REVIEWS

The copathogenesis of influenza

viruses with bacteria in the lung
Jonathan A. McCullers1,2

Abstract | Concern that a highly pathogenic virus might cause the next influenza pandemic
has spurred recent research into influenza and its complications. Bacterial superinfection in
the lungs of people suffering from influenza is a key element that promotes severe disease
and mortality. This copathogenesis is characterized by complex interactions between
coinfecting pathogens and the host, leading to the disruption of physical barriers,
dysregulation of immune responses and delays in a return to homeostasis. The net effect of this
cascade can be the outgrowth of the pathogens, immune-mediated pathology and increased
morbidity. In this Review, advances in our understanding of the underlying mechanisms are
discussed, and the key questions that will drive the field forwards are articulated.
Influenza pandemic
A worldwide and severe
influenza epidemic caused by
a virus with a novel
haemagglutinin.

Department of Pediatrics,
University of Tennessee
Health Sciences Center,
Memphis, Tennessee 38103,
USA.
2
Department of Infectious
Diseases, St. Jude Childrens
Research Hospital, Memphis,
Tennessee 38105, USA.
e-mail: jmccullers@uthsc.edu
doi:10.1038/nrmicro3231
Published online 3 March 2014

During the 1918 influenza pandemic, more than 50 mil

lion individuals died from pneumonia exacerbated by
bacterial superinfection1,2. Influenza viruses that have a
similar potential to synergize with bacterial pathogens
are currently circulating in the wild bird reservoirs of
the world3. Much has changed since 1918 that might
be expected to ameliorate a severe pandemic, includ
ing improved hygiene, vaccines, antibiotics and better
insights into the interactions between influenza viruses
and bacteria. However, the emergence of a new, highly
pathogenic pandemic influenza virus strain would desta
bilize society, even if it were to cause only a fraction of
the death toll of the 1918 strain. Over the past 10years,
research has increasingly focused on the copathogen
esis of influenza viruses and their bacterial partners in
coinfections. The general outlines of the mechanistic
underpinning of these interactions can now be glimpsed,
but the details remain to be worked out and translated
to humans. This Review focuses on recent advances in
our understanding of the pathogenesis of viralbacterial
pneumonia in the setting of influenza and articulates the
key questions for further research in the field.

phenomenon, using the term excess mortality to indi

cate the number of deaths that are in excess of those
expected for the season and that are temporally linked
to the circulation of influenza viruses4; however, the
greater burden of disease in hospitals and outpatient set
tings, when death does not occur, is very poorly defined.
Despite recent advances in diagnostics, current testing
methods for identifying bacterial pathogens within the
lungs suffer from a lack of sensitivity and specificity,
as the lower respiratory tract is difficult to access and
because many pathogens that can cause pneumonia
(for example, Streptococcus pneumoniae) can colonize
the nasopharynx, which is the typical sampling site for
assessing the aetiology of respiratory tract infections6. In
addition, sequential infections can be more difficult to
accurately diagnose if the preceding pathogen (such as
influenza or another virus) has resolved by the time the
patient presents with secondary bacterial pneumonia7.
Despite these challenges, there is an increasing appre
ciation that many episodes of community-acquired
pneumonia result from co-infections, particularly when
associated with influenza8,9.

Epidemiology and microbiology of coinfections

The epidemiology of coinfections remains difficult to
assess with any accuracy. The attribution of mortality
to influenza or complications of influenza is complex4, as
most deaths are from complications of influenza, rather
than the primary disease, and a precise viral aetiology
is infrequently confirmed by diagnostic testing or con
founded by cocirculating pathogens and co-infections5.
Statistical models have been developed to quantify this

Coinfections during the 1918 pandemic. The 1918

pandemic was comprehensively studied by leading
microbiologists and pathologists of the era (BOX1), and
many of their insights have been collated and reinter
preted in recent years10,11. Estimates from clinical case
and autopsy series suggest that more than 95% of all
severe illnesses and deaths were complicated by bacterial
pathogens, most commonly by S.pneumoniae2. How
ever, other common respiratory pathogens, including

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Box 1 | History of coinfection research
Bacterial pneumonia has been recognized for several centuries as a complication of
respiratory disease epidemics that are consistent with influenza. Descriptions from
autopsies that had convincing features of influenza and bacterial coinfection began to
appear in the eighteenth century141. In 1803, the French physician Ren T. H. Laennec,
who invented the stethoscope, recognized the close association between purulent
bacterial pneumonia and influenza both in clinical observations and in a series of
autopsies142. Formal research into the association began in earnest with the devastating
1918 pandemic. Epidemiological and clinical investigations into coinfections were
primarily conducted by the armed forces, as infectious disease was a disruptive factor
during the First World War and structures were in place to assess and measure its
impact10,13. In addition, animal studies of coinfections were reported for the first time
during this pandemic and showed that mice, rats and guinea pigs would develop
bacterial pneumonia when exposed to unfiltered sputum from patients with influenza143.
The first controlled laboratory experiment to show synergism between influenza virus
and bacterial pathogens was reported by Shope in 1931 (REF.144). After isolating the
first swine influenza virus, he showed that the induction of severe disease in pigs
required coinoculation of both the virus and the pig pathogen Haemophilus influenzae
suis. This work was expanded into mouse models in the 1940s by Francis and Torregrosa,
who carried out a series of coinfection experiments in mice using the mouse-adapted
influenza virus strain A/Puerto Rico/8/1934 (also known as PR8; an H1N1 subtype
strain), together with Streptococcus pneumoniae, Staphylococcus aureus or
H.influenzae145. Today, most laboratory models of viralbacterial coinfection are
derived from a sequential infection model that uses influenza virus PR8 followed by a
laboratory strain of S.pneumoniae, which faithfully recapitulates many of the hallmarks
of the clinical syndrome of severe secondary bacterial pneumonia76. Much of the data
on mechanisms for the copathogenesis of pneumonia during viralbacterial
coinfections that are discussed in this Review are derived from studies that have been
carried out in these models; there is a dearth of data in humans to highlight which of
these are most relevant and important.

