Cell Host & Microbe

Review

Influenza Virus Evolution, Host Adaptation,

and Pandemic Formation
Jeffery K. Taubenberger1,* and John C. Kash1
1Viral Pathogenesis and Evolution Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases,

National Institutes of Health, Bethesda, MD 20892, USA

*Correspondence: [email protected]
DOI 10.1016/j.chom.2010.05.009

Newly emerging or ‘‘re-emerging’’ viral diseases continue to pose significant global public health threats.
Prototypic are influenza viruses that are major causes of human respiratory infections and mortality. Influenza
viruses can cause zoonotic infections and adapt to humans, leading to sustained transmission and emer-
gence of novel viruses. Mechanisms by which viruses evolve in one host, cause zoonotic infection, and adapt
to a new host species remain unelucidated. Here, we review the evolution of influenza A viruses in their reser-
voir hosts and discuss genetic changes associated with introduction of novel viruses into humans, leading to
pandemics and the establishment of seasonal viruses.

Introduction independent selective pressures (Taubenberger and Morens,

Zoonotic infections, in which a pathogen adapted to another 2009).
animal species causes disease in a human, may be sporadic,
dead-end infections without adaptation to humans (e.g., Ebola Overview of Influenza Pandemics
and Hanta viruses). Other zoonotic infections can stably adapt Influenza viruses are among the most common causes of human
to humans, leading to sustained person-to-person transmission respiratory infections and among the most significant because
(e.g., HIV and SARS). Influenza A viruses (IAVs) fall into this latter they cause high morbidity and mortality. Influenza outbreaks
category, in which multiple stable host switch events have been have apparently occurred since at least the Middle Ages, if not
reported. The mechanisms by which viruses stably adapt to new since ancient times (Taubenberger and Morens, 2009). In the
host species, often distantly related, are still largely uneluci- elderly, infants, and people with chronic diseases, influenza is
dated. Like IAVs, many of the viruses that have demonstrated associated with especially high mortality. In the United States,
an ability to cause zoonotic infections and/or transmit between influenza results in approximately 200,000 hospitalizations
host species have RNA genomes (Holmes, 2010). and 36,000 deaths in a typical endemic season. In addition to
With a low-fidelity viral RNA polymerase lacking exonuclease annual winter outbreaks, pandemic IAVs occasionally emerge,
proofreading capability and inherently high error rate, IAVs exist as they have every 8–41 years for at least several centuries.
as populations of quasispecies (Domingo et al., 1998). Random IAV pandemics are global outbreaks due to viruses with novel
mutations can be rapidly selected for or against, depending antigenic subtypes. Up to 50% of the population can be infected
upon the evolutionary pressures applied, including novel host in a single pandemic year and can be associated with a dramatic
environment (Landolt and Olsen, 2007), response to pre-existing increase in number of deaths.
immunity leading to antigenic drift (Smith et al., 2004), or antiviral In the last 500 years, since 1510, there have been approxi-
drug pressure leading to resistance (Ong and Hayden, 2007). mately 14 IAV pandemics; in the past 120 years, there were
The major natural host species of IAVs include wild aquatic pandemics in 1889, 1918, 1957, 1968, 1977, and 2009 (Tauben-
waterfowl and shorebirds (Webster et al., 1992). IAVs have berger and Morens, 2009). In 1918, the worst pandemic in
been able to adapt stably to a wide variety of animals, including recorded history caused approximately 675,000 total deaths in
avian and mammalian species, and novel human-adapted the United States and killed up to 50 million people worldwide
IAVs have emerged to cause pandemics several times in the (Johnson and Mueller, 2002). The 1957 and 1968 pandemics
last 100 years (Taubenberger and Morens, 2009). Each of the caused approximately 70,000 and 34,000 excess deaths in the
viruses causing these pandemics has emerged in a different United States, respectively (Noble, 1982). While it is too early
way, making generalizations about zoonotic IAV adaptation to to assess the full impact of the 2009 pandemic, in its first year,
humans difficult. As data have accumulated, it has become there have been approximately 12,000 deaths in the United
clear that IAV host switch events are polygenic and represent States, of which the vast majority have been in individuals under
different solutions to the common problem of replication and age 65.
transmission in a host (Taubenberger and Morens, 2009). The
relative paucity of stable IAV host switch events from the avian Overview of the Biology of IAVs
reservoir to humans and domestic animals further supports Influenza viruses (family Orthomyxoviridae) are enveloped nega-
the complexity of the process and severely limits our ability to tive-strand RNA viruses with segmented genomes containing
predict future emergence. Host switch/adaptation, transmissi- 7–8 gene segments. The five genera in the family include influen-
bility, and pathogenicity/virulence are each complex multifacto- zavirus A, influenzavirus B, influenzavirus C, thogotovirus, and
rial interactions between virus and host and are likely under isavirus (infectious salmon anemia virus) (Palese and Shaw,

440 Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc.
Cell Host & Microbe

Review

2007; Wright et al., 2007). The three influenzavirus genera differ

in host range and pathogenicity and are likely to have diverged
evolutionarily at least several thousand years ago. Influenza A
and B viruses have a similar structure, whereas influenza C is
more divergent. Influenza A- and B-type viruses contain eight
discrete single-stranded RNA gene segments, each encoding
at least one protein. Only IAVs pose a significant risk of zoonotic
infection, host switch, and the generation of pandemic IAVs.
IAVs are enveloped with a host cell-derived lipid membrane.
The eight gene segments encode at least 11 open reading
frames (ORFs) (Figure 1). IAVs are covered with projections of
three proteins: hemagglutinin (HA), neuraminidase (NA), and
matrix 2 (M2). HA is a glycosylated type I integral membrane
protein with functions both as the viral receptor-binding protein
and fusion protein. The matrix 1 (M1) protein is found under the
membrane and interacts with cytoplasmic domains of the
surface glycoproteins and also with the viral ribonucleoprotein
Figure 1. Diagrammatic Representation of an Influenza A Virus
(RNP) complexes. HA recognizes sialic acid (SA) (N-acetyl The two major surface glycoproteins, hemagglutinin (HA) and neuraminidase
neuraminic acid) bound to underlying sugars on the tips of (NA), along with small numbers of the matrix 2 (M2) ion channel protein, are
host cell glycoproteins. IAVs have HAs with different specific- embedded in a lipid bilayer. The matrix 1 (M1) protein underlies the envelope
and interacts with the surface proteins and also with the ribonucleoproteins
ities for the disaccharide consisting of SAs and the penultimate (RNPs). RNPs consist of the eight negative-stranded RNA segments and
sugar (galactose or N-Acetylgalactosamine [GalNAc]) with nucleoprotein (NP) and the polymerase complex heterotrimer (PB2, PB1,
different glycosidic bond isomerization. IAVs adapted to birds and PA). The nuclear export protein (NEP, or nonstructural protein 2, NS2) is
contained within the virion, but the nonstructural protein 1 (NS1) is not.
have an HA receptor-binding specificity for a2-3 SA, while
HAs from IAVs adapted to humans have higher specificity for
a2-6 SA (see below). After binding its receptors, the virus is Each IAV RNA segment is encapsidated by nucleoprotein
internalized, and the acidic pH in the endosomal compartment (NP). In virions, viral RNA are wrapped around NP monomers
leads to a conformational change in HA, mediating fusion of and packaged into viral RNPs, along with the three polymerase
the viral and endosomal membranes, allowing release of viral proteins (polymerase basic protein 2 [PB2], PB1 polymerase
RNPs into the cytoplasm. Mature HA is a trimer, and each basic protein 1 [PB1], and polymerase acidic protein [PA]).
monomer undergoes proteolytic cleavage to generate disul- The polymerase proteins form a heterotrimer bound to a short
fide-bonded HA1 and HA2 polypeptide chains prior to activa- hairpin structure formed by the partially complementary terminal
tion. IAV requires exogenous serine proteases (trypsin-like 50 and 30 untranslated regions (UTRs) of each RNA segment. PB1
enzymes) for activation, recognizing a conserved Q/E-X-R motif serves as the RNA-dependent RNA polymerase. PB2 functions
found at the HA cleavage site (Chen et al., 1998). In humans and in mRNA synthesis by binding host mRNA caps. While PA
other mammals, this is likely to be the tryptase Clara produced is necessary for a functional polymerase complex, including
by cells of the bronchiolar epithelium. Cleavage activation of HA endonucleolytic cleavage of host RNAs, its biological roles
likely requires similar proteases in avian intestinal and/or remain poorly understood. It may have additional proteolytic
respiratory cells. IAV of H5 and H7 subtypes can acquire inser- activity and may also act as an elongation factor during RNA
tional mutations at the HA cleavage site, changing their protease synthesis. NP acts primarily as a single-strand RNA-binding
recognition site to a furin-like recognition sequence, R-X-R/K-R. protein and serves as the structural protein in the RNPs. In addi-
This polybasic cleavage site broadens protease specificity, tion, it plays an important role in transcription and in the traf-
allowing intracellular cleavage activation and systemic replica- ficking of RNPs between the cytoplasm and nucleus. IAV RNA
tion, resulting in a highly pathogenic avian influenza (HPAI). transcription and replication occur in the host nucleus, since
Prototypic is the Eurasian lineage of H5N1 HPAI, which has IAV is dependent on the RNA processing machinery of the host
circulated widely in the last decade, causing high mortality cell. The processes of being imported into the nucleus, exported
in domestic poultry as well as causing human infections and back out to the cytoplasm, and then prevented from re-entering
death. the nucleus also all appear to depend on the interaction of NP
NA is a type II integral membrane glycoprotein with sialidase with host proteins.
enzymatic activity required for cleavage of both host cell SAs, The nonstructural 1 (NS1) protein has multiple functional
allowing release of newly produced virions, and SAs on viral domains, including N-terminal dsRNA binding (1–73) containing
glycoproteins to prevent aggregation of nascent viral particles. a nuclear localization signal (NLS), a central effector domain
The complementary functions between SA binding by HA and (73–207) containing a nuclear export signal, and a C-terminal
SA removal by NA likely require evolutionary coadaptation. region (207–230) containing a PDZ domain (Hale et al., 2008). NS1
Both HA and NA are the major antigenic targets of the humoral has pleiotropic functions, including dsRNA binding, enhance-
immune response to IAV, and NA is the target of the antiviral ment of viral mRNA translation, inhibition of host mRNA process-
drugs oseltamivir and zanamivir. The small protein M2 is a proton ing, and type I interferon antagonism (Palese and Shaw, 2007).
channel necessary for viral replication and is the target of the The NS2 protein (also referred to as nuclear export protein,
adamantane class of antiviral drugs. NEP) is found in virions and facilitates nuclear export of viral

