in the absence of B cells. J Virol. 199872:882&ndash5. PubMed

19. Wood JM, Robertson JS. From lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza. Nat Rev Microbiol. 20042:842&ndash7 . PubMed

20. Wraith DC, Vessey AE, Askonas BA. Purified influenza virus nucleoprotein protects mice from lethal infection. J Gen Virol. 198768:433&ndash40 . PubMed

21. Zaunders JJ, Dyer WB, Wang B, Munier ML, Miranda-Saksena M, Newton R, et al. Identification of circulating antigen-specific CD4+ T lymphocytes with a CCR5+, cytotoxic phenotype in an HIV-1 long-term nonprogressor and in CMV infection. Blood. 2004103:2238&ndash47. PubMed

Dr Thomas works in the Department of Immunology at St. Jude Children's Research Hospital in Memphis, Tennessee. His primary research involves the use of reverse genetics&ndashengineered influenza viruses to understand the dynamics of CD8+ and CD4+ T-cell responses.


Jeffery K. Taubenberger 4

Department of Molecular Pathology

Armed Forces Institute of Pathology


Influenza A viruses are negative strand RNA viruses of the genus Orthomyxoviridae. They continually circulate in humans in yearly epidemics (mainly in the winter in temperate climates) and antigenically novel virus strains emerge sporadically as pandemic viruses (Cox and Subbarao, 2000). In the United States, influenza is estimated to kill 30,000 people in an average year (Simonsen et al., 2000 Thompson et al., 2003). Every few years, a more severe influenza epidemic occurs, causing a boost in the annual number of deaths past the average, with 10,000 to 15,000 additional deaths. Occasionally, and unpredictably, influenza sweeps the world, infecting 20 to 40 percent of the population in a single year. In these pandemic years, the numbers of deaths can be dramatically above average. In 1957�, a pandemic was estimated to cause 66,000 excess deaths in the United States (Simonsen et al., 1998). In 1918, the worst pandemic in recorded history was associated with approximately 675,000 total deaths in the United States (U.S. Department of Commerce, 1976) and killed at least 40 million people worldwide (Crosby, 1989 Patterson and Pyle, 1991 Johnson and Mueller, 2002).

Influenza A viruses constantly evolve by the mechanisms of antigenic drift and shift (Webster et al., 1992). Consequently they should be considered emerging infectious disease agents, perhaps 𠇌ontinually” emerging pathogens. The importance of predicting the emergence of new circulating influenza virus strains for subsequent annual vaccine development cannot be underestimated (Gensheimer et al., 1999). Pandemic influenza viruses have emerged three times in this century: in 1918 (“Spanish” influenza, H1N1), in 1957 (𠇊sian” influenza, H2N2), and in 1968 (“Hong Kong” influenza, H3N2) (Cox and Subbarao, 2000 Webby and Webster, 2003). Recent circulation of highly pathogenic avian H5N1 viruses in Asia from 1997 to 2004 has caused a small number of human deaths (Claas et al., 1998 Subbarao et al., 1998 Tran et al., 2004 Peiris et al., 2004). How and when novel influenza viruses emerge as pandemic virus strains and how they cause disease is still not understood.

Studying the extent to which the 1918 influenza was like other pandemics may help us to understand how pandemic influenzas emerge and cause disease in general. On the other hand, if we determine what made the 1918 influenza different from other pandemics, we may use the lessons of 1918 to predict the magnitude of public health risks a new pandemic virus might pose.

Origin of Pandemic Influenza Viruses

The predominant natural reservoir of influenza viruses is thought to be wild waterfowl (Webster et al., 1992). Periodically, genetic material from avian virus strains is transferred to virus strains infectious to humans by a process called reassortment. Human influenza virus strains with recently acquired avian surface and internal protein-encoding RNA segments were responsible for the pandemic influenza outbreaks in 1957 and 1968 (Scholtissek et al., 1978a Kawaoka et al., 1989). The change in the hemagglutinin subtype or the hemagglutinin (HA) and the neuraminidase (NA) subtype is referred to as antigenic shift. Because pigs can be infected with both avian and human virus strains, and various reassortants have been isolated from pigs, they have been proposed as an intermediary in this process (Scholtissek, 1994 Ludwig et al., 1995). Until recently there was only limited evidence that a wholly avian influenza virus could directly infect humans, but in 1997 18 people were infected with avian H5N1 influenza viruses in Hong Kong, and 6 died of complications after infection (Claas et al., 1998 Subbarao et al., 1998 Scholtissek, 1994 Ludwig et al., 1995). Although these viruses were very poorly transmissible or non-transmissible (Claas et al., 1998 Subbarao et al., 1998 Scholtissek, 1994 Ludwig et al., 1995 Katz et al., 1999), their isolation from infected patients indicates that humans can be infected with wholly avian influenza virus strains. In 2003�, H5N1 outbreaks in poultry have become widespread in Asia (Tran et al., 2004), and at least 32 people have died of complications of infection in Vietnam and Thailand (World Health Organization, 2004). In 2003, a highly pathogenic H7N7 outbreak occurred in poultry farms in The Netherlands. This virus caused infections (predominantly conjunctivitis) in 86 poultry handlers and 3 secondary contacts. One of the infected individuals died of pneumonia (Fouchier et al., 2004 Koopmans et al., 2004 World Health Organization, 2004). In 2004, an H7N3 influenza outbreak in poultry in Canada also resulted in the infection of a single individual (World Health Organization, 2004), and a patient in New York was reported to be sick following infection with an H7N2 virus (Lipsman, 2004). Therefore, it may not be necessary to invoke swine as the intermediary in the formation of a pandemic virus strain because reassortment between an avian and a human influenza virus could take place directly in humans.

While reassortment involving genes encoding surface proteins appears to be a critical event for the production of a pandemic virus, a significant amount of data exists to suggest that influenza viruses must also acquire specific adaptations to spread and replicate efficiently in a new host. Among other features, there must be functional HA receptor binding and interaction between viral and host proteins (Weis et al., 1988). Defining the minimal adaptive changes needed to allow a reassortant virus to function in humans is essential to understanding how pandemic viruses emerge.

Once a new virus strain has acquired the changes that allow it to spread in humans, virulence is affected by the presence of novel surface protein(s) that allow the virus to infect an immunologically naïve population (Kilbourne, 1977). This was the case in 1957 and 1968 and was almost certainly the case in 1918. While immunological novelty may explain much of the virulence of the 1918 influenza, it is likely that additional genetic features contributed to its exceptional lethality. Unfortunately not enough is known about how genetic features of influenza viruses affect virulence. The degree of illness caused by a particular virus strain, or virulence, is complex and involves host factors like immune status, and viral factors like host adaptation, transmissibility, tissue tropism, or viral replication efficiency. The genetic basis for each of these features is not yet fully characterized, but is most likely polygenic in nature (Kilbourne, 1977).

Prior to the analyses on the 1918 virus described in this review, only two pandemic influenza virus strains were available for molecular analysis: the H2N2 virus strain from 1957 and the H3N2 virus strain from 1968. The 1957 pandemic resulted from the emergence of a reassortant influenza virus in which both HA and NA had been replaced by gene segment closely related to those in avian virus strains (Scholtissek et al., 1978b Schafer et al., 1993 Webster et al., 1995). The 1968 pandemic followed with the emergence of a virus strain in which the H2 subtype HA gene was exchanged with an avian-derived H3 HA RNA segment (Scholtissek et al., 1978b Webster et al., 1995), while retaining the N2 gene derived in 1957. More recently it has been shown that the PB1 gene was replaced in both the 1957 and the 1968 pandemic virus strains, also with a likely avian derivation in both cases (Kawaoka et al., 1989). The remaining five RNA segments encoding the PA, PB2, nucleoprotein, matrix and non-structural proteins, all were preserved from the H1N1 virus strains circulating before 1957. These segments were likely the direct descendants of the genes present in the 1918 virus. Because only the 1957 and 1968 influenza pandemic virus strains have been available for sequence analysis, it is not clear what changes are necessary for the emergence of a virus strain with pandemic potential. Sequence analysis of the 1918 influenza virus allows us potentially to address the genetic basis of virulence and human adaptation.

Historical Background

The influenza pandemic of 1918 was exceptional in both breadth and depth. Outbreaks of the disease swept not only North America and Europe, but also spread as far as the Alaskan wilderness and the most remote islands of the Pacific. It has been estimated that one-third of the world's population may have been clinically infected during the pandemic (Frost, 1920 Burnet and Clark, 1942). The disease was also exceptionally severe, with mortality rates among the infected of more than 2.5 percent, compared to less than 0.1 percent in other influenza epidemics (Marks and Beatty, 1976 Rosenau and Last, 1980). Total mortality attributable to the 1918 pandemic was probably around 40 million (Crosby, 1989 Johnson and Mueller, 2002 Patterson and Pyle, 1991).

Unlike most subsequent influenza virus strains that have developed in Asia, the 𠇏irst wave” or “spring wave” of the 1918 pandemic seemingly arose in the United States in March 1918 (Barry, 2004 Crosby, 1989 Jordan, 1927). However, the near simultaneous appearance of influenza in March𠄺pril 1918 in North America, Europe, and Asia makes definitive assignment of a geographic point of origin difficult (Jordan, 1927). It is possible that a mutation or reassortment occurred in the late summer of 1918, resulting in significantly enhanced virulence. The main wave of the global pandemic, the �ll wave” or “second wave,” occurred in September–November 1918. In many places, there was yet another severe wave of influenza in early 1919 (Jordan, 1927).