Staphylococcus aureus, Haemophilus influenzae and

Streptococcus pyogenes (group A Streptococcus) were all
identified as the predominant pathogen in various indi
vidual studies (reviewed in REF.12), which suggests that
there was regional variation. Furthermore, pre-existing
immunity after prior exposure to these bacterial patho
gens modified the apparent attack rate and severity of
pandemic disease. This is shown by an inverse correla
tion in members of the Australian army between length
of service and mortality from secondary bacterial pneu
monia13 and a lower case fatality rate in other military
units for established medical care providers compared
with newly arrived providers or patients14. Overall, these
data indicate that severe disease and mortality were
linked to the presence of secondary bacterial invaders,
with factors such as variations in which bacterial strains
were endemic being modified by personal or population
immunity, dictating the epidemiology in a particular
region9,12,15.

Clonotypes
Groups of staphylococcal
strains that share similar
characteristics, as
distinguished by pulsed-field
gel electrophoresis.

Coinfections during the 1957 and 1968 pandemics.

The patterns of mortality in the next two pandemics
in 1957 and 1968 resembled those of seasonal influ
enza in the respect that bacterial coinfections were
a less likely cause of death than they were during the
1918 pandemic. Still, pneumonia accounted for a higher
percentage of deaths in 19571958 (44%)16 than during
most of the preceding inter-pandemic years (~20%;
range 444%)17,18, but did not approach the 95% of 1918
(REF.2). In addition, most deaths resulting from pneu
monia were in people with chronic medical conditions19.

In 19571958, S.aureus was the most frequent second

ary invader to cause fatal pneumonia2024. The clinical
course was often fulminant, with death occurring in
less than 7days, and severe pulmonary oedema and
haemorrhage were commonly found on autopsy25. The
higher incidence of S.aureus was a striking departure
from previous pandemics and seasonal epidemics and
was initially blamed on the use of antibiotics that were
effective against S.pneumoniae, S.pyogenes and many
strains of H.influenzae but not against emerging anti
biotic-resistant strains of S.aureus that were prevalent
in hospitals at the time24,26. However, the data fromthe
1968 pandemic suggested that antibiotics were not
the only reason for the changes to the microbiology. In
19681969, the incidence of pneumonia was comparable
to that of 1957 and most mortality was observed in peo
ple with underlying chronic diseases, but S.pneumoniae
was once again the predominant pathogen2730. Thus, it
is more likely that strain-related differences in either the
virus or the bacterial copathogens are responsible for
these shifts in aetiology between pandemics.
Recent epidemiology of coinfections. Although S.pneumoniae was considered to be the most common cause
of secondary pneumonia in the decades after the 1968
pandemic, S.aureus is emerging as a cause of fulminant
pneumonia in association with influenza in many parts
of the world31,32. The USA300 and USA400 clonotypes of
S.aureus seem to be particularly likely to cause second
ary pneumonia with influenza, compared with other
circulating strains, for unclear reasons that are prob
ably related to the altered expression or regulation of
particular bacterial virulence factors, such as cytotoxins
or adherence factors9,33. H.influenzae has become less
prominent as a cause of secondary bacterial pneumonia
following the introduction of the H.influenzae type B
conjugate vaccine in 1985, although it remains important
in regions of the world that have poor vaccine coverage34,
and non-typeable strains that are not covered by the vac
cine continue to be seen in a minority of cases in adults35.
Group A Streptococcus is entirely absent from many case
series that describe community-acquired pneumonia in
association with viruses and is typically third in incidence
when it does appear36. Viruses other than influenza also
participate in viralbacterial coinfections, including
respiratory syncytial virus (RSV), parainfluenzaviruses,
rhinoviruses and adenoviruses3743 (BOX2).
In 2009, a novel H1N1 virus emerged from swine
and caused the first pandemic in more than 40years44.
In contrast to the 1957 and 1968 pandemics, mortality
rates were similar to recent seasonal epidemics and most
deaths occurred in young adults, often with no under
lying chronic conditions 45. Respiratory deaths that
were associated with an aberrant immune response to
the virus accounted for most of this mortality in previ
ously healthy people4547. The precise effect of second
ary bacterial disease remains unclear; some estimates
put excess mortality from influenza and pneumonia as
low as 10% (REF.48), which is lower than that of many
seasonal influenza epidemics49. In careful studies of
severe or fatal cases, bacterial pneumonia was found to