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RNP complexes. Another small viral protein, PB1-F2, is variably ing has been described and has played a role in changing the
encoded within the PB1 gene by an alternative reading frame, virulence or fitness of some IAVs (Wright et al., 2007).
targets the mitochondrial inner membrane, and may play a role
in apoptosis during IAV infection. Recently, the PB1 gene has IAVs in Wild Birds
also been reported to encode a third polypeptide expressed via Genetically and antigenically diverse IAVs are widely distributed
differential AUG codon usage, termed N40 (Wise et al., 2009). in wild avian species around the world. They are maintained
Type B and C influenza viruses are adapted to and isolated predominantly by asymptomatic infections (termed low patho-
almost exclusively from humans, although influenza B viruses genic avian influenza [LPAI]), most frequently documented in
have been isolated from seals and influenza C viruses have aquatic birds of the orders Anseriformes (ducks, geese, swans,
been isolated from pigs and dogs (Wright et al., 2007). In etc.) and Charadriiformes (gulls, terns, etc.). At least 105 species
contrast, however, IAVs infect a wide variety of warm-blooded of wild birds have been identified as harboring IAVs (Munster
animals, including birds, swine, horses, and humans. Avian IAV et al., 2007). IAVs in wild aquatic birds tend to be predominantly
in aquatic birds likely serves as the predominant natural reservoir transmitted via a fecal-oral route and to infect epithelial cells in
for all known subtypes and probably is the ultimate source of all the lower intestinal tract, where they cause little to no apparent
human pandemic IAV strains (Webster et al., 1992). disease.
IAVs are subdivided by antigenic characterization of the HA The distribution of HA and NA subtypes is not uniform among
and NA surface glycoproteins. Sixteen HA and nine NA subtypes wild bird IAV isolates, with some HA subtypes being more
are known, all of which have been isolated from avian hosts. common than others. While most HA subtypes have been iso-
Theoretically, therefore, 144 possible HA-NA subtype combina- lated from the Anseriformes, HA subtypes H13 and H16 have
tions are possible, and IAVs expressing at least 116 of these been isolated predominantly from Charadriiformes (Munster
subtype combinations have been isolated in birds (Krauss et al., 2007). IAVs have been isolated from other orders of birds
et al., 2004; Munster et al., 2007). World Health Organization (e.g., the large order Passeriformes, containing over half the
guidelines for the nomenclature of influenza viruses are as bird species), but most surveillance efforts have focused on
follows. First, the type of virus is designated (A, B, or C), then Anseriformes (aquatic waterfowl) and to a lesser extent Chara-
the host (if nonhuman), place of isolation, isolation number, driiformes (shorebirds) because of the high prevalence of IAV
and year of isolation (separated by slashes). For IAV, HA (H1– infection in these species, especially in dabbling ducks. There
H16) and NA (N1–N9) subtypes are noted in parentheses. For are little available data to assess whether particular IAV subtypes
example, strains included in the most recent trivalent vaccine or strains possess adaptations to particular wild bird host
for the 2010–2011 season in the United States are: A/Califor- species outside of the observation that IAVs with H13 and H16
nia/7/2009 (H1N1), A/Perth/16/2009 (H3N2), and B/Brisbane/ HA subtypes are isolated predominantly from gulls. Ecological
60/2008. differences, like differences in feeding behavior, geographic
localization, and migratory patterns, likely all play roles in the
Antigenic Drift and Antigenic Shift complex ecobiology of IAV in birds.
IAVs are evolutionarily dynamic viruses and have high mutation Two evolutionary models can explain the global pattern of IAV
rates (ranging from approximately 1 3 103 to 8 3 103 substi- diversity in wild birds, analogous to the allopatric and sympatric
tutions per site per year) (Chen and Holmes, 2006). Mutations models of speciation, and it is likely that both have played roles in
that change amino acids in the antigenic portions of the surface IAV evolution in wild birds (Dugan et al., 2008). Phylogenetic
glycoproteins HA and NA may produce selective advantages for analyses demonstrate that while all IAV HA subtypes had a
viral strains by allowing them to evade pre-existing immunity. common ancestor, the HA subtypes did not originate in a single
This is especially important in IAVs adapted to humans, which radiation and include higher-order clustering. Intersubtype
are subjected to strong population immunologic pressures. genetic diversity is high, but intrasubtype diversity is quite low.
Antibodies against the HA protein prevent receptor binding, Hence, the genetic structure of avian IAV HA is characterized
can be neutralizing, and are effective at preventing reinfection by highly divergent subtypes that harbor relatively little internal
with the same strain (Murphy and Clements, 1989). Such selec- genetic diversity. This is also the case for the evolution of the
tive mutation in the mapped antigenic domains of HA and NA has nine NA subtypes. Interestingly, analyses suggest that this diver-
been termed antigenic drift. sity reflects a very recent origin, with ranges for the most recent
Because the IAV genome consists of eight discrete RNA common ancestors (TMRCAs) of the different HA subtypes in the
segments, coinfection of one host cell with two different IAVs period of the last several hundred years (Chen and Holmes,
can result in progeny viruses containing gene segments of 2010). The NS gene segment in bird IAV is characterized by
both parental viruses. When this process of genetic reassort- a deep divergence between the A and B alleles, strongly sug-
ment involves the gene segments encoding the HA and/or NA gesting that the two alleles are subject to some form of balancing
genes it has been termed antigenic shift. There are theoretically selection (Dugan et al., 2008). Far less genetic diversity is
256 (28) possible combinations of the eight gene segments from observed in the five remaining IAV gene segments in wild birds
reassortment between two parental viruses. Reassortment has (PB2, PB1, PA, NP, and M). Phylogenetic analyses also reveal
been shown to be both common and important in IAV evolution a clear separation of avian IAV sequences from eastern and
(Dugan et al., 2008; Holmes et al., 2005) and host switch events western hemispheres, supporting allopatric evolutionary pres-
(Garten et al., 2009; Scholtissek et al., 1978). Homologous sures (Dugan et al., 2008; Munster and Fouchier, 2009). Mixed
recombination is not common in negative-sense RNA viruses infection and reassortment has also been shown to be extremely
like IAV (Boni et al., 2008), but recombination by template switch- common in IAVs in wild birds (Wang et al., 2008) with little