Three extensive outbreaks of influenza within 1 year is unusual, and may point to unique features of the 1918 virus that could be revealed in its sequence. Interpandemic influenza outbreaks generally occur in a single annual wave in the late winter. The severity of annual outbreaks is affected by antigenic drift, with an antigenically modified virus strain emerging every 2 to 3 years. Even in pandemic influenza, while the normal late winter seasonality may be violated, the successive occurrence of distinct waves within a year is unusual. The 1890 pandemic began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early 1890. The second wave peaked in spring 1891 (over a year after the first wave) and the third wave in early 1892 (Jordan, 1927). As in 1918, subsequent waves seemed to produce more severe illness so that the peak mortality was reached in the third wave of the pandemic. The three waves, however, were spread over more than 3 years, in contrast to less than 1 year in 1918. It is unclear what gave the 1918 virus this unusual ability to generate repeated waves of illness. Perhaps the surface proteins of the virus drifted more rapidly than other influenza virus strains, or perhaps the virus had an unusually effective mechanism for evading the human immune system.

The influenza epidemic of 1918 killed an estimated 675,000 Americans, including 43,000 servicemen mobilized for World War I (Crosby, 1989). The impact was so profound as to depress average life expectancy in the United States by more than 10 years (Grove and Hetzel, 1968) (Figure 1-1) and may have played a significant role in ending the World War I conflict (Crosby, 1989 Ludendorff, 1919).


Life expectancy in the United States, 1900�, showing the impact of the 1918 influenza pandemic. SOURCES: U.S. Department of Commerce (1976) Grove and Hetzel (1968) Linder and Grove (1943).

Many individuals who died during the pandemic succumbed to secondary bacterial pneumonia (Jordan, 1927 LeCount, 1919 Wolbach, 1919) because no antibiotics were available in 1918. However, a subset died rapidly after the onset of symptoms often with either massive acute pulmonary hemorrhage or pulmonary edema, often in less than 5 days (LeCount, 1919 Winternitz et al., 1920 Wolbach, 1919). In the hundreds of autopsies performed in 1918, the primary pathologic findings were confined to the respiratory tree and death was due to pneumonia and respiratory failure (Winternitz et al., 1920). These findings are consistent with infection by a well-adapted influenza virus capable of rapid replication throughout the entire respiratory tree (Reid and Taubenberger, 1999 Taubenberger et al., 2000). There was no clinical or pathological evidence for systemic circulation of the virus (Winternitz et al., 1920).

Furthermore, in the 1918 pandemic most deaths occurred among young adults, a group that usually has a very low death rate from influenza. Influenza and pneumonia death rates for 15- to 34-year-olds were more than 20 times higher in 1918 than in previous years (Linder and Grove, 1943 Simonsen et al., 1998) (Figure 1-2). The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza mortality was higher in those younger than age 65 than in those older than 65. Strikingly, persons less than 65 years old accounted for more than 99 percent of all excess influenza-related deaths in 1918� (Simonsen et al., 1998). In contrast, the less-than-65 age group accounted for only 36 percent of all excess influenza-related mortality in the 1957 H2N2 pandemic and 48 percent in the 1968 H3N2 pandemic. Overall, nearly half of the influenza-related deaths in the 1918 influenza pandemic were young adults aged 20 to 40 (Simonsen et al., 1998) (Figure 1-2). Why this particular age group suffered such extreme mortality is not fully understood (see below).


Influenza and pneumonia mortality by age, United States. Influenza and pneumonia specific mortality by age, including an average of the interpandemic years 1911� (dashed line), and the pandemic year 1918 (solid line). Specific death rate is (more. )

The 1918 influenza had another unique feature: the simultaneous infection of both humans and swine. Interestingly, swine influenza was first recognized as a clinical entity in that species in the fall of 1918 (Koen, 1919) concurrently with the spread of the second wave of the pandemic in humans (Dorset et al., 1922�). Investigators were impressed by clinical and pathological similarities of human and swine influenza in 1918 (Koen, 1919 Murray and Biester, 1930). An extensive review by the veterinarian W.W. Dimoch of the diseases of swine published in August 1918 makes no mention of any swine disease resembling influenza (Dimoch, 1918�). Thus, contemporary investigators were convinced that influenza virus had not circulated as an epizootic disease in swine before 1918 and that the virus spread from humans to pigs because of the appearance of illness in pigs after the first wave of the 1918 influenza in humans (Shope and Lewis, 1931).

Thereafter the disease became widespread among swine herds in the U.S. midwest. The epizootic of 1919� was as extensive as in 1918�. The disease then appeared among swine in the midwest every year, leading to Shope's isolation of the first influenza virus in 1930, A/swine/ Iowa/30 (Shope and Lewis, 1931), 3 years before the isolation of the first human influenza virus, A/WS/33 by Smith, Andrewes, and Laidlaw (Smith et al., 1933). Classical swine viruses have continued to circulate not only in North American pigs, but also in swine populations in Europe and Asia (Brown et al., 1995 Kupradinun et al., 1991 Nerome et al., 1982).

During the fall and winter of 1918�, severe influenza-like outbreaks were noted not only in swine in the United States, but also in Europe and China (Beveridge, 1977 Chun, 1919 Koen, 1919). Since 1918 there have been many examples of both H1N1 and H3N2 human influenza A virus strains becoming established in swine (Brown et al., 1998 Castrucci et al., 1993 Zhou et al., 2000), while swine influenza A virus strains have been isolated only sporadically from humans (Gaydos et al., 1977 Woods et al., 1981).

The unusual severity of the 1918 pandemic and the exceptionally high mortality it caused among young adults have stimulated great interest in the influenza virus strain responsible for the 1918 outbreak (Crosby, 1989 Kolata, 1999 Monto et al., 1997). Because the first human and swine influenza A viruses were not isolated until the early 1930s (Shope and Lewis, 1931 Smith et al., 1933), characterization of the 1918 virus strain previously has had to rely on indirect evidence (Kanegae et al., 1994 Shope, 1958).

Serology and Epidemiology of the 1918 Influenza Virus

Analyses of antibody titers of 1918 influenza survivors from the late 1930s suggested correctly that the 1918 virus strain was an H1N1-subtype influenza A virus, closely related to what is now known as 𠇌lassic swine” influenza virus (Dowdle, 1999 Philip and Lackman, 1962 Shope, 1936). The relationship to swine influenza is also reflected in the simultaneous influenza outbreaks in humans and pigs around the world (Beveridge, 1977 Chun, 1919 Koen, 1919). Although historical accounts described above suggest that the virus spread from humans to pigs in the fall of 1918, the relationship of these two species in the development of the 1918 influenza has not been resolved.

Which influenza A subtype(s) circulated before the 1918 pandemic is not known for certain. In a recent review of the existing archaeoserologic and epidemiologic data, Walter Dowdle concluded that an H3-subtype influenza A virus strain circulated from the 1889� pandemic to 1918, when it was replaced by the novel H1N1 virus strain of the 1918 pandemic (Dowdle, 1999).

It is reasonable to conclude that the 1918 virus strain must have contained a hemagglutinin gene encoding a novel subtype such that large portions of the population did not have protective immunity (Kilbourne, 1977 Reid and Taubenberger, 1999). In fact, epidemiological data collected between 1900 and 1918 on influenza prevalence by age in the population provide good evidence for the emergence of an antigenically novel influenza virus in 1918 (Jordan, 1927). Jordan showed that from 1900 to 1917, the 5 to 15 age group accounted for 11 percent of total influenza cases in this series while the 㹥 age group similarly accounted for 6 percent of influenza cases. In 1918 the 5- to 15-year-old group jumped to 25 percent of influenza cases, compatible with exposure to an antigenically novel virus strain. The 㹥 age group only accounted for 0.6 percent of the influenza cases in 1918. It is likely that this age group accounted for a significantly lower percentage of influenza cases because younger people were so susceptible to the novel virus strain (as seen in the 1957 pandemic [Ministry of Health, 1960 Simonsen et al., 1998]), but it is also possible that this age group had pre-existing H1 antibodies. Further evidence for pre-existing H1 immunity can be derived from the age-adjusted mortality data in Figure 1-2. Those individuals 㹵 years had a lower influenza and pneumonia case mortality rate in 1918 than they had for the prepandemic period of 1911�.

When 1918 influenza case rates by age (Jordan, 1927) are superimposed on the familiar “W”-shaped mortality curve (seen in Figure 1-2), a different perspective emerges (Figure 1-3). As shown, those 㰵 years of age in 1918 accounted for a disproportionately high influenza incidence by age. Interestingly, the 5 to 14 age group accounted for a large fraction of 1918 influenza cases, but had an extremely low case mortality rate compared to other age groups (Figure 1-3). Why this age group had such a low case fatality rate cannot yet be fully explained. Conversely, why the 25 to 34 age group had such a high influenza and pneumonia mortality rate in 1918 remains enigmatic, but it is one of the truly unique features of the 1918 influenza pandemic.