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Box 2 | Copathogenesis of respiratory viruses other than influenza with bacteria
It is clear from codetection studies that bacterial pneumonia is temporally associated with infections from respiratory
viruses other than influenza viruses, including respiratory syncytial virus (RSV) , parainfluenzaviruses, rhinoviruses and
adenoviruses3943. However, most studies with viruses other than influenza A virus have focused on potential associations
with Streptococcuspneumoniae, and little data on the frequency or outcomes of coinfections that involve other
pathogen pairs are available. Animal models have shown synergistic interactions between S.pneumoniae and multiple
viruses, including RSV, parainfluenza viruses and human metapneumovirus99101. As other respiratory viruses share many
common virulence traits with influenza viruses, such as the physiological effects that occur during lower respiratory tract
infection, it is expected that many of the same mechanisms for synergy are in effect. The parainfluenzaviruses encode a
neuraminidase, which can affect receptor use by bacteria in a similar manner to that of influenza viruses101. RSV and
parainfluenza viruses can upregulate receptors, such as ICAM1 (intracellular adhesion molecule 1), CEACAM1
(carcinogenic embryonic adhesion molecule 1) and PAFR (platelet-activating factor receptor), which can be used by
non-typable Haemophilusinfluenzae to initiate infection146. RSV infection activates typeI interferons96, which have
multiple potential downstream effects on antibacterial immunity, including augmentation of NOD2 (nucleotide-binding
oligomerization domain-containing protein 2) signalling and the promotion of inflammation upon recognition of
Gram-positive bacterial cell wall components147. Many respiratory viruses, including RSV and human metapneumovirus,
produce interferon antagonists to blunt the host response during invasion of the respiratory tract148; it is probable that
viral infection would be facilitated during bacterial infection via the synergistic inhibition of innate immunity in a
manner that is analogous to that which occurs during influenza viruspneumococcal coinfections.

complicate between one-quarter and one-half of infec

tions5054. S.pneumoniae and S.aureus were the most
common aetiologies of secondary bacterial pneumonia,
with regional variations in which pathogen was more
frequent51,52,55,56. The prominence of S.aureus in 2009
2010 was probably due to strain-specific features of the
recently emerged USA300 clonotype, such as Panton
Valentine leukotoxin (PVL) expression31, coupled with
increased penetrance of pneumococcal vaccination in the
past decade57. Of note, the role of PVL in the pathogenesis
of pneumonia is controversial, and its expression might
instead be a USA300 genotype marker that commonly
assorts with as yet unknown virulence factors that facili
tate pneumonia in virus-infected hosts58. S.pyogenes was
absent as a secondary pathogen from many case series
but was surprisingly frequent in others, which suggests
that there are further regional differences in common copathogens51,59. In some carefully conducted studies, no
serious bacterial superinfections were seen at all, despite
extensive sampling60. However, when present, it was clear
that bacterial superinfections from S.pneumoniae or
S.aureus resulted in worse outcomes55,56,61,62.

Dead space
An area of lung that has a
mismatch between aeration
and perfusion, such that gas
exchange cannot take place.

Mechanisms of copathogenesis
Although mostly based on animal-model data, it is clear
that copathogenesis between influenza and superinfect
ing bacteria has a multifactorial basis63,64 (FIG.1). Several
virulence factors that are expressed by the virus have
viral strain-specific effects on the host that enable bacte
ria to cause disease. Multiple host pathways are affected,
and specific host states, or factors such as the timing
between exposures to the virus and the bacterium, might
favour certain outcomes. Although much less is known
about the importance of specific bacterial virulence
factors, there is growing evidence that strain-specific dif
ferences in expression are also important on the bacterial
side of the equation.
Dysfunction of lung physiology. Influenza virus infec
tion causes multiple changes in the lungs that can facili
tate secondary bacterial invasion. Epithelial damage,

disruption of surfactant and the sloughing of cells into

the airways provide access and a rich source of nutri
ents, promoting rapid bacterial growth65. Combined
with the release of fibrinous materials and the secre
tion of mucins, small airways become obstructed,
leading to dead space, decreased oxygen and carbon
dioxide diffusion capacities and lung dysfunction66,67.
Ciliary beat frequency is decreased and ciliary motion
becomes uncoordinated68. The physiological effects
of these functional changes in the respiratory tract are
decreased oxygen exchange, airway hyper-reactivity
and decreased mechanical clearance of bacteria. These
changes are most problematic in hosts with pre-existing
conditions that limit pulmonary function, such as
patients with chronic obstructive pulmonary disease,
who are more likely to have exacerbations, chronic bron
chitis and pneumonia during the course of an influenza
virus infection69.
Increased receptor availability. The prevailing dogma
in the field since 1918 has been that the respiratory epi
thelial sloughing that is associated with highly patho
genic influenza viruses provides a foothold for bacteria
by exposing sites for attachment70,71. Bacteria express a
range of virulence factors, such as pneumococcal sur
face protein A (PsaP), choline-binding protein A (CbpA)
and pneumococcal serine-rich repeat protein (PsrP)
in S.pneumoniae72, and members of the MSCRAMM
(microbial surface components recognizing adhesive
matrix molecules) family (for example, fibronectinbinding protein A (FnBPA), FnBPB, clumping factorA
(ClfA) and ClfB) and non-MSCRAMM members of
the serineaspartate dipeptide repeat-containing (Sdr)
family in S.aureus73, that can be used for adherence to
the basement membrane or elements of the extracellular
matrix such as fibrin, fibrinogen and collagen74,75 (FIG.1c).
Virulent viruses, such as the mouse-adapted influenza
virus strain PR8, cause substantial epithelial cell death
invivo, which exposes sites for adherence in the tracheo
bronchial tree70,76. In humans, autopsy studies show the
adherence of bacteria to sites of epithelial damage during