442 Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc.
Cell Host & Microbe

Review

evidence of genetic linkage among specific segments. The large resulting in a polybasic amino acid cleavage site within the HA
number of different HA-NA subtype combinations recovered (Wright et al., 2007). The current Asian-lineage HPAI H5N1 pan-
also highlights the frequency of reassortment in avian IAVs zootic appears to be unique in the era of modern influenza
and provides little evidence for the elevated fitness of specific virology (Webster et al., 2007), resulting in the deaths of millions
HA-NA combinations. In contrast to the extensive genetic diver- of poultry in 64 countries on three continents, either from infec-
sity seen in HA, NA, and NS, the five remaining internal gene tion or culling. There are also significant zoonotic implications
segments encode proteins that are highly conserved at the of this panzootic, with 498 documented cases in humans, result-
amino acid level, indicating that they are subject to widespread ing in 294 deaths in 15 countries since 2003 as of May 2010.
purifying selection. The fitness landscape for these genes is
therefore not determined by cross-immunity but by functional IAVs in Mammals
viability, with less selective pressure to fix advantageous muta- IAVs have been isolated from numerous mammalian host
tions. Given such strong conservation of amino acid sequence, species, including humans, domestic pigs, horses, and dogs,
large-scale reassortment likely involves the exchange of func- as well as such diverse hosts as pinnipeds (seals), cetaceans
tionally equivalent segments, with little impact on overall fitness. (toothed whales), mink, and anteaters, among others (Landolt
Dugan et al. hypothesized that IAV in wild birds exists as a large and Olsen, 2007). Phylogenetic evidence suggests that all
pool of functionally equivalent and so often interchangeable mammalian IAV strains ultimately derive from the avian IAV
gene segments that form transient genome constellations, pool (Wright et al., 2007). IAV strains have been consistently
without the strong selective pressure to be maintained as linked isolated from pigs and horses, but only sporadically from other
genomes (Dugan et al., 2008). domestic and wild mammals. It is unclear if there are stable
IAVs maintained in wild birds have been associated with stable wild mammalian IAV hosts or if occasional zoonotic infections
host switch events to novel hosts, including domestic gallina- leads to localized outbreaks that do not persist. Only additional
ceous poultry, horses, swine, and humans, leading to the emer- surveillance can answer this question.
gence of viral lineages transmissible in the new host. Adaptation IAV in Swine
to domestic poultry species is the most frequent (Wright et al., Swine IAV was first clinically detected in 1918 in association with
2007). Stable host switching likely involves the acquisition of the 1918 pandemic (Taubenberger et al., 2001), and it is unclear if
a number of mutations, depending on the virus and the species, IAV infections in swine occurred prior to that time. Since then,
that serve to separate an individual clonally derived IAV strain swine IAV has been continuously recognized and is a disease
from the large wild bird IAV gene pool. Because adaptation to of major economic and public health importance (Landolt and
a new host likely limits the ability of these viruses to return to Olsen, 2007). IAVs of a number of subtypes have been isolated
the wild bird IAV gene pool (Swayne, 2007), these emergent from swine globally, a few causing enzootic infections and
viruses must evolve as distinct eight-segment genome constel- many causing only limited outbreaks without continued circula-
lations within the new host (Dugan et al., 2008; Taubenberger tion. Since the isolation of the first IAV by Shope from pigs in
and Morens, 2009). the 1930s, these ‘‘classical’’ swine H1N1 viruses, likely derived
from the 1918 pandemic IAV (Taubenberger et al., 2001), caused
IAVs in Domestic Galliform Poultry enzootic seasonal disease in pigs in North America and world-
Domesticated birds of the order Galliformes (e.g., turkeys, wide (Reperant et al., 2009). From 1998, several different line-
chickens, quails, etc.) are not a reservoir host of avian IAV, but ages of ‘‘triple’’ reassortant viruses of H3N2, H1N2, and H1N1
are nonetheless susceptible to infection with wild-bird-derived subtypes, containing genes from the classical swine H1N1,
IAV after adaptation. Once IAVs are adapted to gallinaceous human H3N2, and avian IAVs, emerged to cause enzootic
poultry, they rarely circulate in the wild bird IAV viral pool disease in pigs in the U.S. and globally (Olsen, 2002). In Europe,
(Swayne, 2007). A dramatic exception to this trend is the recent a novel lineage of H1N1 emerged in the late 1970s by adaptation
Eurasian-lineage HPAI H5N1 viruses that have been isolated in of an avian IAV to swine, leading to enzootic disease in Eurasia
wild bird populations in Europe and Asia. (Dunham et al., 2009; Pensaert et al., 1981). Other fully avian or
The molecular features of host adaptation to domestic gallina- fully human IAV-derived isolates or reassortant viruses contain-
ceous poultry are not yet fully elucidated, but include positive ing genes of avian, swine, and/or human IAV origin have been
selection for mutations in both HA and NA (Campitelli et al., associated with less widespread disease in swine (Reperant
2004; Perez et al., 2003) and viral RNP proteins (Wasilenko et al., 2009).
et al., 2008). IAVs isolated from domestic poultry generally Since swine have been shown to be susceptible to infection
maintain an HA receptor-binding specificity for a2-3 SA (Wright with both avian and human IAV strains, this host species has
et al., 2007). Another characteristic feature of poultry-adapted been considered a prime ‘‘mixing vessel’’ or intermediate host
IAV is an in-frame deletion of approximately 20 amino acids for the generation of IAVs of pandemic potential to humans
from the stalk region of the NA (Blok and Air, 1982). NA stalk (Webster et al., 1992). This dual susceptibility has been associ-
deletion has been associated with reduced enzymatic activity ated with the presence of both a2-3 and a2-6 SA linkages on
of the NA (Baigent and McCauley, 2001) and may be a compen- the glycocalyx of epithelial cells lining the pig trachea, but recent
satory adaptive change to reduced HA receptor-binding activity lectin histochemistry studies have shown little a2-3 SA in the
of wild bird IAV adapted to replicate in the respiratory tract of nasal, tracheal, and bronchial epithelium (Van Poucke et al.,
poultry. 2010), with a SA receptor pattern not unlike the human respira-
Sporadically, strains of poultry-adapted H5 or H7 IAV evolve tory tree (Nicholls et al., 2007). These data, along with evidence
into HPAI, usually through acquisition of an insertional mutation, of limited replication and transmission of avian IAV in swine,

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Figure 2. Genetic Relationships between Human and Relevant Swine Influenza Viruses, 1918–2009
Gray arrows reflect derivation of one or more gene segments from the avian IAV gene pool (although the timing and mechanism of emergence in each case
remains unknown). The dashed red arrow indicates a period without circulation of H1N1 in humans. Solid red arrows indicate the evolutionary paths of human
IAV lineages; solid black arrows, of swine IAV lineages; and the black-to-red arrow, of the swine-origin 2009 human H1N1 pandemic IAV. IAVs contain eight gene
segments (as shown in Figure 1). The dashed, descending black arrows reflect human zoonotic infections with swine IAVs. Figure is modified from (Morens et al.,
2009).

further complicate our understanding of the mechanisms of IAV IAVs in Humans

avian-to-mammalian host switch events. Clearly, changes in HA Our understanding of pandemic IAV at the viral level is limited.
receptor-binding specificity play a significant role, but the Pandemic viruses have been isolated from 1957 onward, and
constellation of genes that control efficient replication and trans- the 1918 pandemic virus was reconstructed using an ‘‘archae-
mission in the new host is also vitally important. virologic’’ approach (Taubenberger et al., 2007), but we do not
IAV in Horses yet know the subtypes or genetic makeup of the pandemics
Unlike swine IAV, equine IAV has been recognized for hundreds before 1918. All the pandemic viruses since 1918 contain gene
of years (Taubenberger and Morens, 2009): interestingly, often in segments derived from the 1918 virus (Figure 2), and conse-
close association with human IAV epidemics or pandemics. quently, it may be considered that the past 91 years are part of
In the virologic era, the first IAV isolated from horses occurred a larger ‘‘pandemic era’’ (Morens et al., 2009).
in an epizootic (an outbreak in animals) in 1956. This lineage of The 1918–1919 Pandemic Virus
equine H7N7 is now likely extinct, but an H3N8 equine IAV The ‘‘Spanish’’ influenza pandemic killed an estimated 50 million
lineage first detected in the early 1960s continues to circulate or more people (Johnson and Mueller, 2002). Reconstruction of
enzootically (endemically in animals) and cause a high disease the viral genome from the tissues of several victims has demon-
and economic burden on the horse industry. In 1989, an strated that the causative agent was an avian-descended H1N1
independent, avian-like H3N8 virus was isolated following an virus (Rabadan et al., 2006; Taubenberger et al., 2005). As no
epizootic in horses in China, but this lineage has not persisted. human pre-1918 IAV sequences are currently available, the
Available phylogenetic evidence suggests that all these equine origin of the pandemic virus, including timing of its emergence
lineages were derived from avian IAV (Horimoto and Kawaoka, in humans and whether an intermediate host was involved,
2001). remains unresolved (Smith et al., 2009a). The high mortality

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associated with the 1918 virus appears to have been a result ness of the 1968 pandemic was the result of the retention of
of bacterial pneumonia, but the copathogenic mechanisms the previously circulating N2 NA (Kilbourne, 1997). The 1968
responsible for such fatal bacterial diseases remain unknown pandemic was so mild in its mortality impact that in some loca-
(Morens et al., 2008). Epidemiological features of the pandemic tions, fewer deaths occurred than in certain nonpandemic years
were also unprecedented, including its appearance in up to three (Morens et al., 2009). As had been the case in 1957, the virus
waves within the first year and a ‘‘W-shaped’’ (trimodal) age- quickly became endemic and seasonal in its appearance and
specific mortality curve that featured an unexplained peak in has now circulated globally for 42 years. Paradoxically, the
healthy young adults. morbidity and mortality burden of antigenic drift variants of the
By about 1920, the virus began to circulate in a pattern of H3N2 lineage over the past decades has been very high (Morens
seasonal endemic recurrences, and it remained so as it ‘‘drifted’’ et al., 2009). With the emergence of the novel H1N1 pandemic in
antigenically for nearly 40 years. When the next pandemic 2009, little evidence of H3N2 circulation was noted. However, as
appeared in 1957, the H1N1 virus disappeared from circulation. of May 2010, H3N2 continues to be isolated at low levels around
However, 20 years later, in 1977, it returned to circulation and the world.
caused a (low-grade) pandemic that disproportionately affected The 1977–1978 Pandemic Virus
persons of less than 20 years old. The 1918 H1N1-lineage IAV The re-emergence in 1977 of a descendant of the 1918 H1N1
continues to cocirculate globally today, along with H3N2 IAV virus that had been absent from circulation for 20 years consti-
descended from the 1968 pandemic and. since 2009, with the tutes a pandemic by definition, but it is usually regarded as
new H1N1 pandemic virus. It is remarkable not only that direct a ‘‘technical’’ pandemic that represents an unusual coda to the
(all-eight-gene segment) descendants of the 1918 virus still 1918 pandemic. It is curious that the same virus that disap-
circulate in humans as epidemic H1N1 viruses and in epizootic peared on its own in 1957 has, after reintroduction, been able
form as classical swine H1N1 viruses, but that for the past 50 to survive for over 30 years in the face of immunity pressures
years the original virus and its progeny have continually donated thought to be as great or greater than those associated with its
genes to new viruses to cause new pandemics, epidemics, and disappearance (i.e., high population immunity from natural infec-
epizootics (Figure 2). The novel H1N1 virus associated with the tion and additional immunity from annual vaccination, which is
ongoing 2009 pandemic is a fourth-generation descendant of much more common now than it was in 1957). It is considered
the 1918 virus (Morens et al., 2009). unlikely that human IAVs could have been maintained in nature
The 1957–1958 Pandemic Virus for 20 years without accumulating mutations, suggesting that
The H2N2 ‘‘Asian’’ pandemic virus that emerged in 1957 was the 1977 epidemic resulted from the release of a frozen strain
a lineal descendant of the 1918 H1N1 pandemic virus that from the 1950s. Molecular genetic analyses confirmed that the
acquired three novel gene segments by reassortment with an 1977 strain was very similar to early 1950s H1N1 strains in all
unknown avian virus. The gene segments encoding HA and NA eight gene segments (Nakajima et al., 1978). The H3N2 and
were replaced by an avian-like H2 subtype HA and an N2 H1N1 viruses cocirculated endemically for over 30 years in the
subtype NA (Scholtissek et al., 1978), respectively, with the other face of high population immunity (Rambaut et al., 2008). Coinfec-
five gene segments retained from the 1918-derived H1N1 tion with both subtypes has been reported, together with the
lineage. The gene segment encoding the PB1 polymerase was circulation of occasional reassortant H1N2 viruses. Like H3N2,
also replaced with an avian-like gene segment (Kawaoka et al., seasonal H1N1 also continues to be isolated at low levels (as
1989). Even though this pandemic occurred in an era when of May 2010) despite the emergence of the novel 2009 H1N1
experimental influenza virology was active, identity of the host virus. Whether seasonal H3N2 and/or H1N1 viruses will continue
in which reassortment event(s) occurred remains unknown. It is circulate along with derivatives of the 2009 pandemic H1N1 virus
also not known how long it took from the initial reassortment is unknown.
event(s) for the virus to evolve into the efficiently transmissible, The 2009–2010 Pandemic Virus
human-adapted IAV that caused the pandemic. Its pathology The current pandemic, caused by a novel H1N1 virus derived
and clinical appearance were similar or identical to those of from two unrelated swine H1N1 viruses (Garten et al., 2009),
the 1918 virus, although the unusual epidemiologic features of one of them a ‘‘classical’’ swine derivative of the 1918 human
the 1918 pandemic noted above were not seen in 1957. As virus and the other the European avian-like H1N1 lineage
was true for the 1918 pandemic, after about two years the virus (Dunham et al., 2009), adds to the complexity of pandemic virus
became seasonally endemic and sporadic, disappearing entirely emergence (Figure 2). The current H1N1 pandemic derived its
within 11 years. To date, it has not returned. NA and M gene segments from the European avian-like H1N1
The 1968 Pandemic Virus lineage and its remaining six gene segments (PB2, PB1, PA,
Like the pandemic that preceded it, the 1968 H3N2 ‘‘Hong HA, NP, and NS) from the North American swine H1N2 ‘‘triple’’
Kong’’ pandemic was caused by a reassortment event between reassortant lineage. The HA, NP, and NS gene segments of
a circulating human H2N2 virus and an avian IAV, acquiring novel this lineage are derived from the classical swine H1N1 (1918
HA (H3 subtype) and PB1 gene segments (Kawaoka et al., 1989; origin) lineage, while the polymerase gene segments have
Scholtissek et al., 1978). The other six gene segments, including different origins: PB2 and PA were derived from an avian IAV
the NA gene segment, were retained from the 1957 H2N2 virus source and PB1 from a human seasonal H3N2 virus when the
(including five segments—PB2, PA, NP, M, and NS—retained ‘‘triple’’ reassortant swine lineage emerged in the late 1990s
from the 1918 H1N1 lineage). Antibodies to NA, while not pre- (Smith et al., 2009b). Despite the recent explosion in IAV surveil-
venting infection, have been shown to reduce the duration and lance and gene sequencing, we do not know when and where
severity of illness. It has been suggested that the relative mild- the novel pandemic virus originated or the species in which the

Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc. 445
 Cell Host & Microbe

Review

reassortment event took place, although evolution in swine is ment with other avian IAVs (Chen et al., 2006). It is not yet clear
a favored hypothesis. TMRCA analyses suggest that the which of these many changes are associated with lethality in
pandemic virus was circulating in humans for at least several wild birds or with pathogenicity and transmissibility in poultry
months prior to its detection in early 2009 (Smith et al., 2009b). or other species. At the same time, adaptation of H5N1 HPAI
strains associated with asymptomatic, endemic infection of
IAV Host Switch Events domestic ducks is probably contributing to continuing spillover
Avian-to-Mammalian Host Switch Events into poultry, leading to the maintenance of a pool of pathogenic
The mechanisms by which avian IAVs cross species barriers to viruses to which humans will be continually exposed (Sturm-
infect humans or other mammals, either causing dead-end Ramirez et al., 2005). Nevertheless, there are limited data relating
infections or leading to subsequent transmission in the novel to whether or not any H5N1 IAV strain is evolving in the direction
mammalian host, are unknown. Moreover, the properties of of human adaptation.
IAVs that have the greatest medical and public health relevance, Avian H7 and H9 Subtype Infections
such as human infectivity, transmissibility, and pathogenicity, Although overshadowed by the spread of H5N1, during the
appear to be complex and polygenic and are poorly understood past decade at least eight other major poultry epizootics have
(Parrish et al., 2008; Taubenberger and Morens, 2009). occurred, caused either by emergence of novel H5 or H7 sub-
Avian IAV Infections in Swine and Horses type HPAI viruses unrelated to Asian H5N1 viruses or, in one
Although a number of fully avian IAVs (without reassortment with case, by an H9N2 LPAI virus (Alexander, 2007). Some of these
a human or swine IAV) of various subtypes have been isolated epizootics have featured human infections with few human
from swine populations globally (Brown, 2000), most of these deaths. Since the mid-1990s, strains of H9N2 LPAI have become
transmission events have been self-limited and not led to stable, enzootic in domestic poultry populations on several continents
long-term circulation of novel swine-adapted IAVs. The Euro- (Alexander, 2007), leading to a small number of human infec-
pean avian-like lineage of swine H1N1 is an important exception, tions. Like H5N1, different genetic lineages of H9N2 have been
having circulated in swine since the late 1970s (Dunham et al., established, some of which share with H5N1 viruses closely
2009) and also playing a role in the emergence of the 2009 related internal gene segments. Some H9N2 viruses have even
pandemic H1N1 lineage (Garten et al., 2009). Both of the stable acquired enhanced specificity for the human form of the HA
equine IAV lineages are thought to have derived from avian sour- receptor (Matrosovich et al., 2001). In 2003, an H7N7 HPAI virus
ces, and an epizootic of an avian-derived H3N8 virus was caused a poultry epizootic in the Netherlands and spread region-
detected in horses in China in 1989. ally. Before the epizootic was contained, at least 86 poultry
Avian IAV Infections in Humans workers and three of their contacts had become infected and
In the past decade, a large number of documented zoonotic developed conjunctivitis with or without an influenza-like illness;
avian IAV infections of humans have occurred, predominantly one of them died (Fouchier et al., 2004). Similarly, two persons
in association with HPAI epizootics of H5N1 in Eurasia and Africa developed influenza conjunctivitis during an outbreak of H7N3
(Peiris et al., 2007) and in smaller epizootics with H7N7 HPAI in HPAI in Canada in 2004 (Tweed et al., 2004). The H5N1 epizo-
the Netherlands (Fouchier et al., 2004), H7N3 HPAI in Canada otics are unique, however, in causing infections and deaths in
(Tweed et al., 2004), and LPAI infections with H9N2 (Lin et al., a large number of wild bird species (Alexander, 2007), occasional
2000), all without evidence of stable adaptation or sustained infections in wild and domestic mammals, severe and fatal
human-to-human transmission. Prior to this, there was limited human spillover infections (Peiris et al., 2007), and in rare
evidence of direct avian-to-human IAV exposure, based on a instances, limited human-to-human transmission.
small number of experimental human volunteer infections with Mammalian-to-Mammalian Host Switch Events
LPAI (Beare and Webster, 1991) or by serological surveillance The historical medical literature frequently described equine IAV
(Malik Peiris, 2009). epizootics in relation to IAV epidemics in humans (Taubenberger
H5N1 Infections and Morens, 2009) but also to IAV infections in dogs. Recently,
The continuing spread of H5N1 HPAI viruses into poultry popu- equine H3N8 viruses have adapted to dogs in a stable host
lations on several continents, associated with a growing number switch event (Crawford et al., 2005), where they are now evolving
of human zoonotic infections, has been associated with intense by antigenic drift. Equine H3N8 viruses were also recently iso-
public health interest and concern for a future pandemic (Wright lated from pigs in China, without evidence of stable host switch
et al., 2007). H5N1 HPAI viruses initially caused a 1996 poultry (Tu et al., 2009).
epizootic in southern China, followed within a year by an epizo- Swine-adapted IAVs have also been associated with zoonotic
otic in Hong Kong that produced 18 human cases and six deaths infections in humans, with 37 documented cases between 1958
(Claas et al., 1998; Subbarao et al., 1998). H5N1 strains con- and 2005 with several reported fatalities (Myers et al., 2007),
tinued to circulate thereafter in China, and they reappeared in including an outbreak of classical swine H1N1 infection in
epizootic form in 2003 and spread widely thereafter. Evidence soldiers at Fort Dix, NJ, in 1976 (Gaydos et al., 2006) with one
suggests that H5N1 viruses are evolving rapidly; however, the death. The ability of a swine-adapted IAV to result in a stable
direction of this evolution, which is driven by incompletely under- host switch event to humans was proven only with the emer-
stood selection pressures, is unclear. While current strains of gence of the 2009 H1N1 pandemic virus (Garten et al., 2009;
Southeast Asian H5N1 HPAI viruses are descendants of the Smith et al., 2009b). It is not known why zoonotic infection with
1996 Chinese epizootic virus, significant genetic and antigenic previous swine-adapted IAV strains, including the 1976 Fort
evolution has since occurred (Guan et al., 2004), involving anti- Dix strain (Kilbourne, 2006) in which human-to-human transmis-
genic drift in the H5 HA, mutations in other genes, and reassort- sion was demonstrated, did not result in a stable host switch and