Influenza and pneumonia mortality by age (solid line), with influenza morbidity by age (dashed line) superimposed. Influenza and pneumonia mortality by age as in Figure 1-2. Specific death rate per age group, left ordinal axis. Influenza morbidity presented (more. )

One theory that may explain these data concerns the possibility that the virus had an intrinsically high virulence that was only tempered in those patients who had been born before 1889. It can be speculated that the virus circulating prior to 1889 was an H1-like virus strain that provided partial protection against the 1918 virus strain (Ministry of Health, 1960 Simonsen et al., 1998 Taubenberger et al., 2001). Short of this cross-protection in patients older than 29 years of age, the pandemic of 1918 might have been even more devastating (Zamarin and Palese, 2004). A second possibility remains that the high mortality of young adults in the 20 to 40 age group may have been a consequence of immune enhancement in this age group. Currently, however, the absence of pre-1918 human influenza samples and the lack of pre-1918 sera samples for analysis makes it impossible to test this hypothesis.

Thus, it seems clear that the H1N1 virus of the 1918 pandemic contained an antigenically novel hemagglutinin to which most humans and swine were susceptible in 1918. Given the severity of the pandemic, it is also reasonable to suggest that the other dominant surface protein, NA, also would have been replaced by antigenic shift before the start of the pandemic (Reid and Taubenberger, 1999 Taubenberger et al., 2000). In fact, sequence and phylogenetic analyses suggest that the genes encoding these two surface proteins were derived from an avian-like influenza virus shortly before the start of the 1918 pandemic and that the precursor virus did not circulate widely in either humans or swine before 1918 (Fanning et al., 2002 Reid et al., 1999, 2000) (Figure 1-4). It is currently unclear what other influenza gene segments were novel in the 1918 pandemic virus in comparison to the previously circulating virus strain. It is possible that sequence and phylogenetic analyses of the gene segments of the 1918 virus may help elucidate this question.


Phylogenetic tree of the influenza virus hemagglutinin gene segment. Amino acid changes in three lineages of the influenza virus hemagglutinin protein segment, HA1. The tree shows the numbers of unambiguous changes between these sequences, with branch (more. )

Genetic Characterization of the 1918 Virus

Sequence and Functional Analysis of the Hemagglutinin and Neuraminidase Gene Segments

Samples of frozen and fixed lung tissue from five second-wave influenza victims (dating from September 1918 to February 1919) have been used to examine directly the genetic structure of the 1918 influenza virus. Two of the cases analyzed were U.S. Army soldiers who died in September 1918, one in Camp Upton, New York, and the other in Fort Jackson, South Carolina. The available material consists of formalin-fixed, paraffin-embedded autopsy tissue, hematoxylin and eosin-stained microscopic sections, and the clinical histories of these patients. A third sample was obtained from an Alaskan Inuit woman who had been interred in permafrost in Brevig Mission, Alaska, since her death from influenza in November 1918. The influenza virus sequences derived from these three cases have been called A/ South Carolina/1/18 (H1N1), A/New York/1/18 (H1N1), and A/Brevig Mission/1/18 (H1N1), respectively. To date, five RNA segment sequences have been published (Basler et al., 2001 Reid et al., 1999, 2000, 2002, 2004). More recently, the HA sequences of two additional fixed autopsy cases of 1918 influenza victims from the Royal London Hospital were determined (Reid et al., 2003). The HA sequences from these five cases show 㺙 percent sequence identity, but differ at amino acid residue 225 (see below).

The sequence of the 1918 HA is most closely related to that of the A/ swine/Iowa/30 virus. However, despite this similarity the sequence has many avian features. Of the 41 amino acids that have been shown to be targets of the immune system and subject to antigenic drift pressure in humans, 37 match the avian sequence consensus, suggesting there was little immunologic pressure on the HA protein before the fall of 1918 (Reid et al., 1999). Another mechanism by which influenza viruses evade the human immune system is the acquisition of glycosylation sites to mask antigenic epitopes. The HAs from modern H1N1 viruses have up to five glycosylation sites in addition to the four found in all avian HAs. The HA of the 1918 virus has only the four conserved avian sites (Reid et al., 1999).

Influenza virus infection requires binding of the HA protein to sialic acid receptors on the host cell surface. The HA receptor binding site consists of a subset of amino acids that are invariant in all avian HAs, but vary in mammalian-adapted HAs. Human-adapted influenza viruses preferentially bind sialic acid receptors with α(2-6) linkages. Those viral strains adapted to birds preferentially bind α(2-3) linked sugars (Gambaryan et al., 1997 Matrosovich et al., 1997 Weis et al., 1988). To shift from the proposed avian-adapted receptor-binding site configuration (with a preference for α(2-3) sialic acids) to that of swine H1s (which can bind both α(2-3) and α(2-6)) requires only one amino acid change, E190D. The HA sequences of all five 1918 cases have the E190D change (Reid et al., 2003). In fact, the critical amino acids in the receptor-binding site of two of the 1918 cases are identical to that of the A/swine/Iowa/30 HA. The other three 1918 cases have an additional change from the avian consensus, G225D. Because swine viruses with the same receptor site as A/swine/Iowa/30 bind both avian- and mammalian-type receptors (Gambaryan et al., 1997), A/ New York/1/18 virus probably also had the capacity to bind both. The change at residue 190 may represent the minimal change necessary to allow an avian H1-subtype HA to bind mammalian-type receptors (Reid et al., 1999, 2003 Stevens et al., 2004 Gamblin et al., 2004 Glaser et al., 2004), a critical step in host adaptation.

The crystal structure analysis of the 1918 HA (Stevens et al., 2004 Gamblin et al., 2004) suggests that the overall structure of the receptor binding site is akin to that of an avian H5 HA in terms of its having a narrower pocket than that identified for the human H3 HA (Wilson et al., 1981). This provides an additional clue for the avian derivation of the 1918 HA. The four antigenic sites that have been identified for another H1 HA, the A/PR/8/34 virus HA (Caton et al., 1982), also appear to be the major antigenic determinants on the 1918 HA. The X-ray analyses suggest that these sites are exposed on the 1918 HA and thus they could be readily recognized by the human immune system.

The principal biological role of NA is the cleavage of the terminal sialic acid residues that are receptors for the virus's HA protein (Palese and Compans, 1976). The active site of the enzyme consists of 15 invariant amino acids that are conserved in the 1918 NA. The functional NA protein is configured as a homotetramer in which the active sites are found on a terminal knob carried on a thin stalk (Colman et al., 1983). Some early human virus strains have short (11-16 amino acids) deletions in the stalk region, as do many virus strains isolated from chickens. The 1918 NA has a full-length stalk and has only the glycosylation sites shared by avian N1 virus strains (Schulze, 1997). Although the antigenic sites on human-adapted N1 neuraminidases have not been definitively mapped, it is possible to align the N1 sequences with N2 subtype NAs and examine the N2 antigenic sites for evidence of drift in N1. There are 22 amino acids on the N2 protein that may function in antigenic epitopes (Colman et al., 1983). The 1918 NA matches the avian consensus at 21 of these sites (Reid et al., 2000). This finding suggests that the 1918 NA, like the 1918 HA, had not circulated long in humans before the pandemic and very possibly had an avian origin (Reid and Taubenberger, 2003).

Neither the 1918 HA nor NA genes have obvious genetic features that can be related directly to virulence. Two known mutations that can dramatically affect the virulence of influenza virus strains have been described. For viral activation, HA must be cleaved into two pieces, HA1 and HA2, by a host protease (Lazarowitz and Choppin, 1975 Rott et al., 1995). Some avian H5 and H7 subtype viruses acquire a mutation that involves the addition of one or more basic amino acids to the cleavage site, allowing HA activation by ubiquitous proteases (Kawaoka and Webster, 1988 Webster and Rott, 1987). Infection with such a pantropic virus strain can cause systemic disease in birds with high mortality. This mutation was not observed in the 1918 virus (Reid et al., 1999 Taubenberger et al., 1997).

The second mutation with a significant effect on virulence through pantropism has been identified in the NA gene of two mouse-adapted influenza virus strains, A/WSN/33 and A/NWS/33. Mutations at a single codon (N146R or N146Y, leading to the loss of a glycosylation site) appear, like the HA cleavage site mutation, to allow the virus to replicate in many tissues outside the respiratory tract (Li et al., 1993). This mutation was also not observed in the NA of the 1918 virus (Reid et al., 2000).

Therefore, neither surface protein-encoding gene has known mutations that would allow the 1918 virus to become pantropic. Because clinical and pathological findings in 1918 showed no evidence of replication outside the respiratory system (Winternitz et al., 1920 Wolbach, 1919), mutations allowing the 1918 virus to replicate systemically would not have been expected. However, the relationship of other structural features of these proteins (aside from their presumed antigenic novelty) to virulence remains unknown. In their overall structural and functional characteristics, the 1918 HA and NA are avian-like, but they also have mammalian-adapted characteristics.

Interestingly, recombinant influenza viruses containing the 1918 HA and NA and up to three additional genes derived from the 1918 virus (the other genes being derived from the A/WSN/33 virus) were all highly virulent in mice (Tumpey et al., 2004). Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/ NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage (Kash et al., 2004). These findings were unusual because the viruses with the 1918 genes had not been adapted to mice. The completion of the sequence of the entire genome of the 1918 virus and the reconstruction and characterization of viruses with 1918 genes under appropriate biosafety conditions will shed more light on these findings and should allow a definitive examination of this explanation.