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REVIEWS
a Virushost interactions

b Host physiology and immunity

c Bacteriahost interactions

Lung
attachment

Sialic acid
cleavage

Epithelial
damage

Neuraminidase
Haemagglutinin

Inuenza virion
Neuraminidase

Haemagglutinin

RNA
Bacterium
Cytokines

Non-structural proteins
PB1-F2
Cytokine storm
NS1
IFN production

Toxic proteins
Epithelial damage
and inammation

Over-exuberant
inammatory
response

Ligands
Attachment
Macrophage
Capsule proteins

TLR downregulation
Dying macrophage
Alveolar
macrophage
depletion

Lung alveolus

Other virulence factors

Figure 1 | The interplay between virus, host and bacteria in coinfections. a | Several virulence factors that are
Nature Reviews | Microbiology
expressed by influenza viruses can directly interact with the lungs or with the host immune system.
Haemagglutinin
mediates attachment by binding to terminal sialic acids on cell surface proteins and initiating endocytosis. A variety of
extracellular proteins can bind to glycans on haemagglutinin and neutralize or help to eliminate viruses from the lower
respiratory tract. Viruses with a poorly glycosylated haemagglutinin and the ability to engage both 2,3- and 2,6-linked
sialic acids as receptors are able to penetrate deep into the lungs. The sialidase activity of the neuraminidase protein
cleaves sialic acids from the surface of epithelial cells and from mucins that try to bind and eliminate virions this
facilitates bacterial access to receptors. The non-structural proteins PB1F2 and NS1 are made in infected cells. PB1F2
causes cytotoxicity and promotes inflammatory responses to copathogens; NS1 modulates innate pathways, including
interferon signalling. b | These virus-mediated effects engender changes in the physical properties of the lungs and
compromise innate immunity at several levels. Epithelial damage and increased receptor availability enable bacteria to
adhere and grow. Depletion of the specific subset of lung macrophages that is functionally capable of phagocytosing
bacteria enables escape from early innate immunity. Anergy of the primary bacterial sensing apparatus of the immune
system, pattern recognition receptors, such as the Toll-like receptors (TLRs), prolongs this window of susceptibility for
weeks, while a dysfunctional and paradoxically over-exuberant inflammatory response that is characterized by neutrophil
influx and cytokine storm furthers the acute lung injury that has been started by the virus. c | Bacteria that express specific
virulence factors may take advantage of these changes to the host, grow unchecked and cause disease. Adherence
factors, such as pneumococcal surface protein A or staphylococcal MSCRAMMs (microbial surface components
recognizing adhesive matrix molecules), enable bacteria to attach to newly uncovered receptors or to the matrix of
collagen and fibrin that has been laid down as a scaffold for repair. Bacterial cytotoxins synergize with their viral
counterparts to further the physical and immune-mediated damage to the lungs. Specific characteristics of some
bacterial strains, such as a thick, complement-resistant capsule, and a set of unknown proteins whose existence can be
inferred but have not yet been described, enable improved survival, growth and pathology in virus-infected hosts.

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Box 3 | Transition to the lower respiratory tract
There are two mechanisms by which bacteria such as Streptococcuspneumoniae might
access the lung and establish pneumonia. The first is extension of bacterial colonization
from the upper respiratory tract. Pneumococci colonize the posterior nasopharynx and
exist in a state of balance with the mucosal immune system, shuttling back and forth
between the intracellular and extracellular compartments149 or growing in biofilms150.
Extension of colonization down into the lower respiratory tract is prevented by a
combination of physical barriers and immune mechanisms. The glottis prevents the
gross aspiration of liquids and solids that might carry bacteria into the lungs. Cilia
sweep any debris or adventitious pathogens upwards and out of the bronchial tree and
trachea, aided by a series of molecules such as mucins and collagenous lectins that bind
and neutralize pathogens. Immune cells, complement and mucosal antibodies can be
quickly mobilized once a nascent infection is established. Respiratory viruses, such as
influenza virus, disrupt many of these processes, including facilitating the emergence of
bacteria from biofilms151 (see the figure) and also interfere with host immune responses,
enabling direct extension via micro-aspiration into the lower respiratory tract. However,
epidemiological evidence suggests that most invasive infections and pneumonia occur
within a short period of time following the acquisition of a new bacterial strain, as
systemic immunity (for example, pathogen-specific serum IgG) that is raised against
colonizing bacteria limits the ability of these strains to successfully disseminate
(reviewed in REF.138). Studies in ferrets suggest that otitis media and sinusitis can
occur with some regularity in animals that are colonized by S.pneumoniae and infected
with influenza virus, but pneumonia and invasive disease are rarely seen in this
scenario138. Thus, an alternative path to infection of the lower respiratory tract may be
by the direct inhalation of bacteria from the environment or a short-term colonization
event in the upper respiratory tract before the development of adaptive immune
responses and a homeostatic carriage state. In this scenario, respiratory viruses
contribute to the acquisition of pneumonia by increasing pathogen density in the
nasopharynx and promoting coughing and sneezing in the person from whom the
pneumococci are acquired138, while breaching physical barriers to enable the
penetration of bacteria into the depths of the lungs in the person who is being newly
infected. Of note, animal studies138 and modelling of human incidence data152 both
suggest that effects on the
Release from
susceptibility of the recipient
biolm that
are stronger than effects on
colonizes the
transmission from the donor. In
posterior
nasopharynx
either scenario, altered
receptor availability as a result
of virus-mediated local damage Inhaled
bacteria
to the epithelial layer and
increased receptor display in
response to inflammatory
Damaged
signals and dysfunction of early
lung after viral
innate immune defences
infection
combine to create a zone
where infection can be initiated
once bacteria have transitioned
to an affected site.
Nature Reviews | Microbiology