446 Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc.
Cell Host & Microbe

Review

emergence of a novel pandemic IAV (Shinde et al., 2009; Van binding to SA in determining host range (Nicholls et al., 2007;
Reeth and Nicoll, 2009). Taubenberger, 2006).
The HA receptor-binding domain (RBD) is conserved in avian
Molecular Correlates of Human Adaptation HAs, but those IAVs adapted to humans have mutations in
Despite significant research, fundamental questions about how several key residues of the RBD, including sites 138, 190, 194,
IAVs switch hosts remain unanswered. Also poorly understood 225, 226, and 228 (H3 numbering) (Wright et al., 2007). It is
are the viral genetic changes that underlie human adaptation, thought that mutations at these sites increase binding from
human-to-human transmissibility, and pathogenesis. Biological a2-3 to a2-6 SA. For H1-subtype IAV adapting to humans or
barriers to the fitness of viruses with various gene segment swine, E190D and G225D (H3 numbering) were shown to be
combinations are still poorly understood; however, virulence/ crucial changes for enhanced a2-6 binding, while for H2- and
pathogenicity, host adaptation, and host-to-host transmissibility H3-subtype HAs, Q226L was shown to be crucial (Wright et al.,
are likely independent properties that are associated with 2007). IAV with H13 and H16 HA subtypes have been isolated
different and possibly competing mutations. The role of virulence from gulls rather than ducks, and this may reflect a host adapta-
and pathogenicity in virus-host relationships is therefore unclear; tion, as both H13 and H16 HAs bear mutations in the RBD
pandemic viruses of comparatively low (e.g., 1968 and 2009), (Matrosovich et al., 2009). The HA RBD requirements for host
intermediate (e.g., 1889 and 1957), and high (e.g., 1918) patho- switch have been further complicated by recent studies. The
genicity have all adapted to humans and exhibited efficient majority of H5N1 virus infections have occurred in viruses with
transmissibility. RBD specificity for a2-3 SA, although some isolates have
Another complicating factor stems from the fact that 5 of the 8 acquired mutations giving them enhanced a2-6 SA binding
gene segments introduced with the 1918 IAVs (PB2, PA, NP, M, (Wright et al., 2007). Some avian IAV of H7N2, H7N3 (Belser
and NS) were retained in the reassortment events leading to the et al., 2008), and H9N2 subtypes (Matrosovich et al., 2001)
1957 H2N2 and 1968 H3N2 pandemics (Figure 2) and thus have have shown enhanced specificity for a2-6 SA and yet have
circulated continuously in both pandemic and seasonal human only caused occasional zoonotic infections in humans.
H1N1, H2N2, and H3N2 IAVs from 1918 to 2010 (Morens et al., Sequence analysis of 1918 pandemic HA (directly from clinical
2009). A number of groups have performed comparative anal- material) has shown that isolates differed at HA residue 225
yses to propose mutations that are signatures of human IAV (with three cases having the human aspartic acid and two cases
adaptation (Finkelstein et al., 2007; Taubenberger et al., 2005) retaining the avian glycine) (Reid et al., 2003). Glycan array and
that relate back to this single host switch event in 1918. Exami- structural analyses (Chandrasekaran et al., 2008; Stevens
nation of independent avian-to-mammalian host switch events, et al., 2006) showed that the 1918 variant with a glycine at 225
however, suggests a lack of parallel evolution (Dunham et al., had a blended a2-3/a2-6 SA-binding specificity while those
2009). The unexpected emergence of the 2009 H1N1 pandemic sequences with an aspartic acid at 225 had a a2-6 SA-binding
virus further supports the independent and polygenic nature of specificity, yet there was no appreciable difference in clinical
these host adaptation events in IAV (Herfst et al., 2010; Jagger course or autopsy findings between the cases (Reid et al.,
et al., 2010; Mehle and Doudna, 2009). 2003). Clearly, enhanced binding to a2-6 SA is not in itself
Changes in IAV Genome GC Content adequate for host switch of an avian-adapted IAV to humans,
Rabadan and colleagues have shown that avian-origin IAVs have nor is it a requirement for human infections. IAVs bearing the
a higher GC content than IAVs adapted to humans (Greenbaum 1918 HA with different RBD configurations (a2-3, a2-6, and
et al., 2008; Rabadan et al., 2006). Gene segments from the 1918 blended a2-3/a2-6 specificities) were all shown to be virulent
pandemic demonstrated a nucleotide composition and guanine- in mice (Qi et al., 2009) and ferrets, but viral constructs with
cytosine (GC) content similar to avian IAV, supporting an ultimate the 1918 HA having only a2-3 specificity did not transmit
avian origin for this pandemic virus (Rabadan et al., 2006). Similar between ferrets (Tumpey et al., 2007). Interestingly, 2009 H1N1
changes in nucleotide composition with a decline in GC content pandemic viruses with variable amino acids at the 225 site
were also shown for swine-adapted IAV (Dunham et al., 2009). have also been isolated.
The biological basis for these observations is not yet clear, Clearly, a number of fundamental questions remain unan-
but hypotheses include core temperature differences (with swered. Structural studies have shown that it is inadequate to
higher core temperatures in avian hosts), a mutational bias in consider only the linkage of the terminal SA in regard to deter-
the cellular components needed for RNA replication (Rabadan mining the specificity of different IAV HAs. What is the distribu-
et al., 2006), a human defense mechanism against RNA viruses tion of these diverse glycans on the respiratory tree? How
operating functionally analogous to the Apobec family of deam- much variability in expression patterns is observed between indi-
inases against HIV infection, or avoidance of immune detection viduals, or are there changes in glycan expression with age or
by mimicry of host mRNA composition (Greenbaum et al., 2008). other physiological states? What about HA subtypes other
HA Receptor-Binding Specificity than H1–H3? Several mutations have been reported to enhance
IAV infection is initiated by viral attachment mediated by HA the binding of H5 to a2-6, but fixation of such mutations has not
proteins binding to SA-bearing cell surface receptors. The occurred after at least 11 years of exposure of thousands of
binding interactions are very weak, and high avidity is achieved humans to H5N1, suggesting that changes in HA RBD during
by binding interactions between multiple HA molecules on the host adaptation are extremely complex and must differ from
virus and the SA receptors on the target cell (Matrosovich subtype to subtype (Ayora-Talavera et al., 2009).
et al., 2009). HA glycoproteins bind to certain SA isomers on The HA of H5 or H7 HPAI viruses with a polybasic cleavage site
the tips of host glycoproteins, strongly supporting a role for HA serves as a primary virulence factor in poultry. The HA of the

Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc. 447
 Cell Host & Microbe

Review

1918 pandemic virus does not have a polybasic cleavage site, human adaptation, the so-called ‘‘SR’’ polymorphism, involving
but nonetheless has been shown to be a virulence factor in residues 590–591. Both strategies may operate by preserving
mammalian models (Kash et al., 2004, 2006; Kobasa et al., a large, positively charged PB2 surface domain, implying that
2004). There is no evidence, however, linking HA virulence in this region is involved in interactions with undetermined host-
a mammalian model either with or without a polybasic cleavage specific factors (Mehle and Doudna, 2008).
site with host adaptation. Remaining IAV Genes
IAV RNP Complex Much fewer experimental data exist to evaluate the significance
Changes in the constellations of gene segments as well as muta- of proposed human-adaptive changes in IAV in the NA-, M-, and
tions in the RNP genes have been long implicated in the adapta- NS-encoded genes. Similarly, mutations associated with host
tion of avian IAV to humans (Wright et al., 2007). The PB1 gene adaptation in the NS gene are unclear. Although birds have
segment was shown to be very avian IAV-like in the 1918 virus two NS alleles (A and B), mammalian viruses contain only allele
(Taubenberger et al., 2005) and was replaced by reassortment A, and substitution of an allele A NS with an avian allele B NS
with an avian IAV in the 1957 and 1968 pandemics (Wright significantly restricted viral replication in nonhuman primates
et al., 2007). These reassortment events suggest that an avian (Treanor et al., 1989). The NS gene from the 1918 virus has
IAV-derived PB1 segment can function in an otherwise mamma- been reported to be a very potent inhibitor of antiviral and type
lian-adapted IAV without prior adaptation, as has been shown I IFN responses in cultured human lung cells (Geiss et al.,
experimentally. The PB1-F2 protein encoded from a +1 ORF 2002), but has >98% identity with NS1 from a number of LPAI
within the PB1 ORF (Chen et al., 2001) is highly variable in coding sequences. Moreover, the 1918 NS gene attenuated a lethal
sequence in wild bird IAV (Holmes et al., 2006) and is also varia- mouse-adapted virus (Basler et al., 2001); yet the 1918 virus is
bly encoded in human and swine IAV. Mutations in PB1-F2 have extremely virulent in mice (Tumpey et al., 2005). Several H5N1
been mapped as virulence factors in the 1918 virus and in some NS1 mutations have been associated with replication and
H5N1 HPAI viruses (Conenello et al., 2007), but there are no clear increased virulence, but their role in host switch is unclear. Seo
data linking PB1-F2 with host adaptation (Taubenberger et al., et al. reported that increased virulence in pigs of a human
2005). The PA and NP proteins have been associated with host H1N1 virus containing the NS gene of an H5N1 HPAI required
restriction, and a number of mutations have been proposed for substitution of aspartic acid for glutamine at amino acid 92
human adaptation (Wright et al., 2007). However, further exper- (Seo et al., 2002), and this mutation has been associated with
imental data mapping the host range restriction associated with increased resistance to interferon (Lipatov et al., 2005). Addi-
specific mutations are needed. tional mutations have been observed in H5N1 HPAI viruses
The PB2 gene segment, and in particular PB2 residue 627, that have been associated with increased virulence, but their
has been identified as an important determinant of host range significance for host switch to humans remains unclear (Hale
restriction (Subbarao et al., 1993) and virulence in animal models. et al., 2008).
Avian IAVs generally encode glutamine at this site, while human
isolates from 1918–2009 typically encoded lysine. Residues Conclusions
701–702, residing in a region of PB2 implicated in nuclear locali- Host switch events in IAV, i.e., the formation of pandemic IAVs or
zation (Gabriel et al., 2008; Tarendeau et al., 2007), have similarly the emergence of novel swine IAVs lineages, have been shown to
been identified as a host-adaptive locus (Gabriel et al., 2005), be independently evolving polygenic processes. Evolutionary
with the D701N mutation increasing both replication in mice analyses have demonstrated little evidence of either parallel or
(Li et al., 2005) and transmission in guinea pigs (Steel et al., convergent mutations in such events, suggesting a strong role
2009). Independent examples of selection of the PB2 D701N for historical contingency in the origination of any particular
mutation have also been observed, for example, in the nonpan- host switch genotype. Instead, mutations identified as important
demic avian-origin European swine H1N1 viruses (Dunham in one host switch event may or may not be observed in other
et al., 2009), as well as in some HPAI H5N1 viruses. Several other such events, depending on the viral and host genetic context
PB2 mutations have been proposed to play a role in human of each event. The utility of identifying sets of mutations as
adaptation (Taubenberger et al., 2005), but notably, the 2009 proxies to define whether a future IAV is acquiring changes
H1N1 pandemic virus PB2 (derived independently of the 1918 important in mammalian adaptation might thus be limited. Future
host switch event) shows little evidence of parallel evolution. progress will require a much better understanding of the struc-
For example, Finkelstein et al. proposed ten amino acid changes ture-function relationship of all the IAV proteins.
in PB2 as persistent human IAV host markers, but the 2009
pandemic virus only shares one, T271A. This change was, ACKNOWLEDGMENTS
however, not present in the 1918 pandemic virus and thus was
likely not required for human adaptation. This work was supported by the Intramural Research Program of the NIH and
There has been concern that if the 2009 pandemic virus the NIAID.
acquired some of these previously identified human IAV-associ-
REFERENCES
ated changes, it might increase its virulence or transmissibility
(Herfst et al., 2010; Jagger et al., 2010; Mehle and Doudna,
Alexander, D.J. (2007). An overview of the epidemiology of avian influenza.
2009). Herfst et al. and Jagger et al. recently showed no Vaccine 25, 5637–5644.
enhanced replication or virulence when the E627K or D701N
Ayora-Talavera, G., Shelton, H., Scull, M.A., Ren, J., Jones, I.M., Pickles, R.J.,
was added to viruses bearing the 2009 RNP complex. Mehle and Barclay, W.S. (2009). Mutations in H5N1 influenza virus hemagglutinin that
and Doudna recently proposed an alternate strategy for PB2 confer binding to human tracheal airway epithelium. PLoS ONE 4, e7836.