Antigenic analysis of recombinant viruses possessing the 1918 HA and NA by hemagglutination inhibition tests using ferret and chicken antisera suggested a close relationship with the A/swine/Iowa/30 virus and H1N1 viruses isolated in the 1930s (Tumpey et al., 2004), further supporting data of Shope from the 1930s (Shope, 1936). Interestingly, when mice were immunized with different H1N1 virus strains, challenge studies using the 1918-like viruses revealed partial protection by this treatment, suggesting that current vaccination strategies are adequate against a 1918-like virus (Tumpey et al., 2004). In fact, the data may even allow us to suggest that the human population, having experienced a long period of exposure to H1N1 viruses, may be partially protected against a 1918-like virus (Tumpey et al., 2004).

Because virulence (in the immunologically naïve person) has not yet been mapped to particular sequence motifs of the 1918 HA and NA genes, what can gene sequencing tell us about the origin of the 1918 virus? The best approach to analyzing the relationships among influenza viruses is phylogenetics, whereby hypothetical family trees are constructed that take available sequence data and use them to make assumptions about the ancestral relationships between current and historical influenza virus strains (Fitch et al., 1991 Gammelin et al., 1990 Scholtissek et al., 1993) (Figure 1-5). Because influenza viruses possess eight discrete RNA segments that can move independently between virus strains by the process of reassortment, these evolutionary studies must be performed independently for each gene segment.


Change in hemagglutinin (HA) and neuraminidase (NA) proteins over time. The number of amino acid changes from a hypothetical ancestor was plotted versus the date of viral isolation for viruses isolated from 1930 to 1993. Open circles, human HA closed (more. )

A comparison of the complete 1918 HA (Figure 1-5) and NA genes with those of numerous human, swine, and avian sequences demonstrates the following: Phylogenetic analyses based on HA nucleotide changes (either total or synonymous) or HA amino acid changes always place the 1918 HA with the mammalian viruses, not with the avian viruses (Reid et al., 1999). In fact, both synonymous and nonsynonymous changes place the 1918 HA in the human clade. Phylogenetic analyses of total or synonymous NA nucleotide changes also place the 1918 NA sequence with the mammalian viruses, but analysis of nonsynonymous changes or amino acid changes places the 1918 NA with the avian viruses (Reid et al., 2000). Because the 1918 HA and NA have avian features and most analyses place HA and NA near the root of the mammalian clade (close to an ancestor of the avian genes), it is likely that both genes emerged from an avian-like influenza reservoir just prior to 1918 (Reid et al., 1999, 2000, 2003 Fanning and Taubenberger, 1999 Fanning et al., 2000) (Figure 1-4). Clearly, by 1918 the virus had acquired enough mammalian-adaptive changes to function as a human pandemic virus and to form a stable lineage in swine.

Sequence and Functional Analysis of the Non-Structural Gene Segment

The complete coding sequence of the 1918 non-structural (NS) segment was completed (Basler et al., 2001). The functions of the two proteins, NS1 and NS2 (NEP), encoded by overlapping reading frames (Lamb and Lai, 1980) of the NS segment, are still being elucidated (O'Neill et al., 1998 Li et al., 1998 Garcia-Sastre et al., 1998 Garcia-Sastre, 2002 Krug et al., 2003). The NS1 protein has been shown to prevent type I interferon (IFN) production by preventing activation of the latent transcription factors IRF-3 (Talon et al., 2000) and NF-㮫 (Wang et al., 2000). One of the distinctive clinical characteristics of the 1918 influenza was its ability to produce rapid and extensive damage to both the upper and lower respiratory epithelium (Winternitz et al., 1920). Such a clinical course suggests a virus that replicated to a high titer and spread quickly from cell to cell. Thus, an NS1 protein that was especially effective at blocking the type I IFN system might have contributed to the exceptional virulence of the 1918 virus strain (Garcia-Sastre et al., 1998 Talon et al., 2000 Wang et al., 2000). To address this possibility, transfectant A/WSN/33 influenza viruses were constructed with the 1918 NS1 gene or with the entire 1918 NS segment (coding for both NS1 and NS2 [NEP] proteins) (Basler et al., 2001). In both cases, viruses containing 1918 NS genes were attenuated in mice compared to wild-type A/WSN/33 controls. The attenuation demonstrates that NS1 is critical for the virulence of A/WSN/33 in mice. On the other hand, transcriptional profiling (microarray analysis) of infected human lung epithelial cells showed that a virus with the 1918 NS1 gene was more effective at blocking the expression of IFN-regulated genes than the isogenic parental mouse-adapted A/WSN/33 virus (Geiss et al., 2002), suggesting that the 1918 NS1 contributes virulence characteristics in human cells, but not murine ones. The 1918 NS1 protein varies from that of the WSN virus at 10 amino acid positions. The amino acid differences between the 1918 and A/WSN/33 NS segments may be important in the adaptation of the latter virus strain to mice and likely account for the observed differences in virulence in these experiments. Recently, a single amino acid change (D92E) in the NS1 protein was associated with increased virulence of the 1997 Hong Kong H5N1 viruses in a swine model (Seo et al., 2002). This amino acid change was not found in the 1918 NS1 protein.

Sequence and Functional Analysis of the Matrix Gene Segment

The coding region of influenza A RNA segment 7 from the 1918 pandemic virus, consisting of the open reading frames of the two matrix genes, M1 and M2, has been sequenced (Reid et al., 2002). Although this segment is highly conserved among influenza virus strains, the 1918 sequence does not match any previously sequenced influenza virus strains. The 1918 sequence matches the consensus over the M1 RNA-binding domains and nuclear localization signal and the highly conserved transmembrane domain of M2. Amino acid changes that correlate with high yield and pathogenicity in animal models were not found in the 1918 virus strain.

Influenza A virus RNA segment 7 encodes two proteins, the matrix proteins M1 and M2. The M1 mRNA is colinear with the viral RNA, while the M2 mRNA is encoded by a spliced transcript (Lamb and Krug, 2001). The proteins encoded by these mRNAs share their initial 9 amino acids and also have a stretch of 14 amino acids in overlapping reading frames. The M1 protein is a highly conserved 252-amino-acid protein. It is the most abundant protein in the viral particle, lining the inner layer of the viral membrane and contacting the ribonucleoprotein (RNP) core. M1 has been shown to have several functions (Lamb and Krug, 2001), including regulation of nuclear export of vRNPs, both permitting the transport of vRNP particles into the nucleus upon infection and preventing newly exported vRNP particles from reentering the nucleus. The 97-amino-acid M2 protein is a homotetrameric integral membrane protein that exhibits ion-channel activity and is the target of the drug amantadine (Hay et al., 1985). The ion-channel activity of M2 is important both during virion uncoating and during viral budding (Lamb and Krug, 2001).

Five amino acid sites have been identified in the transmembrane region of the M2 protein that are involved in resistance to the antiviral drug amantadine: sites 26, 27, 30, 31, and 34 (Holsinger et al., 1994). The 1918 influenza M2 sequence is identical at these positions to that of the amantadine-sensitive influenza virus strains. Thus, it was predicted that the M2 protein of the 1918 influenza virus would be sensitive to amantadine. This was recently demonstrated experimentally. A recombinant virus possessing the 1918 matrix segment was inhibited effectively both in tissue culture and in vivo by the M2 ion-channel inhibitors amantadine and rimantadine (Tumpey et al., 2002).

The phylogenetic analyses suggest that the 1918 matrix genes, while more avian-like than those of other mammalian influenza viruses, were mammalian adapted (Reid et al., 2002). For example, the extracellular domain of the M2 protein contains four amino acids that differ consistently between the avian and mammalian clades (M2 residues #14, 16, 18, and 20). The 1918 sequence matches the mammalian sequence at all four of these residues (Reid et al., 2002), suggesting that the matrix segment may have been circulating in human virus strains for at least several years before 1918.

Sequence and Functional Analysis of the Nucleoprotein Gene Segment

The nucleoprotein gene (NP) of the 1918 pandemic influenza A virus has been amplified and sequenced from archival material (Reid et al., 2004). The NP gene is known to be involved in many aspects of viral function and to interact with host proteins, thereby playing a role in host specificity (Portela and Digard, 2002). NP is highly conserved, with a maximum amino acid difference of 11 percent among virus strains, probably because it must bind to multiple proteins, both viral and cellular. Numerous studies suggest that NP is a major determinant of host specificity (Scholtissek et al., 1978a, 1985). The 1918 NP amino acid sequence differs at only six amino acids from avian consensus sequences, consistent with reassortment from an avian source shortly before 1918. However, the 1918 NP nucleotide sequence has more than 170 differences from avian consensus sequences, suggesting substantial evolutionary distance from known avian sequences. Both the 1918 NP gene and protein sequences fall within the mammalian clade upon phylogenetic analysis.