Cytokine storm
A state characterized by
cytokine levels that are higher
than physiological norms; it is
associated with inflammation
and organ dysfunction.

2,3 or 2,6 linkage

Linkages between terminal
sialic acids and galactose on
host cells; influenza viruses
preferentially attach to
different sialic acids depending
on linkage type.

pandemic influenza2,19,21,25,51. As virulence in influenza

viruses is a multigenic trait, any mutations that increase
viral fitness or cytotoxic potential can contribute to
epithelial damage and subsequent secondary bacterial
infection. In particular, the viral cytotoxin PB1F2 (see
below) can cause cell death and a cytokine storm7779.
However, other receptor-mediated mechanisms must
also be used as most seasonal influenza strains do not
cause severe lung injury but can still facilitate bacterial
superinfection although to a lesser degree80. Three
additional mechanisms might increase receptor avail
ability. First, the influenza virus neuraminidase cleaves
sialic acids, which can expose cryptic receptors for pneu
mococcal adherence on host cells and disrupt sialylated

mucins that can function as decoy receptors for the

bacteria81,82 (FIG.1a). Bacteria that can cause productive
infections in the lungs, such as S.pneumoniae, often
produce neuraminidases themselves in order to access
receptors and avoid host defences by cleaving sialic acids
from protective mucins, thereby preventing aggregation
and removal via the ciliary ladder83 . The presence of
influenza virus in the lungs before bacterial incursion
can thus facilitate access to the lower respiratory tract by
providing a zone in which bacteria that are inhaled from
the environment or that are transitioning from a coloniz
ing state in the upper respiratory tract can establish an
initial infection81 (BOX 3). Second, the host inflammatory
response to viral infections can alter the regulatory state
and surface display of multiple proteins, including some,
such as the platelet activating factor receptor, that can
be used to facilitate pneumococcal invasion84,85. Third,
changes in the airway during wound healing might pro
vide adherence sites during recovery71,86,87. Injured cells or
cells that are in an intermediate differentiation state may
express apical receptors, including asialylated glycans (for
example, GalNac1-4Gal) or 51 integrins, to which
bacteria such as S.aureus or Pseudomonas aeruginosa
can attach (reviewed in REF.88). Areas of incomplete heal
ing, where basement membrane elements, such as laminin
or typeI and type IV collagen, are exposed, or fibrin
and fibrinogen deposition have taken place, might also
lead to more avid bacterial binding by S.pneumoniae,
H.influenzae or S.aureus, mediated by traditional bac
terial adhesins. This contributes to the clinical observa
tion that some secondary bacterial infections occur as the
patient begins to recover from the primary illness89.
Altered tropism of the virus. Viral polymorphisms that
alter the tropism of influenza viruses from the upper to
lower respiratory tract facilitate the copathogenesis of
bacteria in the lungs. Two features of the influenza virus
haemagglutinin dictate its tropism within the respira
tory tract (FIG.1a). The first feature is receptor use; influ
enza viruses can bind to terminal sialic acids that are
attached via either an 2,3 or 2,6 linkage90 and, depend
ing on the configuration of the receptor-binding site,
specific viruses might bind to one more efficiently than
the other. Although both types of sialic acids are present
in both the upper and lower respiratory tract, a prefer
ence for 2,3linked receptors is thought to favour deep
lung infections owing to poorly understood differences
in the prevalence and distribution of the receptors on
specific cells types91. Lower respiratory tract infection by
the virus then enables deep lung infection by the bacteria
by the other mechanisms that are discussed above. Sec
ondly, the glycosylation status of haemagglutinin affects
lung tropism. Collagenous lectins in surfactant can bind
to haemagglutinin proteins that are highly glycosylated,
and thus highly glycosylated viruses are eliminated from
the airways via the ciliary ladder, which limits infection
to the upper respiratory tract92,93. A poorly glycosylated
virus that has an ability to bind to 2,3linked sialic acid
receptors, such as the 1918 pandemic strain, is likely
to be most capable of causing deep lung infection and
facilitating secondary bacterial pneumonia.