448 Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc.
Cell Host & Microbe

Review
Baigent, S.J., and McCauley, J.W. (2001). Glycosylation of haemagglutinin and European avian-like and classical swine H1N1 influenza A viruses. J. Virol.
stalk-length of neuraminidase combine to regulate the growth of avian influ- 83, 5485–5494.
enza viruses in tissue culture. Virus Res. 79, 177–185.
Finkelstein, D.B., Mukatira, S., Mehta, P.K., Obenauer, J.C., Su, X., Webster,
Basler, C.F., Reid, A.H., Dybing, J.K., Janczewski, T.A., Fanning, T.G., Zheng, R.G., and Naeve, C.W. (2007). Persistent host markers in pandemic and
H., Salvatore, M., Perdue, M.L., Swayne, D.E., Garcı́a-Sastre, A., et al. (2001). H5N1 influenza viruses. J. Virol. 81, 10292–10299.
Sequence of the 1918 pandemic influenza virus nonstructural gene (NS)
segment and characterization of recombinant viruses bearing the 1918 NS Fouchier, R.A., Schneeberger, P.M., Rozendaal, F.W., Broekman, J.M.,
genes. Proc. Natl. Acad. Sci. USA 98, 2746–2751. Kemink, S.A., Munster, V., Kuiken, T., Rimmelzwaan, G.F., Schutten, M.,
Van Doornum, G.J., et al. (2004). Avian influenza A virus (H7N7) associated
Beare, A.S., and Webster, R.G. (1991). Replication of avian influenza viruses in with human conjunctivitis and a fatal case of acute respiratory distress
humans. Arch. Virol. 119, 37–42. syndrome. Proc. Natl. Acad. Sci. USA 101, 1356–1361.

Belser, J.A., Blixt, O., Chen, L.M., Pappas, C., Maines, T.R., Van Hoeven, N., Gabriel, G., Dauber, B., Wolff, T., Planz, O., Klenk, H.D., and Stech, J. (2005).
Donis, R., Busch, J., McBride, R., Paulson, J.C., et al. (2008). Contemporary The viral polymerase mediates adaptation of an avian influenza virus to
North American influenza H7 viruses possess human receptor specificity: a mammalian host. Proc. Natl. Acad. Sci. USA 102, 18590–18595.
Implications for virus transmissibility. Proc. Natl. Acad. Sci. USA 105,
7558–7563. Gabriel, G., Herwig, A., and Klenk, H.D. (2008). Interaction of polymerase
subunit PB2 and NP with importin alpha1 is a determinant of host range of
Blok, J., and Air, G.M. (1982). Variation in the membrane-insertion and ‘‘stalk’’ influenza A virus. PLoS Pathog. 4, e11.
sequences in eight subtypes of influenza type A virus neuraminidase.
Biochemistry 21, 4001–4007. Garten, R.J., Davis, C.T., Russell, C.A., Shu, B., Lindstrom, S., Balish, A.,
Sessions, W.M., Xu, X., Skepner, E., Deyde, V., et al. (2009). Antigenic and
Boni, M.F., Zhou, Y., Taubenberger, J.K., and Holmes, E.C. (2008). Homolo- genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circu-
gous recombination is very rare or absent in human influenza A virus. J. Virol. lating in humans. Science 325, 197–201.
82, 4807–4811.
Gaydos, J.C., Top, F.H., Jr., Hodder, R.A., and Russell, P.K. (2006). Swine
Brown, I.H. (2000). The epidemiology and evolution of influenza viruses in pigs. influenza a outbreak, Fort Dix, New Jersey, 1976. Emerg. Infect. Dis. 12, 23–28.
Vet. Microbiol. 74, 29–46.
Geiss, G.K., Salvatore, M., Tumpey, T.M., Carter, V.S., Wang, X., Basler, C.F.,
Campitelli, L., Mogavero, E., De Marco, M.A., Delogu, M., Puzelli, S., Frezza, Taubenberger, J.K., Bumgarner, R.E., Palese, P., Katze, M.G., and Garcı́a-
F., Facchini, M., Chiapponi, C., Foni, E., Cordioli, P., et al. (2004). Interspecies Sastre, A. (2002). Cellular transcriptional profiling in influenza A virus-infected
transmission of an H7N3 influenza virus from wild birds to intensively reared lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of
domestic poultry in Italy. Virology 323, 24–36. the host innate defense and its potential contribution to pandemic influenza.
Proc. Natl. Acad. Sci. USA 99, 10736–10741.
Chandrasekaran, A., Srinivasan, A., Raman, R., Viswanathan, K., Raguram, S.,
Tumpey, T.M., Sasisekharan, V., and Sasisekharan, R. (2008). Glycan topology Greenbaum, B.D., Levine, A.J., Bhanot, G., and Rabadan, R. (2008). Patterns
determines human adaptation of avian H5N1 virus hemagglutinin. Nat. of evolution and host gene mimicry in influenza and other RNA viruses. PLoS
Biotechnol. 26, 107–113. Pathog. 4, e1000079.