Phylogenetic analyses of NP sequences from many virus strains result in trees with two main branches, one consisting of mammalian-adapted virus strains and one of avian-adapted virus strains (Gammelin et al., 1990 Gorman et al., 1991 Shu et al., 1993). The NP gene segment was not replaced in the pandemics of 1957 and 1968, so it is likely that the sequences in the mammalian clade are descended from the 1918 NP segment. The mammalian branches, unlike the avian branch, show a slow but steady accumulation of changes over time. Extrapolation of the rate of change along the human branch back to a putative common ancestor suggests that this NP entered the mammalian lineage sometime after 1900 (Gammelin et al., 1990 Gorman et al., 1991 Shu et al., 1993). Separate analyses of synonymous and nonsynonymous substitutions also placed the 1918 virus NP gene in the mammalian clade (Reid et al., 2004). When synonymous substitutions were analyzed, the 1918 virus gene was placed within and near the root of swine viruses. When nonsynonymous viruses were analyzed, the 1918 virus gene was placed within and near the root of the human viruses.

The evolutionary distance of the 1918 NP from avian and mammalian sequences was examined using several different parameters. There are at least three possibilities for the origin of the 1918 NP gene segment (Reid et al., 2004). First, it could have been retained from the previously circulating human virus, as was the case with the 1957 and 1968 pandemic virus strains, whose NP segments are descendants of the 1918 NP. The large number of nucleotide changes from the avian consensus and the placement of the 1918 sequence in the mammalian clade are consistent with this hypothesis. Neighbor-joining analyses of nonsynonymous nucleotide sequences or of amino acid sequences place the 1918 sequence within and near the root of the human clade. The 1918 NP has only a few amino acid differences from most bird virus strains, but this consistent group of amino acid changes is shared by the 1918 NP and its subsequent mammalian descendants and is not found in any birds, resulting in the 1918 sequence being placed outside the avian clade (Reid et al., 2004). One or more of these amino acid substitutions may be important for adaptation of the protein to humans. However, the very small number of amino acid differences from the avian consensus argues for recent introduction from birds� years after 1918, the NP genes of human influenza virus strains have accumulated more than 30 additional amino acid differences from the avian consensus (a rate of 2.3 amino acid changes per year). Thus it seems unlikely that the 1918 NP, with only six amino acid differences from the avian consensus, could have been in humans for many years before 1918. This conclusion is supported by the regression analysis that suggests that the progenitor of the 1918 virus probably entered the human population around 1915 (Reid et al., 2004).

A second possible origin for the 1918 NP segment is direct reassortment from an avian virus. The small number of amino acid differences between 1918 and the avian consensus supports this hypothesis. While 1918 varies at many nucleotides from the nearest avian virus strain, avian virus strains are quite diverse at the nucleotide level. Synonymous/nonsynonymous ratios between 1918 and avian virus strains are similar to the ratios between avian virus strains, opening the possibility that avian virus strains may exist that are more closely related to 1918. The great evolutionary distance between the 1918 sequence and the avian consensus suggests that no avian virus strain similar to those in the currently identified clades could have provided the 1918 virus strain with its NP segment.

A final possibility is that the 1918 gene segment was acquired shortly before 1918 from a source not currently represented in the database of influenza sequences. There may be a currently unknown influenza host that, while similar to currently characterized avian virus strains at the amino acid level, is quite different at the nucleotide level. It is possible that such a host was the source of the 1918 NP segment (Reid et al., 2004).

Future Work

Five of the eight RNA segments of the 1918 influenza virus have been sequenced and analyzed. Their characterization has shed light on the origin of the virus and strongly supports the hypothesis that the 1918 virus was the common ancestor of both subsequent human and swine H1N1 lineages. Sequence analysis of the genes to date offers no definitive clue as to the exceptional virulence of the 1918 virus strain. Thus, experiments testing models of virulence using reverse genetics approaches with 1918 influenza genes have begun.

In future work it is hoped that the 1918 pandemic virus strain can be placed in the context of influenza virus strains that preceded it and followed it. The direct precursor of the pandemic virus, the first or “spring” wave virus strain, lacked the exceptional virulence of the fall wave virus strain. Identification of an influenza RNA-positive case from the first wave would have tremendous value in deciphering the genetic basis for virulence by allowing differences in the sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would clarify which gene segments were novel in the 1918 virus.

In many respects, the 1918 influenza pandemic was similar to other influenza pandemics. In its epidemiology, disease course, and pathology, the pandemic generally was different in degree but not in kind from previous and subsequent pandemics. Furthermore, laboratory experiments using recombinant influenza viruses containing genes from the 1918 virus suggest that the 1918 and 1918-like viruses would be as sensitive to the Food and Drug Administration-approved anti-influenza drugs rimantadine and oseltamivir as other virus strains (Tumpey et al., 2002). However, there are some characteristics of the pandemic that appear to be unique: Mortality was exceptionally high, ranging from 5 to 20 times higher than normal. Clinically and pathologically, the high mortality appears to be the result of a higher proportion of severe and complicated infections of the respiratory tract, not with systemic infection or involvement of organ systems outside the influenza virus's normal targets. The mortality was concentrated in an unusually young age group. Finally, the waves of influenza activity followed each other unusually rapidly, resulting in three major outbreaks within a year's time. Each of these unique characteristics may find their explanation in genetic features of the 1918 virus. The challenge will be in determining the links between the biological capabilities of the virus and the known history of the pandemic.

Research Agenda for the Future

The work on the 1918 influenza virus, especially its origin, has led to the support of more comprehensive influenza virus surveillance and genomics initiatives for both human and animal influenza A viruses. We believe significant advancement in the understanding of influenza biology and ecology can be made by the generation of full genomic sequences of a large number of influenza viruses from different hosts. In conclusion, some of the questions that need to be addressed in pandemic influenza include the following:

American Experience

Much has changed since the influenza pandemic of 1918, yet our responses to COVID-19 must still rely on many of the century-old lessons.

The source of the influenza illness remained a mystery to scientists as viruses were too small and obscure for the optical microscopes available in 1918. Credit: Naval Historical Society

Pandemic. The word originates from the Greek word pandēmos – meaning from ‘all’ (pan) ‘people’ (dēmos). Today, the word conjures many frightening images but holds similar meaning – a geographically widespread or global malady, generally with regard to infectious disease outbreaks. Indeed, for centuries, novel diseases and pathogens have emerged to produce pandemics in human populations, causing widespread illness and death, as well as economic, social and political disruptions.

The ancient Greeks believed diseases to be of a spiritual origin – a punishment from the gods for wrongdoings. In the 5th century BCE, an outbreak characterized by sore throat, aches and respiratory distress was noted by Hippocrates and named "The Cough of Perinthus." In doing so, Hippocrates may have provided the first documented experience of perhaps the most notorious pandemic pathogen: influenza. Although it is unlikely that the Cough of Perinthus was the first influenza outbreak in humans, it is the first chapter or at least the prologue to a dramatic history of significant human influenza outbreaks. Medical historians believe large scale influenza outbreaks occurred in 1510 and 1557 that may have been pandemics, but an outbreak in 1580 marks what is widely regarded as the first true influenza pandemic. This pandemic caused upwards of 8,000 deaths in Rome and devastated cities in Spain – literally decimating some by killing one out of every 10 residents. Since that time, we have documented two additional influenza pandemics in the 18th century, another two in the 19th century, three in the 20th century and one thus far in the 21st century.

Of these, an influenza pandemic occurring in 1918 is the most infamous. Fueled by the transport of soldiers in the final stages of World War I, the outbreak quickly spread around the world in three distinct waves, infecting up to one-third of the people on earth and killing an estimated 50 to 100 million people. Infections were complicated with high rates of bacterial pneumonia, and the pandemic was characterized by a uniquely high mortality rate in young adults between 20 and 40 years of age. By the time the pandemic ended in 1920, it was the worst acute infectious disease outbreak in modern history and the greatest mortality event in the world since the Black Death – a 14th-century pandemic caused by the plague.

When young, healthy soldiers began getting sick by the dozens in March, 1918, military physicians were baffled by what might be causing it. Credit: National Archives and Records Administration

The 1918 pandemic had profound impacts on life in the United States. In October of 1918, some 195,000 Americans were killed by the outbreak. By the time it ended, over 600,000 had lost their lives, and thousands of children were orphaned. So dire was the situation that many cities including Boston, Richmond, St. Louis and others mandated quarantines and social-distancing measures. In San Francisco and Seattle, laws were passed forcing people to wear masks covering their mouths and noses while in public. The public health commissioner in Chicago told police to arrest anyone seen sneezing without covering their face in public.

These horrors were exacerbated by a number of factors. Many physicians and nurses were enlisted in the armed forces to aid in the efforts to win the First World War, leaving a depleted healthcare workforce. The outbreak stoked nativist reactions and the stigmatization of certain ethnic groups – such as anti-Italian sentiments in Denver – which limited the effectiveness of efforts to curtail the outbreak. There were also restrictions on communication and the flow of information. President Woodrow Wilson created the Committee on Public Information when the United States entered World War I, and at Wilson’s urging, Congress passed the Sedition Act in 1918, which allowed for up to 20 years of imprisonment for criticizing the government or spreading information that could hamper the production of materials necessary for the war effort. To this end, the government printed materials urging people to report anyone “who spreads pessimistic stories. cries for peace, or belittles our effort to win the war” to the Justice Department. These greatly limited communication and delayed the public health response to the emerging health crisis [1]. For example, newspapers in Washington, D.C. did not begin reporting on the outbreak until the last week of August – months after it had begun.