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Virus effects on immunity to bacteria. Bacteria such
as pneumococci activate, modulate and are eventu
ally controlled by multiple responses of the immune
system during invasion of the lungs94,95. Respiratory
viruses including influenza virus, RSV, human meta
pneumovirus and parainfluenzaviruses also manipulate
many of these pathways by the expression of multi
functional accessory proteins, such as the influenza
virus non-structural protein 1 (NS1) (REFS 96,97), which
potentially interfere with lung immune responses to
bacterial invaders42,98101 (BOX2). Early innate responses
to bacteria have been shown to be compromised by
the preceding induction of interferons 102104,104,105,
which participate in immune sensing and the con
trol of Gram-positive pathogens106,107. This apparently
paradoxical finding is mediated by typeI interferon,
which is produced following the recognition of viral
nucleic acids by Toll-like receptors 104, and probably
functions by suppressing the macrophage and neutro
phil responses that normally assist in the clearance of
bacteria from the lungs102,105. Inhibition of acute proinflammatory cytokines by the impairment of natural
killer cell responses108 and the direct suppression of
chemokines, which is mediated by the antiviral state
promoted by typeI interferon105, depress the normal
phagocytic activity of macrophages and neutrophils. In
addition, influenza viruses specifically deplete the air
way-resident alveolar macrophages that are responsible
for early bacterial clearance, which leads to a deficit in
early bacterial surveillance and killing109 (FIG.1b). These
alveolar macrophages die during the initial stages of
infection and are replaced over the next 2weeks by
the proliferation and differentiation of macrophages
from other classes, which creates a window of primary
susceptibility that extends beyond the immediate viral
infection. During the clearance of influenza virus and
the onset of wound healing, a general anti-inflamma
tory state that is orchestrated by systems dedicated to
the restoration of lung immune homeostasis110,111 and
characterized by increased interleukin-10 (IL10) pro
duction112 broadly suppresses multiple mechanisms
that are involved in pathogen recognition and clearance
(reviewed in REF.113). For example, increased expres
sion of the receptor for the negative regulatory ligand
CD200 on myeloid cells during viral infections raises
the activation threshold for these cells to superinfecting
bacteria, enabling bacterial outgrowth114. Downregula
tion of other receptors, such as MARCO, on phago
cytic cells might impair phagocytosis and killing102.
Desensitization of pattern recognition receptors that
are used by these phagocytes to detect and respond to
bacteria in this setting can persist for weeks or months
and can contribute to late secondary infections after
apparent recovery from the preceding viral illness115
(FIG.1b); however, there seem to be virus-specific differ
ences in the longevity of this suppression116. Broadly, it
is clear that normal pathogen recognition and effector
responses are globally impaired during, and for some
time after, influenza (and possibly other) viral infec
tions, although the precise mechanisms are still being
elucidated.

Increased inflammation. Pneumonia is an inflamma

tory condition of the lungs. Therefore, viral and bacterial
factors or host responses that increase inflammation in
response to the pathogens contribute to the copatho
genesis of secondary bacterial pneumonia. Some influ
enza A viruses express a cytotoxic accessory protein,
PB1F2 (REF.117), that drives inflammatory responses,
which manifest as increased cellular infiltration of the
lungs and airways, together with cytokine storm77,78,118
(FIG. 1a). This pro-inflammatory activity is strongly
linked to the induction and severity of pneumonia from
superinfecting bacteria, as animal models reveal only
minor pathological effects in the context of single viral
infections but a robust effect on morbidity and mortal
ity during bacterial superinfection33,7779,119. Bacteria also
express cytotoxins that contribute to inflammation120,121,
such as pneumolysin and PVL, and these might synergize
with the effects of PB1F2 via increased cell death related
to pore formation or via increased inflammatory signal
ling, leading to cytokine storm122 (FIG.1c). Multiple innate
immune mechanisms that involve pattern recognition
receptors generate inflammatory responses to respira
tory viruses and/or bacteria (reviewed in REFS94,123).
Many of the inflammatory pathways that are involved
overlap, leading to the synergistic activation of immune
responses, with resulting morbidity95,96,98,124. For exam
ple, viruses such as RSV and influenza virus can activate
Toll-like receptor 4 (TLR4), and RSV also activates TLR2
(REFS125,126). As these are prominent receptors that are
involved in the innate sensing of many Gram-positive
bacteria including S.pneumoniae, coinfection or lysis
of bacteria in the setting of viral pre-infection can gener
ate inflammation and acute lung injury127. In the setting
of depressed phagocytotic activity102,105,108, neutrophilmediated inflammatory damage may occur in the lungs
without effective pathogen control127,128. In concert with
these aberrant responses, the virus might cripple woundrepair mechanisms in the lungs, preventing healing
and prolonging the period of susceptibility to secondary
invasion via the mechanisms discussed above129.
Facilitation of the viral infection. Although most of the
mechanisms that are discussed in this Review involve
viral facilitation of subsequent bacterial superinfection,
it is likely that factors that are expressed by coinfecting
bacteria affect the virus as well. Previous studies have
indicated that there is a directional effect; that is, syner
gism occurs when the viral infection precedes bacterial
exposure and no effect or even protection occurs
when the order of challenge is reversed. Bacterial modu
lation of viral infection may be mediated via direct inter
actions, via bacterial interference with antiviral immunity
or by synergism or complementation by virulence factors
that have similar functions. Animal models consistently
show an increase in influenza virus titres during bacte
rial superinfections in which bacterial challenge fol
lows the viral infection; this increased viral lung load is
typically accompanied by delayed clearance via unclear
mechanisms33,76,119 (FIG.2). However, viral titres may be
suppressed and morbidity may be diminished via the
induction of innate immune responses if the bacterial