Chen, R., and Holmes, E.C. (2006). Avian influenza virus exhibits rapid evolu- Guan, Y., Poon, L.L., Cheung, C.Y., Ellis, T.M., Lim, W., Lipatov, A.S., Chan,
tionary dynamics. Mol. Biol. Evol. 23, 2336–2341. K.H., Sturm-Ramirez, K.M., Cheung, C.L., Leung, Y.H., et al. (2004). H5N1
influenza: a protean pandemic threat. Proc. Natl. Acad. Sci. USA 101,
Chen, R., and Holmes, E.C. (2010). Hitchhiking and the population genetic 8156–8161.
structure of avian influenza virus. J. Mol. Evol. 70, 98–105.
Hale, B.G., Randall, R.E., Ortı́n, J., and Jackson, D. (2008). The multifunctional
Chen, J., Lee, K.H., Steinhauer, D.A., Stevens, D.J., Skehel, J.J., and Wiley, NS1 protein of influenza A viruses. J. Gen. Virol. 89, 2359–2376.
D.C. (1998). Structure of the hemagglutinin precursor cleavage site, a determi-
nant of influenza pathogenicity and the origin of the labile conformation. Cell Herfst, S., Chutinimitkul, S., Ye, J., de Wit, E., Munster, V.J., Schrauwen, E.J.,
95, 409–417. Bestebroer, T.M., Jonges, M., Meijer, A., Koopmans, M., et al. (2010). Intro-
duction of virulence markers in PB2 of pandemic swine-origin influenza virus
Chen, W., Calvo, P.A., Malide, D., Gibbs, J., Schubert, U., Bacik, I., Basta, S., does not result in enhanced virulence or transmission. J. Virol. 84, 3752–3758.
O’Neill, R., Schickli, J., Palese, P., et al. (2001). A novel influenza A virus
mitochondrial protein that induces cell death. Nat. Med. 7, 1306–1312. Holmes, E.C. (2010). Evolution in health and medicine Sackler colloquium: The
comparative genomics of viral emergence. Proc. Natl. Acad. Sci. USA 107
Chen, H., Smith, G.J., Li, K.S., Wang, J., Fan, X.H., Rayner, J.M., Vijaykrishna, (Suppl 1), 1742–1746.
D., Zhang, J.X., Zhang, L.J., Guo, C.T., et al. (2006). Establishment of multiple
sublineages of H5N1 influenza virus in Asia: implications for pandemic control. Holmes, E.C., Ghedin, E., Miller, N., Taylor, J., Bao, Y., St George, K., Grenfell,
Proc. Natl. Acad. Sci. USA 103, 2845–2850. B.T., Salzberg, S.L., Fraser, C.M., Lipman, D.J., and Taubenberger, J.K.
(2005). Whole-genome analysis of human influenza A virus reveals multiple
Claas, E.C., de Jong, J.C., van Beek, R., Rimmelzwaan, G.F., and Osterhaus, persistent lineages and reassortment among recent H3N2 viruses. PLoS
A.D. (1998). Human influenza virus A/HongKong/156/97 (H5N1) infection. Biol. 3, e300.
Vaccine 16, 977–978.
Holmes, E.C., Lipman, D.J., Zamarin, D., and Yewdell, J.W. (2006). Comment
Conenello, G.M., Zamarin, D., Perrone, L.A., Tumpey, T., and Palese, P. (2007). on ‘‘Large-scale sequence analysis of avian influenza isolates’’. Science 313,
A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses 1573.
contributes to increased virulence. PLoS Pathog. 3, 1414–1421.
Horimoto, T., and Kawaoka, Y. (2001). Pandemic threat posed by avian influ-
Crawford, P.C., Dubovi, E.J., Castleman, W.L., Stephenson, I., Gibbs, E.P., enza A viruses. Clin. Microbiol. Rev. 14, 129–149.
Chen, L., Smith, C., Hill, R.C., Ferro, P., Pompey, J., et al. (2005). Transmission
of equine influenza virus to dogs. Science 310, 482–485. Jagger, B.W., Memoli, M.J., Sheng, Z.M., Qi, L., Hrabal, R.J., Allen, G.L.,
Dugan, V.G., Wang, R., Digard, P., Kash, J.C., et al. (2010). The PB2 E627K
Domingo, E., Baranowski, E., Ruiz-Jarabo, C.M., Martı́n-Hernández, A.M., mutation attenuates viruses containing the 2009 H1N1 influenza pandemic
Sáiz, J.C., and Escarmı́s, C. (1998). Quasispecies structure and persistence polymerase. mBio 1, e00067–10.
of RNA viruses. Emerg. Infect. Dis. 4, 521–527.
Johnson, N.P., and Mueller, J. (2002). Updating the accounts: global mortality
Dugan, V.G., Chen, R., Spiro, D.J., Sengamalay, N., Zaborsky, J., Ghedin, E., of the 1918-1920 ‘‘Spanish’’ influenza pandemic. Bull. Hist. Med. 76, 105–115.
Nolting, J., Swayne, D.E., Runstadler, J.A., Happ, G.M., et al. (2008). The
evolutionary genetics and emergence of avian influenza viruses in wild birds. Kash, J.C., Basler, C.F., Garcı́a-Sastre, A., Carter, V., Billharz, R., Swayne,
PLoS Pathog. 4, e1000076. D.E., Przygodzki, R.M., Taubenberger, J.K., Katze, M.G., and Tumpey, T.M.
(2004). Global host immune response: pathogenesis and transcriptional
Dunham, E.J., Dugan, V.G., Kaser, E.K., Perkins, S.E., Brown, I.H., Holmes, profiling of type A influenza viruses expressing the hemagglutinin and neur-
E.C., and Taubenberger, J.K. (2009). Different evolutionary trajectories of aminidase genes from the 1918 pandemic virus. J. Virol. 78, 9499–9511.

Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc. 449
 Cell Host & Microbe

Review
Kash, J.C., Tumpey, T.M., Proll, S.C., Carter, V., Perwitasari, O., Thomas, M.J., Nakajima, K., Desselberger, U., and Palese, P. (1978). Recent human influenza
Basler, C.F., Palese, P., Taubenberger, J.K., Garcı́a-Sastre, A., et al. (2006). A (H1N1) viruses are closely related genetically to strains isolated in 1950.
Genomic analysis of increased host immune and cell death responses induced Nature 274, 334–339.
by 1918 influenza virus. Nature 443, 578–581.
Nicholls, J.M., Bourne, A.J., Chen, H., Guan, Y., and Peiris, J.S. (2007). Sialic
Kawaoka, Y., Krauss, S., and Webster, R.G. (1989). Avian-to-human transmis- acid receptor detection in the human respiratory tract: evidence for wide-
sion of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. spread distribution of potential binding sites for human and avian influenza
J. Virol. 63, 4603–4608. viruses. Respir. Res. 8, 73.

Kilbourne, E.D. (1997). Perspectives on pandemics: a research agenda. Noble, G.R. (1982). Epidemiological and clinical aspects of influenza. In Basic
J. Infect. Dis. 176 (Suppl 1), S29–S31. and Applied Influenza Research, A.S. Beare, ed. (Boca Raton: CRC Press),
pp. 11–50.
Kilbourne, E.D. (2006). Influenza pandemics of the 20th century. Emerg. Infect.
Olsen, C.W. (2002). The emergence of novel swine influenza viruses in North
Dis. 12, 9–14.
America. Virus Res. 85, 199–210.
Kobasa, D., Takada, A., Shinya, K., Hatta, M., Halfmann, P., Theriault, S., Ong, A.K., and Hayden, F.G. (2007). John F. Enders lecture 2006: antivirals for
Suzuki, H., Nishimura, H., Mitamura, K., Sugaya, N., et al. (2004). Enhanced influenza. J. Infect. Dis. 196, 181–190.
virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic
virus. Nature 431, 703–707. Palese, P., and Shaw, M.L. (2007). Orthomyxoviridae: the viruses and their
replication. In Fields Virology, D.M. Knipe and P.M. Howley, eds. (Philadelphia:
Krauss, S., Walker, D., Pryor, S.P., Niles, L., Chenghong, L., Hinshaw, V.S., Lippincott, Williams & Wilkins), pp. 1647–1690.
and Webster, R.G. (2004). Influenza A viruses of migrating wild aquatic birds
in North America. Vector Borne Zoonotic Dis. 4, 177–189. Parrish, C.R., Holmes, E.C., Morens, D.M., Park, E.C., Burke, D.S., Calisher,
C.H., Laughlin, C.A., Saif, L.J., and Daszak, P. (2008). Cross-species virus
Landolt, G.A., and Olsen, C.W. (2007). Up to new tricks - a review of cross- transmission and the emergence of new epidemic diseases. Microbiol. Mol.
species transmission of influenza A viruses. Anim. Health Res. Rev. 8, 1–21. Biol. Rev. 72, 457–470.

Li, Z., Chen, H., Jiao, P., Deng, G., Tian, G., Li, Y., Hoffmann, E., Webster, R.G., Peiris, J.S., de Jong, M.D., and Guan, Y. (2007). Avian influenza virus (H5N1):
Matsuoka, Y., and Yu, K. (2005). Molecular basis of replication of duck H5N1 a threat to human health. Clin. Microbiol. Rev. 20, 243–267.
influenza viruses in a mammalian mouse model. J. Virol. 79, 12058–12064.
Pensaert, M., Ottis, K., Vandeputte, J., Kaplan, M.M., and Bachmann, P.A.
Lin, Y.P., Shaw, M., Gregory, V., Cameron, K., Lim, W., Klimov, A., Subbarao, (1981). Evidence for the natural transmission of influenza A virus from wild
K., Guan, Y., Krauss, S., Shortridge, K., et al. (2000). Avian-to-human transmis- ducts to swine and its potential importance for man. Bull. World Health Organ.
sion of H9N2 subtype influenza A viruses: relationship between H9N2 and 59, 75–78.
H5N1 human isolates. Proc. Natl. Acad. Sci. USA 97, 9654–9658.
Perez, D.R., Webby, R.J., Hoffmann, E., and Webster, R.G. (2003). Land-
Lipatov, A.S., Andreansky, S., Webby, R.J., Hulse, D.J., Rehg, J.E., Krauss, S., based birds as potential disseminators of avian mammalian reassortant
Perez, D.R., Doherty, P.C., Webster, R.G., and Sangster, M.Y. (2005). Patho- influenza A viruses. Avian Dis. 47(3, Suppl), 1114–1117.
genesis of Hong Kong H5N1 influenza virus NS gene reassortants in mice: the
Qi, L., Kash, J.C., Dugan, V.G., Wang, R., Jin, G., Cunningham, R.E., and
role of cytokines and B- and T-cell responses. J. Gen. Virol. 86, 1121–1130.
Taubenberger, J.K. (2009). Role of sialic acid binding specificity of the 1918
influenza virus hemagglutinin protein in virulence and pathogenesis for mice.
Malik Peiris, J.S. (2009). Avian influenza viruses in humans. Rev. Sci. Tech. 28,
J. Virol. 83, 3754–3761.
161–173.
Rabadan, R., Levine, A.J., and Robins, H. (2006). Comparison of avian and
Matrosovich, M.N., Krauss, S., and Webster, R.G. (2001). H9N2 influenza A human influenza A viruses reveals a mutational bias on the viral genomes.
viruses from poultry in Asia have human virus-like receptor specificity. Virology J. Virol. 80, 11887–11891.
281, 156–162.
Rambaut, A., Pybus, O.G., Nelson, M.I., Viboud, C., Taubenberger, J.K., and
Matrosovich, M., Stech, J., and Klenk, H.D. (2009). Influenza receptors, Holmes, E.C. (2008). The genomic and epidemiological dynamics of human
polymerase and host range. Rev. Sci. Tech. 28, 203–217. influenza A virus. Nature 453, 615–619.

Mehle, A., and Doudna, J.A. (2008). An inhibitory activity in human cells Reid, A.H., Janczewski, T.A., Lourens, R.M., Elliot, A.J., Daniels, R.S., Berry,
restricts the function of an avian-like influenza virus polymerase. Cell Host C.L., Oxford, J.S., and Taubenberger, J.K. (2003). 1918 influenza pandemic
Microbe 4, 111–122. caused by highly conserved viruses with two receptor-binding variants.
Emerg. Infect. Dis. 9, 1249–1253.
Mehle, A., and Doudna, J.A. (2009). Adaptive strategies of the influenza virus
polymerase for replication in humans. Proc. Natl. Acad. Sci. USA 106, Reperant, L.A., Rimmelzwaan, G.F., and Kuiken, T. (2009). Avian influenza
21312–21316. viruses in mammals. Rev. Sci. Tech. 28, 137–159.