Much has changed for the better since 1918. For one, after Dr. Richard Shope isolated the influenza virus in his laboratory in 1931, we now know that influenza is caused by a virus. We no longer need to exclusively rely on non-pharmaceutical interventions to respond to influenza pandemics because we have developed and refined our ability to create and produce safe and efficacious vaccines. Sir Alexander Fleming discovered penicillin in 1928, which opened the door for developing antibiotics that can help treat complications from influenza, such as pneumonia. There is now an entire field of diplomacy dedicated to health and responding to the global threats posed by infectious diseases. And we no longer name pandemics after geographic locations, people, animals or cultural references in efforts to avoid stigmatization.

However, there have also been changes since 1918 that complicate the responses to pandemics. A growing body of evidence suggests pandemics may occur more frequently due to changes in land use, exploitation of the natural environment and demographic trends like urbanization—all of which increase the risk of infectious disease outbreaks. Today’s society is also undoubtedly more globalized than that of 1918. The World Health Organization estimates that our world is so interconnected, a pathogen could conceivably spread around the world in 36 hours. Simulations suggest that if a highly contagious and lethal pandemic similar to the 1918 influenza were to occur today, approximately 33 million people could die in 6 months.

Presently, we find ourselves in the midst of another pandemic. In December of 2019, a novel virus emerged in China and quickly spread throughout the country and the world, causing a disease called COVID-19, which stands for Coronavirus Disease 2019. Similar to the 1918 influenza, COVID-19 is a respiratory disease and pneumonia can be a complication. It has emerged in a time characterized by rising sentiments of nationalism and isolationism, and one in which the role of the media is in the spotlight. The spread of the disease has been fueled by the transportation of people around the world. And while we currently do not know exactly how contagious or deadly COVID-19 is, because we do not yet know the true number of persons infected, estimates based on available data suggest that it has the potential to rival the 1918 influenza.

The United States Public Health Service issued this pamphlet in October of 1918 as part of a public education campaign to slow the progress of the disease. Credit: Library of Congress, Rare Book and Special Collections Division

In the absence of pharmaceutical treatments and therapies for COVID-19, the response to the virus has relied heavily on non-pharmaceutical interventions and supportive treatment, much like the response to the 1918 influenza. Similarly, the response to COVID-19 has also witnessed the implementation of dramatic social distancing measures in cities, the passing of new policies designed to curtail the spread of disease and the stigmatization of people and culture that hinder the public health response.

The 1918 influenza pandemic was a pandemic in every sense of the word – global and affecting all people, from poor factory workers to world leaders like President Wilson. However, for all of the horrors that the 1918 influenza pandemic brought with it, the outbreak eventually came to an end and brought opportunities to learn and prepare for future pandemics. While our current situation is frightening, COVID-19 can be controlled through public health interventions, much as the influenza pandemic was eventually contained. The century-old lessons are clear: we must act swiftly, intentionally and implement multiple interventions simultaneously to curb the spread of disease.

Matthew Boyce

Matthew Boyce (@mattrbo) is a Senior Research Associate at the Center for Global Health Science & Security at Georgetown University. His research is focused on global health security, sustainable capacity development, and public health preparedness—especially at the local and sub-national levels.

Rebecca Katz

Dr. Rebecca Katz (@RebeccaKatz5) is a Professor and Director of the Center for Global Health Science and Security at Georgetown University Medical Center. She teaches courses on global health diplomacy, global health security, and emerging infectious diseases in the School of Foreign Service. Since 2007, much of her work has been on the domestic and global implementation of the International Health Regulations as well as global governance of public health emergencies.


Management of acute influenza has traditionally relied on supportive measures such as control of fever, symptomatic treatment, rehydration and treatment of complications, such as bacterial pneumonia, if they occur. Supportive care is still the mainstay of management of uncomplicated influenza. However, as described above, several antiviral agents have been developed for influenza or are in active clinical development. Furthermore, given the natural animal reservoirs of influenza (Fig. 3), prevention of transmission from these reservoirs to humans is key. Water treatment, indoor raising and biosecurity are the primary management goals these strategies are followed by quarantine, stamping out and the ultimate use of animal vaccination.

Human influenza

People with clinical symptoms of influenza virus infection should remain home and avoid contact with non-infected people until symptoms alleviate. As the level of virus replication is correlated with the clinical severity of illness, strategies to limit viral replication with antiviral agents would be expected to be effective in acute influenza illness. However, adults with an intact immune system who have had previous influenza infections rapidly limit the replication of these viruses without intervention, so the opportunity to attenuate viral replication with antiviral agents is limited. Effective use of antivirals for seasonal influenza in otherwise healthy adults requires early initiation of therapy, generally within the first 48 hours of symptom onset. However, decisions regarding treatment should consider the somewhat small benefit demonstrated in clinical trials and the overall low risk of complications 177 . The benefits of later therapy in otherwise uncomplicated influenza are unclear.

Patients with underlying immune defects, those with more-severe disease or those who have a higher risk of influenza complications 200 may benefit from antiviral administration, even if initiated after 48 hours of symptom onset 201 if they are still shedding virus. Antiviral therapy is strongly considered if the illness is progressing and symptoms are worsening, particularly if the patient continues to test positive for influenza virus. Indeed, prescription of antivirals for high-risk outpatients with acute respiratory illness is beneficial 202 . Furthermore, a diagnosis of influenza in patients who have been hospitalized with community-acquired pneumonia should be confirmed to increase antiviral treatment rates in patients 203 . Studies suggest that, since the 2009 pandemic, the use of antivirals in patients who have been hospitalized with influenza has increased 204 , which is an improvement. Complications of influenza virus infection might require specific treatments, as is the case with secondary bacterial pneumonia, which should be treated with appropriate antibiotics.

Finally, consideration should also be given to the possible use of immunotherapy aimed at controlling exacerbated pro-inflammatory responses in patients with severe symptoms, especially in combination with antiviral therapy. This approach is supported by recent clinical studies suggesting beneficial effects when inhibitors of cytokine production and leukocyte recruitment were added to NA inhibitor therapy 205 .

Animal influenza

As influenza in wild and domestic waterfowl is primarily spread through contaminated water, water treatment (with, for example, chlorine and ultraviolet light), biosecurity and indoor raising of animals are the main strategies to prevent transmission. Biosecurity includes placing poultry houses and piggeries on high ground away from high-density and backyard farms, live poultry markets and bodies of water to minimize contact with waterfowl and passerine birds. Risk assessment and training of personnel to change outdoor clothing, disinfect foot baths and treat food and water are also important aspects of biosecurity.

After a novel influenza virus has breached biosecurity, a strategy that includes rapid detection and stamping out is optimal 206 . Stamping out is easier when animals display signs of severe infection, but these can go unnoticed when LPAI infection occurs. After initial emergence of HPAI H5 or H7 in poultry, quarantine, culling, decontamination and compensation of farmers with restricted zones of movement are imposed. This classic strategy was successful in the majority of Eurasian countries to which HPAI H5N1 spread 206 . However, late detection and widespread dissemination of HPAI H5N1 in China, Indonesia, Vietnam, Bangladesh and Egypt resulted in endemicity, making virus eradication difficult 206 . One difficulty in eradication is inadequate provision of compensation to farmers, who will be incentivized to hide infected animals.

Poultry vaccines use inactivated whole virus in oil emulsion, recombinant DNA or vectorized vaccines. These vaccines can be highly effective at reducing disease signs and inter-species spread, but they do not induce sterilizing immunity and have not resulted in eradication from endemic countries. Poultry vaccination is highly contentious both the United Nations Food and Agriculture Organization (FAO) and the World Organisation for Animal Health (OIE) state that long-term vaccination without additional measures is not recommended 207 . Although poultry vaccination dramatically reduced human cases of H5N1 in Vietnam in 2006, it did not eradicate the virus and likely contributed to the antigenic diversity. On the other hand, vaccination used in combination with unvaccinated sentinel chickens and stamping out can achieve control and local eradication 208 .

Live poultry markets have been recognized as a breeding ground of influenza viruses. Changes to live poultry marketing achieved by implementing so-called clean days (that is, days when stalls are cleared of unsold poultry and disinfected) and no carry-over (that is, no infectious material remains when the next live birds arrive after disinfection) of live poultry improved hygiene and reduced virus spread 209,210 . A definitive solution would be the closing of all live poultry markets. Indeed, market closure contributed to the reduction of the spread of HPAI H5N1 in Hong Kong and H7N9 transmissions to humans in China 211 . Such changes without public training risk poultry smuggling from unregulated sources.

The management of influenza in pigs or horses is dependent on the use of vaccines. In pigs, influenza is a minor disease problem for which biosecurity is paramount, and vaccination uses primarily whole inactivated virus in oil emulsion. In horses, the problem with vaccination is the short-term protection offered, but antigenically matched adjuvanted inactivated vaccines and LAIVs are efficacious 212 .


Our findings indicate that online influenza surveillance data can be used in conjunction with a simple dynamical model and data assimilation methods to infer many key epidemiological parameters for both seasonal and pandemic influenza. Moreover, this approach provides joint estimates of these key epidemiological variables and parameters that best match influenza activity over the entire season. This model inference framework is able to depict the evolving epidemiological features of influenza across seasons and over the course of each outbreak.