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REVIEWS
Viral titre bump as bacterial
infection is established

Intensity

Depletion of alveolar
macrophages and
impairment of innate
immunity enables
bacterial overgrowth

Impairment
of bacterial
clearance
leads to
overwhelming
infection

Time

Figure 2 | Temporal associations between viral titre, bacterial load and the
Nature Reviews | Microbiology
availability of immune effectors in a model of viralbacterial
coinfection. Influenza
viruses replicate rapidly when a primary infection is established, reaching a peak titre
(blue line) in the lungs 23days after inoculation. Impairment of host defences, including
a swift depletion of alveolar macrophages (green line) over the first 3days of infection
enables superinfecting bacteria to grow rapidly (red line) and cause pneumonia. The
rapid growth of bacteria is associated with a rebound in viral titre via unclear mechanisms.
Failure of secondary immunity to control the coinfection can lead to unchecked
bacterial growth after viral clearance, resulting in morbidity and mortality109,153.

infection precedes viral challenge76,130. It has been pro

posed that bacterial proteases from S.aureus are capable
of cleaving the nascent haemagglutinin from its basal
state to a fusion-active complex131, which would increase
viral titres and spread. A mostly unexplored area is the
effect of bacterial virulence factors on antiviral immu
nity. Although no data are available, it is reasonable to
hypothesize that the bacterial mechanisms that broadly
interfere with innate immune mechanisms95,98 might also
affect the immune response to respiratory viruses. There
is evidence that the composition of the microbiome alters
immune responses to influenza both by changing the acti
vation set-point for antiviral responses and by influenc
ing the development of adaptive immune responses132,133.
Whether pathogens such as S.pneumoniae and S.aureus
have similar effects during colonization is an interest
ing area for exploration. Finally, there is some limited
evidence from animal models that viral and bacterial
gene products that have similar functions (for exam
ple, viral and bacterial neuraminidases and cytotoxins)
might synergize or functionally complement each other
to increase growth, avoid immune surveillance or cause
additional inflammation and damage33,81,119.

Intermediate hosts
Hosts such as pigs or domestic
poultry, in which a virus first
adapts before successfully
jumping to humans.

Reassortment
A process by which segmented
viruses, such as influenza, can
swap genes with one another
while coinfecting a cell.

Spatiotemporal differences in virulence

The epidemiological data reviewed above, such as the
differences in incidence of bacterial superinfections
between seasonal and pandemic viruses, suggest that
viruses differ in their potential to facilitate bacterial
superinfections. All influenza A viruses are zoonoses
that originate in the wild bird reservoir and transition
through other intermediate hosts before emerging as
epidemic or pandemic strains after undergoing gene
reassortment in the different host134. Viruses that suc
cessfully establish themselves in humans then adapt and

evolve over time. Virulence is a multigenic trait in influ

enza viruses, and one would expect that any features of
the viral genome that increase replication, fitness in the
lungs or pathogenicity would have downstream effects
on coinfections via one or several of the mechanisms
described above. However, specific changes that affect
copathogenesis that are distinct from traditional viru
lence mechanisms have also been identified in animal
models. It is becoming increasingly evident that there are
certain gene constellations that favour copathogenesis
with bacteria, such as low glycosylation of haemaggluti
nin, an inflammatory PB1F2 and high neuraminidase
activity (FIG.3). Viruses that have these attributes should
be targeted by surveillance and prioritized for study and
pandemic preparation119.
Many of the features that support synergism with
bacteria are lost or diminished during adaptation and
evolution in mammalian hosts, which creates a gradi
ent between seasonal and pandemic strains9. For exam
ple, the glycosylation state of haemagglutinin increases
over decades of circulation in humans to facilitate escape
from immune pressure; this is associated with decreased
virulence, a shift in tropism to the upper respiratory tract
and diminished support of bacterial lung infections9,93.
Similarly, the inflammatory properties of PB1F2 are lost
during adaptation to mammalian lungs, abrogating its
contribution to secondary bacterial infections78. Although
the timescale for this evolutionary loss of activity is meas
ured in decades, the consistency of the finding suggests
that the cytotoxic or pro-inflammatory activity of PB1F2
is detrimental when expressed in the lungs but not when
expressed in the gut which is thehost niche of these
viruses in birds leading to negative selection after
crossing species3,78. Generally, as zoonotic viruses that
have recently been introduced into humans adapt to their
new hosts, they become less virulent, probably via nega
tive selection for traits that stimulate immune responses,
and their propensity to support bacterial superinfections
decreases. This accounts, in part, for the differing epide
miological patterns of complications and mortality that
are associated with seasonal and pandemic strains.
However, it also seems probable that geographic
differences in the regional distribution of bacteria can
help to determine local epidemiology during influenza
epidemics and pandemics135. Although there are cur
rently little data available, one intriguing hypothesis is
that specific bacterial virulence factors that contribute
to the copathogenesis of lung infections with viruses
are unevenly distributed across genotypes, such that the
local endemicity of bacteria that express these factors
influences outcomes9. In a broad sense, this is likely to
have occurred with the spread of the USA300 clonotype
of S.aureus across North America136; the incidence of
serious influenza and S.aureus coinfections in chil
dren increased in parallel in the United States with the
emergence and dominance of this strain32,137. Animal
model data support the idea that there are serotype- and
clonotype-specific differences in the ability to partici
pate in coinfections, including a propensity for USA300
strains to cause severe disease together with influenza
virus33,119,138. In the United States between 2004 and 2009,