Morens, D.M., Taubenberger, J.K., and Fauci, A.S. (2008). Predominant role of Scholtissek, C., Rohde, W., Von Hoyningen, V., and Rott, R. (1978). On the
bacterial pneumonia as a cause of death in pandemic influenza: implications origin of the human influenza virus subtypes H2N2 and H3N2. Virology 87,
for pandemic influenza preparedness. J. Infect. Dis. 198, 962–970. 13–20.

Morens, D.M., Taubenberger, J.K., and Fauci, A.S. (2009). The persistent Seo, S.H., Hoffmann, E., and Webster, R.G. (2002). Lethal H5N1 influenza
legacy of the 1918 influenza virus. N. Engl. J. Med. 361, 225–229. viruses escape host anti-viral cytokine responses. Nat. Med. 8, 950–954.

Shinde, V., Bridges, C.B., Uyeki, T.M., Shu, B., Balish, A., Xu, X., Lindstrom, S.,
Munster, V.J., and Fouchier, R.A. (2009). Avian influenza virus: of virus and bird
Gubareva, L.V., Deyde, V., Garten, R.J., et al. (2009). Triple-reassortant swine
ecology. Vaccine 27, 6340–6344.
influenza A (H1) in humans in the United States, 2005-2009. N. Engl. J. Med.
360, 2616–2625.
Munster, V.J., Baas, C., Lexmond, P., Waldenström, J., Wallensten, A.,
Fransson, T., Rimmelzwaan, G.F., Beyer, W.E., Schutten, M., Olsen, B., Smith, D.J., Lapedes, A.S., de Jong, J.C., Bestebroer, T.M., Rimmelzwaan,
et al. (2007). Spatial, temporal, and species variation in prevalence of influenza G.F., Osterhaus, A.D., and Fouchier, R.A. (2004). Mapping the antigenic and
A viruses in wild migratory birds. PLoS Pathog. 3, e61. genetic evolution of influenza virus. Science 305, 371–376.
Murphy, B.R., and Clements, M.L. (1989). The systemic and mucosal immune Smith, G.J., Bahl, J., Vijaykrishna, D., Zhang, J., Poon, L.L., Chen, H., Webster,
response of humans to influenza A virus. Curr. Top. Microbiol. Immunol. 146, R.G., Peiris, J.S., and Guan, Y. (2009a). Dating the emergence of pandemic
107–116. influenza viruses. Proc. Natl. Acad. Sci. USA 106, 11709–11712.

Myers, K.P., Olsen, C.W., and Gray, G.C. (2007). Cases of swine influenza in Smith, G.J., Vijaykrishna, D., Bahl, J., Lycett, S.J., Worobey, M., Pybus, O.G.,
humans: a review of the literature. Clin. Infect. Dis. 44, 1084–1088. Ma, S.K., Cheung, C.L., Raghwani, J., Bhatt, S., et al. (2009b). Origins and

450 Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc.
Cell Host & Microbe

Review
evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Tu, J., Zhou, H., Jiang, T., Li, C., Zhang, A., Guo, X., Zou, W., Chen, H., and Jin,
Nature 459, 1122–1125. M. (2009). Isolation and molecular characterization of equine H3N8 influenza
viruses from pigs in China. Arch. Virol. 154, 887–890.
Steel, J., Lowen, A.C., Mubareka, S., and Palese, P. (2009). Transmission of
influenza virus in a mammalian host is increased by PB2 amino acids 627K Tumpey, T.M., Basler, C.F., Aguilar, P.V., Zeng, H., Solórzano, A., Swayne,
or 627E/701N. PLoS Pathog. 5, e1000252. D.E., Cox, N.J., Katz, J.M., Taubenberger, J.K., Palese, P., and Garcı́a-Sastre,
A. (2005). Characterization of the reconstructed 1918 Spanish influenza
Stevens, J., Blixt, O., Glaser, L., Taubenberger, J.K., Palese, P., Paulson, J.C.,
pandemic virus. Science 310, 77–80.
and Wilson, I.A. (2006). Glycan microarray analysis of the hemagglutinins from
modern and pandemic influenza viruses reveals different receptor specific-
ities. J. Mol. Biol. 355, 1143–1155. Tumpey, T.M., Maines, T.R., Van Hoeven, N., Glaser, L., Solórzano, A.,
Pappas, C., Cox, N.J., Swayne, D.E., Palese, P., Katz, J.M., and Garcı́a-
Sturm-Ramirez, K.M., Hulse-Post, D.J., Govorkova, E.A., Humberd, J., Seiler, Sastre, A. (2007). A two-amino acid change in the hemagglutinin of the 1918
P., Puthavathana, P., Buranathai, C., Nguyen, T.D., Chaisingh, A., Long, H.T., influenza virus abolishes transmission. Science 315, 655–659.
et al. (2005). Are ducks contributing to the endemicity of highly pathogenic
H5N1 influenza virus in Asia? J. Virol. 79, 11269–11279. Tweed, S.A., Skowronski, D.M., David, S.T., Larder, A., Petric, M., Lees, W., Li,
Y., Katz, J., Krajden, M., Tellier, R., et al. (2004). Human illness from avian
Subbarao, E.K., London, W., and Murphy, B.R. (1993). A single amino acid in influenza H7N3, British Columbia. Emerg. Infect. Dis. 10, 2196–2199.
the PB2 gene of influenza A virus is a determinant of host range. J. Virol. 67,
1761–1764. Van Poucke, S.G., Nicholls, J.M., Nauwynck, H.J., and Van Reeth, K. (2010).
Replication of avian, human and swine influenza viruses in porcine respiratory
Subbarao, K., Klimov, A., Katz, J., Regnery, H., Lim, W., Hall, H., Perdue, M.,
explants and association with sialic acid distribution. Virol. J. 7, 38.
Swayne, D., Bender, C., Huang, J., et al. (1998). Characterization of an avian
influenza A (H5N1) virus isolated from a child with a fatal respiratory illness.
Science 279, 393–396. Van Reeth, K., and Nicoll, A. (2009). A human case of swine influenza virus
infection in Europe–implications for human health and research. Euro Surveill.
Swayne, D.E. (2007). Understanding the complex pathobiology of high patho- 14, 19124–19126.
genicity avian influenza viruses in birds. Avian Dis. 51(1, Suppl), 242–249.
Wang, R., Soll, L., Dugan, V., Runstadler, J., Happ, G., Slemons, R.D., and
Tarendeau, F., Boudet, J., Guilligay, D., Mas, P.J., Bougault, C.M., Boulo, S., Taubenberger, J.K. (2008). Examining the hemagglutinin subtype diversity
Baudin, F., Ruigrok, R.W., Daigle, N., Ellenberg, J., et al. (2007). Structure and among wild duck-origin influenza A viruses using ethanol-fixed cloacal swabs
nuclear import function of the C-terminal domain of influenza virus polymerase and a novel RT-PCR method. Virology 375, 182–189.
PB2 subunit. Nat. Struct. Mol. Biol. 14, 229–233.
Wasilenko, J.L., Lee, C.W., Sarmento, L., Spackman, E., Kapczynski, D.R.,
Taubenberger, J.K. (2006). Influenza hemagglutinin attachment to target cells:
Suarez, D.L., and Pantin-Jackwood, M.J. (2008). NP, PB1, and PB2 viral genes
‘birds do it, we do it.’. Future Virol 1, 415–418.
contribute to altered replication of H5N1 avian influenza viruses in chickens.
Taubenberger, J.K., and Morens, D.M. (2009). Pandemic influenza—including J. Virol. 82, 4544–4553.
a risk assessment of H5N1. Rev. Sci. Tech. 28, 187–202.
Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., and Kawaoka, Y.
Taubenberger, J.K., Reid, A.H., Janczewski, T.A., and Fanning, T.G. (2001). (1992). Evolution and ecology of influenza A viruses. Microbiol. Rev. 56,
Integrating historical, clinical and molecular genetic data in order to explain 152–179.
the origin and virulence of the 1918 Spanish influenza virus. Philos. Trans.
R. Soc. Lond. B Biol. Sci. 356, 1829–1839. Webster, R.G., Hulse-Post, D.J., Sturm-Ramirez, K.M., Guan, Y., Peiris, M.,
Smith, G., and Chen, H. (2007). Changing epidemiology and ecology of highly
Taubenberger, J.K., Reid, A.H., Lourens, R.M., Wang, R., Jin, G., and Fanning, pathogenic avian H5N1 influenza viruses. Avian Dis. Suppl. 51, 269–272.
T.G. (2005). Characterization of the 1918 influenza virus polymerase genes.
Nature 437, 889–893.
Wise, H.M., Foeglein, A., Sun, J., Dalton, R.M., Patel, S., Howard, W.,
Taubenberger, J.K., Hultin, J.V., and Morens, D.M. (2007). Discovery and char- Anderson, E.C., Barclay, W.S., and Digard, P. (2009). A complicated message:
acterization of the 1918 pandemic influenza virus in historical context. Antivir. Identification of a novel PB1-related protein translated from influenza A virus
Ther. 12, 581–591. segment 2 mRNA. J. Virol. 83, 8021–8031.

Treanor, J.J., Snyder, M.H., London, W.T., and Murphy, B.R. (1989). The B Wright, P.F., Neumann, G., and Kawaoka, Y. (2007). Orthomyxoviruses. In
allele of the NS gene of avian influenza viruses, but not the A allele, attenuates Fields Virology, D.M. Knipe and P.M. Howley, eds. (Philadelphia: Lippincott,
a human influenza A virus for squirrel monkeys. Virology 171, 1–9. Williams & Wilkins), pp. 1691–1740.

Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc. 451