The inference approach presented here used a simple well-mixed, single-age class SIRS model. The model includes simple distributions for sojourn times in compartments, in particular an exponential distribution for the infectious period, with fixed infectiousness until recovery, two assumptions that are probably not accurate and will bias estimates of the reproductive number (15, 27, 33). Such model misspecification is a potential source of error that suggests caution when interpreting these findings. However, the filters, through their continued adjustment of the model state, are able to partially compensate for model misspecification (14). Tests using synthetic data indicate the inference framework is able to accurately estimate key epidemiological parameters despite its simplicity (SI Appendix).

The model-filter framework also accommodates signals of second peaks in incidence, which would not be possible in an unforced SIRS model. Such simulation occurs, in principle, in two different ways: either by attributing the second peak to a humidity-driven increase in R0 large enough to compensate for depletion of susceptibles or by revising its estimates of state variables (such as the proportion susceptible) upward in light of data suggesting increasing incidence after a first peak. We summarize our parameter estimates by their values at the week of maximum epidemic forcing, but the actual values of these parameters vary during the simulation, due to the action of the filtering methods. Therefore, as expected, the shapes of the epidemic curves (SI Appendix, Figs. S15 and S16) are more faithful to the data than would be the predictions of an equivalent model with fixed parameters.

The ILI metric in the United States is recorded as the ratio of ILI-related patient visits to total doctor−patient visits the denominator of this ratio can be affected by circulating virus severity (e.g., a more virulent strain, as during the 2012–2013 season) and novelty (e.g., the 2009 pandemic). Year to year, these issues, as well as changes in the number of participating clinics, can introduce biases in the CDC ILI record. These biases are in part handled by the mapping parameter γ, which provides an estimate of the difference in medical attention-seeking behavior over different seasons. For the GFT data, which are based on online search behavior, γ also reflects the attention accorded an influenza outbreak in the general population. This attention likely varies with influenza virulence or confounding events such as more intensive media coverage that changes online search behavior. By estimating the γ parameter, we are able to compensate for unusual increases in ILI observations such as those seen during the 2012–2013 season due to intense media coverage of influenza. More importantly, the estimates of γ, using either the municipal GFT or regional CDC ILI+ data, are consistent with observationally derived estimates of asymptomatic infection rates (29, 34). The parameter γ therefore also appears to account for asymptomatic infections, although we did not explicitly model this phenomenon.

Our study provides estimates of initial susceptibility for both epidemic and pandemic influenza outbreaks. These estimates provide some interesting insights into the dynamics of influenza transmission over a large population. Susceptibility at the beginning of the spring 2009 pandemic wave, although significantly higher than any of the epidemic seasons, is only 75.6% (72.7–78.8%). It is not surprising to find susceptibility lower than 100%, as the elderly are often less susceptible to a pandemic strain due to prior exposure to structurally similar strains (19, 35, 36). However, initial susceptibility, commonly assumed to decrease over time because a portion of the population would have been infected during the herald wave, is even higher at the beginning of the fall 2009 wave (Fig. 1A). Likewise, we find higher R0/Re at the beginning of the fall wave than the spring wave. In a previous study (37), higher R0 was also estimated for the second 1918 pandemic wave in New York City. One hypothesized explanation is that cross-immunity conferred by recent winter infection with seasonal influenza strains provides partial protection against the initial spring emergence of a pandemic strain. The lower initial population susceptibility and R0 for the first wave of the 2009 pandemic might suggest similar cross-protection from seasonal influenza for the general population.

Pairwise analysis of R0 estimates at the time of maximum epidemic forcing during 2003–2013 reveals a positive correlation among most cities in the United States. Cities in the eastern United States exhibit a greater positive correlation of R0 than those located in the western United States. R0 estimates for Las Vegas and cities in Arizona were negatively correlated with many cities outside this region, suggesting differing transmission dynamics for the desert southwest of the United States.

We did not discriminate strains in the SIRS model. As such, our estimates may confound or blend outcomes due to overlapping outbreaks should there be multiple strains cocirculating. Future studies could address strain-specific inference as strain-specific data become available at the municipal level. Our inference system was run discontinuously for each season consequently, the immunity period, L, in the SIRS model was less constrained and thus not analyzed here. Constraining an increased number of state variables/parameters introduced by more comprehensive models will require data streams with additional information and finer resolution (e.g., serosurvey on population susceptibility and age-structured surveillance records). Such in-depth inference could be achieved in the future, as data of better quality become available to address these issues. For instance, data with finer age structure may allow more detailed inference on the transmission dynamics of pandemic versus epidemic influenza.

In summary, we have shown that the transmission dynamics of influenza among the general population can be inferred using data assimilation methods and big data estimates of incidence. As more people have access to and increasingly rely on online systems worldwide, mining of similar big data from online social networks may provide valuable information on the early spread of diseases (e.g., the early wave of a pandemic) as well as transmission dynamics for a number of other diseases. Such inference will rely heavily on the quality and reliability of these big data observational estimates.

When will the next pandemic take place, and which virus will cause it?

We simply do not know and there is no way of knowing. An influenza pandemic could start this winter, it could start next summer or it might not happen for more than five or ten years from now. Influenza viruses are inherently unpredictable. Scientists have expressed special concern about the A/H5N1 bird flu virus, currently circulating in poultry mostly in East and southeast Asia. However, this is just one of several ways a pandemic could start - as we saw in 2009 when a swine (pig) influenza virus resulted in an A(H1N1) pandemic starting in the Americas. Note there are over 70 types of animal influenza viruses, H7N7, H5N9, H7N4, etc., most of which are not going to end up causing a pandemic. This is work underway now to determine which of the animal viruses are more likely to undergo pandemic change and which should be prepared for. But this is certainly not a matter of making predictions.

Influenza (Flu) Viruses: Types, Symptoms, Naming and Vaccine

Influenza viruses can circulate in all parts of the world. Serious flu infection can result in hospitalisation or even death. People who basically are at risk due to serious flu infection include older people, young children, and people with certain health complications.

Influenza Viruses: Types

There are four types of influenza viruses namely A, B, C and D. Influenza A and B viruses circulate, causes acute respiratory infection and the seasonal epidemic of disease. Or we can say that Human influenza A and B viruses cause seasonal epidemics of disease.

Influenza A viruses: These are the only influenza viruses that cause flu pandemics that is global epidemics of flu disease.

Here the question arises what is a pandemic?

When a new and very different influenza A virus emerges and both infects people. Also, has the ability to spread efficiently among people. Then it is said to occur pandemic.

Influenza A viruses according to the combinations of the hemagglutinin (HA) and the neuraminidase (NA), they are further classified into subtypes. HA and NA are the proteins on the surface of the virus. Influenza viruses that circulated in humans are subtype A(H1N1) and A(H3N2). A(H1N1) is also written as A(H1N1)pdm09 as it caused a pandemic in 2009 and replaced the seasonal influenza virus A(H1N1) that was circulated prior to 2009. Let us tell you that only influenza type A viruses are known to have caused a pandemic.

Note: There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes. Potentially there are 198 different influenzas A subtype combinations, only 131 subtypes have been detected in nature.

Influenza B viruses: They are not classified into subtypes and can be broken down into lineages. Influenza B type virus belongs to either B/Yamagata or B/Victoria lineage.

Influenza C viruses: This type of virus is detected less frequently and usually causes mild infections and does not present public health importance. Or we can say that they do not cause human flu epidemic.

Influenza D viruses: They primarily affect cattle and does not infect or cause illness in people.

The above graph clearly shows that only two types of influenza viruses namely A and B cause most human illness and are responsible for the flu season each year.

Influenza Viruses: Symptoms

As we know that seasonal influenza is characterised by sudden onset of fever, cough (mainly dry), headache, muscle and joint pain, feeling of unwell, sore throat and a runny nose. It is also said that cough can be severe and can last for 2 or more weeks. In fact, most of the people recover from fever and other symptoms within a week without the need of medical attention. But for the people at high risk, influenza can be dangerous and can cause severe illness or death.

Let us tell you that illness range from mild to severe and even death. According to WHO, in the whole world, these annual epidemics are estimated to result in about 3 to 5 million cases of severe illness, and about 290000 to 650000 respiratory deaths.

Most of the deaths in industrialised countries are associated with influenza and occur among people age 65 or older. No doubt epidemics may result in high levels or worker/school absenteeism and productivity losses.

Seasonal influenza spreads easily with the rapid transmission in crowded areas like schools, nursing home, and markets. As we know that when an infected person coughs or sneezes the droplets that contain virus are spread in the air and can even spread up to one meter, and infect persons who are close and also to those who breathe these droplets in. A virus can also spread by hands contaminated with influenza viruses. Therefore it is recommended to prevent transmission people should cover their mouth and nose with a tissue when coughing, and wash their hands regularly.

In a temperate climate, seasonal epidemic mainly occurs during winter and in tropical regions, it can occur throughout the year.

The incubation period is the time from infection to illness that is about 2 days but ranges from one to four days.

Influenza Viruses: Naming

There is an internationally accepted naming convention for influenza viruses. In 1979, this convention was accepted by WHO and published it in February 1980 in the Bulletin of the World Health Organisation, 58(4):585-591 (1980).