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2014 Macmillan Publishers Limited. All rights reserved

REVIEWS

Substrate

Neuraminidase

Haemagglutinin

High
neuraminidase
activity

Average polymerase complex

No glycosylation

Seasonal strain

Truncated or
non-functional
PB1-F2

Cytotoxic and
pro-inammatory
PB1-F2

Low
neuraminidase
activity

Heavy glycosylation
Pandemic strain
RNA

Ecient polymerase complex

Figure 3 | Characteristics of a theoretical pandemic influenza virus strain that would strongly
support| secondary
Nature Reviews
Microbiology
bacterial infections, contrasted with a seasonal strain that would provide minimal copathogenesis. Viruses with
a gene constellation that is predicted to efficiently support secondary bacterial infections exist in the animal reservoirs
of the world119. It can be assumed that, should such a virus cross over into humans and initiate a pandemic, bacterial
superinfections would be the primary cause of mortality in a similar manner to the 1918 pandemic. Once established in
humans, these viruses adapt to their new hosts, changing over time and losing many of the features that most strongly
support copathogenesis with bacteria. Many virulence factors change over decades during adaptation as a side effect
of negative selection for traits that are associated with increased immune recognition. As examples, Nlinked glycans on
the globular head of haemagglutinin accumulate to provide immune escape from antibodies in the human population.
Highly glycosylated viruses are eliminated via the ciliary ladder after being bound by molecules such as collagenous
lectins and neutralized. Poorly glycosylated viruses, such as pandemic strains from the avian reservoir, can evade these
protective measures and cause deeper lung infections. The activity of the neuraminidase is in equilibrium with the
strength of receptor binding of haemagglutinin and decreases to maintain a balance with the decreased affinity of
haemagglutinin related to its adaptation. High-activity neuraminidase strains cleave host sialic acids more effectively,
exposing receptors and disrupting the binding of sialylated mucins, which better supports secondary bacterial
infections. As this neuraminidase activity is lost, the incidence of secondary bacterial infections decreases. A polymerase
complex that provides an appropriate balance, enabling replication and transmission without killing the host, is selected
over time. As viral replication efficiency directly relates to virulence and the associated cytotoxicity of the virus,
decreased polymerase activity has global effects on the synergistic damage that occurs during coinfections. The
cytotoxic and inflammatory PB1F2 contributes to the severity of bacterial superinfection. Over decades during
adaptation, it loses its activity as a result of truncation or mutation via negative selection for a virus that is less likely to
trigger a robust immune response. The net effect of these changes in multiple virulence factors, and their functions that
support bacterial superinfections, is that well-adapted seasonal strains often lack identifiable features associated with
viralbacterial synergism78,93,154.

Serotypes
Groups of pneumococcal
strains that are distinguished
by the antigenicity of their
capsules.

specific serotypes of S.pneumoniae seemed to be associ

ated with influenza in humans in an age-related manner:
serotype 19A dominated coinfections in children less than
5years old, 7F dominated in older children and young
adults of up to 24years old, and other serotypes, mostly
those not contained in the current 13valent vaccine,
were most common in older adults139. Whether these
associations represent baseline prevalence in a highly
vaccinated population at the time of the study or whether
they are tied to specific virulence factors linked to these
serotypes is not clear. Recent data from an influenza virus
and H.influenzae mouse model of coinfection suggest

that certain virulence factors contribute to pathogenesis

only in the context of dual infection140; these genes would
not have been found in traditional virulence screens
during single-agent infections.

Key questions for the field

The study of viralbacterial interactions is expand
ing rapidly with our increased appreciation that most
pneumonia results from coinfections. This growing
recognition that the interplay between multiple patho
gens, the immune system and, more recently, the micro
biome is tremendously complex and is breaking down

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REVIEWS
Molecular signatures
Specific amino acids that are
associated with virulence and
copathogenesis with bacteria.

the linear one-pathogen one-disease paradigm that has

been accepted since the statement of Kochs postulates.
Although much has been done as reviewed here to
understand the specific mechanisms underlying interac
tions such as those between influenza viruses, immunity
and bacterial respiratory pathogens such as S.pneumoniae,
there are key unanswered questions that will drive the
field forward over the next decade. Imperatives forthe
molecular biology laboratory include determining the
specific bacterial factors that enable some bacterial
strains to take advantage of virus-induced changes in
immune pathways and cause disease, understanding the
relationship of timing between encounters with patho
gens on the relevance of different mechanisms of synergy
and discovering host-gene polymorphisms that facilitate
coinfections or favour disease. A great deal of the work
on novel modes of treatment and prevention must take
place in animals before translation to humans. The rel
evance of the composition of the human and animal
microbiome to the acquisition of coinfections and the
development of disease, the role of colonization and the
relevance of the mechanisms that are discussed in this
Review to the upper respiratory tract and the details of
the changes to innate and adaptive immunity in the set
ting of coinfection will be exciting new areas to explore.

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Competing interests statement

The author declares no competing interests.

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