Following components are used:

The antigenic type (like A, B, C, D).

- The host of origin (like swine, chicken, etc.). There is no host of origin designation for human-origin viruses. Examples:

(Duck example): avian influenza A(H1N1), A/duck.Alberta/35/76

- Geographical origin (Like Denver, Taiwan, etc.)

- Strain number (like 7,15, etc.)

- Year of collection (like 57,2009. etc.)

- For influenza A viruses, the hemagglutinin and neuraminidase antigen description are given in parentheses.

- There was a different name assigned to the 2009 pandemic virus that is A(H1N1)pdm09 to differentiate it from seasonal influenza viruses A(H1N1), etc.

- A variant virus is when influenza virus infects humans that normally circulate in swine (pigs). They are designated with a letter 'v'. Like A(H3N2)V virus.

Influenza Viruses: Vaccines

Few influenza viruses are associated with seasons influenza vaccines like one influenza A(H1N1), one influenza A(H3N2) and one or two influenza B viruses. A flu vaccine can protect against flu viruses that is it is like viruses used to make a vaccine. Seasonal flu vaccines don't protect against influenza C or D viruses. Therefore, we can say that flu vaccines will not protect against infection and illness that is caused due to other viruses and causes influenza-like symptoms. There are several other viruses besides influenza that can result in influenza-like illness and can spread during flu season.

Therefore, now you may have come to know about flu or influenza viruses, their types, symptoms, etc.

Systems vaccinology informs influenza vaccine immunogenicity

Vaccines are the most efficient way to control and eradicate infectious diseases. The smallpox vaccine has led to the eradication of variola virus, which has been the cause of a high number of human casualties for many years in the not so distant past. Other viral vaccines that have not yet led to eradication, but have remarkably reduced the burden of viral infections, are the poliovirus, measles virus, mump virus, rubella virus, and yellow fever virus vaccines. More recently, the development of hepatitis B virus, chicken pox, zoster, rotavirus, and human papilloma virus vaccines have highlighted the impact of modern vaccines in controlling viral infections, including those involved in cancer development. Nevertheless, there is room for the improvement of several existing viral vaccines, such as the influenza and dengue virus vaccines, and challenges in the generation of effective vaccines against some specific viruses, including respiratory syncytial virus, several herpesviruses, and HIV. It also might be possible to generate effective vaccines against emergent viral infections, including chikungunya, Hendra, Nipah, Zika, and ebolaviruses, but difficulties include the need for large and costly studies to assess vaccine efficacy and the unpredictability of where the next human infections with such emerging pathogens will occur. Another major scientific challenge in the development of novel and improved virus vaccines is that, despite the previous successes in vaccine development, based on studies assessing whether a vaccine is safe and efficacious, no definitive studies have exposed the immunological mechanisms associated with vaccine efficacy. Thus, we still do not know for the most part how vaccines work. Challenges include limitations associated with animal models and difficulties to access informative human samples from multiple tissues. In this respect, the application of systems biology tools to the study of human vaccines (so-called “systems vaccinology”) gives new hope for the elucidation of the mechanistic details associated with vaccine safety and efficacy. In PNAS, Nakaya et al. (1) use systems vaccinology to find new clues on the immunogenic and transcriptional networks that are associated with robust influenza vaccine responses correlated with protection.

Influenza vaccines were first developed in 1938 in the form of virus-inactivated vaccines. Today, high-yield influenza vaccine strains are grown typically in embryonated eggs, partially purified, inactivated, and formulated for intramuscular vaccination. The seasonal influenza virus vaccines contain at least three different vaccine strains that represent the three antigenically diverse circulating human influenza viruses: H1N1 and H3N2, influenza A viruses, and influenza B viruses. Some current seasonal influenza vaccines have a tetravalent formulation, to better cope with the two current lineages of influenza B viruses, B/Victoria and B/Yamagata. After the use of the first influenza virus vaccines, it was realized that influenza viruses change from year to year as a result of selective pressure to evade preexisting immunity developed after previous exposures to influenza. Therefore, updates on vaccine strain formulation were implemented to reflect the antigenic changes present in the circulating viruses, and yearly vaccination campaigns with updated vaccine strains were established. Today, in addition to egg-grown inactivated influenza virus vaccines, we also have available tissue-culture–grown inactivated vaccines, live attenuated influenza virus vaccines, and even baculovirus-produced recombinant protein influenza virus vaccines. Nevertheless, yearly vaccinations are still required to address the antigenic changes of the virus, and problems of vaccine mismatch occur from time to time when the circulating strains are significantly different from the vaccine strains. In addition, the efficacy of the influenza virus vaccine, even when matching the circulating viruses, is far from perfect, especially in the elderly, who represent one of the most vulnerable populations to severe and often lethal influenza virus infection (2). These problems have increased the interest in the possible use of adjuvants to improve the efficacy of influenza virus vaccines.

Systems vaccinology studies to better understand the immunogenicity (or lack of) of influenza virus vaccines have previously been conducted. In addition to the current PNAS study (1), the same group has studied the transcriptional responses in blood of adults vaccinated with inactivated influenza virus vaccines correlating with increased serum influenza HA antibody responses, considered to be predictors of protection (3). Additional systems vaccinology studies have identified baseline as well as early predictors to influenza vaccine responses in adults, illustrating the human variability in responding to immunization, at least in part caused by differences in the preexisting influenza immunity status (4). Early IFN transcriptional signatures in blood appear to be a hallmark of inactivated influenza virus vaccines that correlate with high antibody responses (5, 6). Early activation of Tfh responses correlated with the magnitude of the B-cell antibody responses in adults (7). In the recent PNAS publication (1), these studies have now been expanded to include immunological naïve infants, and to assess the impact of inclusion of MF59 in the vaccine, an oil-in-water–based adjuvant known to increase the magnitude of the antibody responses to inactivated influenza vaccines (8).

In the present PNAS study (1), samples were taken from 14- to 24-mo-old children enrolled in a phase II clinical trial to compare the immunogenicity of the trivalent inactivated influenza virus vaccine with and without MF59 adjuvant (Fig. 1). Two vaccine doses were given approximately 1 mo apart. Blood draws were taken before immunization and at days 1, 3, 7, and 31 after the second immunization to measure antibody, B-cell, and T-cell responses, as well as global transcriptomic changes. Among the subjects, some—but not all—had previous exposure to influenza, as evidenced by antibody titers before vaccination. As expected, after the last blood draw, antibody responses were higher in the MF59 adjuvanted group. Some of the interesting outcomes of these studies were: (i) the relatively low level of changes in transcriptional profiles induced by vaccination in infants compared with previous studies in adults (ii) the differences in the magnitude and kinetics of the plasmablast response, which appear to be stronger in adults and (iii) the high heterogeneity in responses in infants, with some showing more transcriptional repression than induction. Interestingly, this heterogeneity was not driven by lack or presence of preexisting exposure to influenza, highlighting the need to conduct more studies to determine the basis of the variability of the response to vaccination among humans. Remarkably, the more potent antibody responses generated in the subjects receiving MF59-adjuvanted vaccines correlated with strong transcriptional signatures in blood corresponding Systems vaccinology studies in humans have the potential to identify safe strategies that better mimic the early responses associated with long-lasting and potent immune responses induced by natural infection. to the IFN network, similarly to the responses previously observed in adults receiving nonadjuvanted influenza vaccine. Thus, the inclusion of MF59 in the vaccine in infants led to the induction of responses more similar to those observed in adults. These findings also highlight the importance of an early IFN signature after intramuscular vaccination with inactivated vaccines to induce protective responses. Such a signature most likely assures robust antigen presentation and activation of adaptive immune responses. It will be important to find out how general this observation is in the case of other vaccines, especially in the case of live attenuated influenza virus vaccines, which do not appear to induce high levels of seroconversion despite generating improved protection in children compared with inactivated vaccines (9).

Early predictors of influenza virus immunogenicity in infants. One- to 2-y-old children are immunized with trivalent influenza virus vaccine (TIV) or with TIV adjuvanted with MF59. Blood samples are taken prior and after vaccination to determine immunogenicity of the vaccine as well as the blood transcriptome. The data are analyzed and compared with preexisting data in adults to establish differences between children and adults, and to identify early correlates associated with vaccine efficacy. Drawings in the figure are courtesy of Pilar Garcia.

The main challenge with influenza virus vaccines is the development of vaccines that induce protection not only against currently circulating viruses, but also against future antigenically drifted virus strains, as well as antigenically diverse viruses belonging to multiple HA subtypes present in the animal reservoir, as potential seeds for future pandemic influenza viruses. The discovery of conserved B-cell and T-cell epitopes present in all influenza A virus strains, independent of their subtypes, and the identification of individuals in which responses against these conserved epitopes are present, has opened the possibility for the design of cross-protective “universal” influenza virus vaccines (10). However, in addition to the identification of immunogens that induce the type of cross-protective responses needed for a universal influenza virus vaccine, we need to understand the basis for the induction of long-lasting robust responses, such as those induced by most natural infections. Systems vaccinology studies in humans have the potential to identify safe strategies that better mimic the early responses associated with long-lasting and potent immune responses induced by natural infection. The inclusion of such approaches in clinical trials, as described in this PNAS article (1), could elucidate the immunological reasons for success or failure of vaccines.