Pathogenicity and virulence of influenza

Abstract

Influenza viruses, including four major types (A, B, C, and D), can cause mild-to-severe and lethal diseases in humans and animals. Influenza viruses evolve rapidly through antigenic drift (mutation) and shift (reassortment of the segmented viral genome). New variants, strains, and subtypes have emerged frequently, causing epidemic, zoonotic, and pandemic infections, despite currently available vaccines and antiviral drugs. In recent years, avian influenza viruses, such as H5 and H7 subtypes, have caused hundreds to thousands of zoonotic infections in humans with high case fatality rates. The likelihood of these animal influenza viruses acquiring airborne transmission in humans through viral evolution poses great concern for the next pandemic. Severe influenza viral disease is caused by both direct viral cytopathic effects and exacerbated host immune response against high viral loads. Studies have identified various mutations in viral genes that increase viral replication and transmission, alter tissue tropism or species specificity, and evade antivirals or pre-existing immunity. Significant progress has also been made in identifying and characterizing the host components that mediate antiviral responses, pro-viral functions, or immunopathogenesis following influenza viral infections. This review summarizes the current knowledge on viral determinants of influenza virulence and pathogenicity, protective and immunopathogenic aspects of host innate and adaptive immune responses, and antiviral and pro-viral roles of host factors and cellular signalling pathways. Understanding the molecular mechanisms of viral virulence factors and virus-host interactions is critical for the development of preventive and therapeutic measures against influenza diseases.

Full text

REVIEW ARTICLE
Pathogenicity and virulence of influenza
Yuying Liang
Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA
ABSTRACT
Influenza viruses, including four major types (A, B, C, and D), can cause mild-to-severe and lethal
diseases in humans and animals. Influenza viruses evolve rapidly through antigenic drift (muta-
tion) and shift (reassortment of the segmented viral genome). New variants, strains, and subtypes
have emerged frequently, causing epidemic, zoonotic, and pandemic infections, despite currently
available vaccines and antiviral drugs. In recent years, avian influenza viruses, such as H5 and H7
subtypes, have caused hundreds to thousands of zoonotic infections in humans with high case
fatality rates. The likelihood of these animal influenza viruses acquiring airborne transmission in
humans through viral evolution poses great concern for the next pandemic. Severe influenza viral
disease is caused by both direct viral cytopathic effects and exacerbated host immune response
against high viral loads. Studies have identified various mutations in viral genes that increase viral
replication and transmission, alter tissue tropism or species specificity, and evade antivirals or pre-
existing immunity. Significant progress has also been made in identifying and characterizing the
host components that mediate antiviral responses, pro-viral functions, or immunopathogenesis
following influenza viral infections. This review summarizes the current knowledge on viral
determinants of influenza virulence and pathogenicity, protective and immunopathogenic aspects
of host innate and adaptive immune responses, and antiviral and pro-viral roles of host factors
and cellular signalling pathways. Understanding the molecular mechanisms of viral virulence
factors and virus-host interactions is critical for the development of preventive and therapeutic
measures against influenza diseases.
ARTICLE HISTORY
Received 21 February 2023
Revised 3 June 2023
Accepted 5 June 2023
KEYWORDS
influenza virus; virulence;
immune responses; host
factors; virus-host
interactions; pathogenesis
1. Introduction
Influenza viruses (flu) have a global distribution and are
both human and animal pathogens. Human influenza
viruses cause annual epidemics (seasonal), which are
highly contagious respiratory infections that can lead to
severe illness and life-threatening complications in high-
risk groups [1]. Occasionally, a new human influenza
virus strain would arise from an animal origin and spread
rapidly among human populations that have no pre-
existing immunity, causing excessive mortality and mor-
bidity globally, known as a pandemic. There have been
four influenza pandemics in modern times, the 1918–19
“Spanish” flu, the 1957 “Asia” flu, the 1968 “Hong Kong”
flu, and the 2009 “Swine flu.” The 1918–19 “Spanish” flu
was the most severe pandemic in recent history, which
was estimated to have caused ~ 500 million infections and
50–100 million deaths worldwide [2]. Humans can also be
sporadically infected by animal influenza viruses (zoono-
tic), most often avian and swine flu; however, they have
yet to establish sustained infection in humans. In parti-
cular, avian H5 and H7 influenza A viruses have caused
hundreds to thousands of infections in humans, with
a high case fatality rate (30–50%) (reviewed in [3]). The
likelihood of these avian influenza viruses gaining effi-
cient human-to-human transmissions to cause the next
pandemic poses a significant public health risk.
Current control measures for influenza infections
include vaccines and antiviral drugs. However, influ-
enza viruses have evolved rapidly to escape vaccine
immunity and develop drug resistance. New viral var-
iants exhibit reduced binding of vaccine-elicited neu-
tralizing antibodies, causing vaccine mismatches or
reduced vaccine efficacy. Mutations resistant to US
Food and Drug Administration (FDA)-approved anti-
viral drugs have been detected [4]. Some become pre-
dominant in circulating strains, making the class of
antivirals, such as M2 inhibitors, ineffective. Broadly
protective vaccines and novel therapeutics are urgently
needed to combat ever-changing influenza viruses.
Significant progress has been made in the under-
standing of the molecular mechanisms of influenza
viral replication, transmission, and disease pathogen-
esis. This review summarizes the current knowledge of
viral genetic markers and host responses affecting
CONTACT Yuying Liang liangy@umn.edu
VIRULENCE
2023, VOL. 14, NO. 1, 2223057
https://doi.org/10.1080/21505594.2023.2223057
© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been
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influenza virus pathogenicity and virulence, which will
help in the development of better prevention and treat-
ment options.
2. Mechanism of influenza virus replication and
pathogenesis
2.1. Influenza virus genome and genes
Influenza viruses belong to the Orthomyxoviridae
family and include four major types A, B, C, and
D. Influenza A virus (IAV) can infect a wide range of
avian and mammalian species, including humans,
birds, ducks, chickens, turkeys, pigs, horses, and dogs
[5]. Influenza B virus (IBV) infects humans and seals,
whereas influenza C virus (ICV) infects humans and
pigs [5]. IAV, IBV, and ICV are human pathogens. The
influenza D virus (IDV), discovered in 2011, infects
pigs and cattle, with no human infections reported to
date [6]. This review focuses primarily on IAV as it
causes most human and animal flu infections.
IAV are enveloped RNA viruses with eight single-
stranded negative-sense RNA segments (Figure 1a).
Viral envelope proteins include two major glycopro-
teins, haemagglutinin (HA) and neuraminidase (NA),
and the transmembrane protein M2. Beneath the viral
membrane is the matrix lattice formed by the M1
protein. Inside viral particles are eight viral RNA
(vRNA) segments in the form of the viral ribonucleo-
protein complex (vRNP), of which each vRNA is
encapsidated by nucleoproteins (NPs) and associated
with the PB1/PB2/PA polymerase complex (Figure 1b).
Each vRNA segment encodes one to three proteins
via alternative splicing or translation mechanisms
(Figure 1c). There are a total of ten essential proteins
for viral infection (PB1, PB2, PA, NP, HA, NA, M1,
M2, NS1, and NEP/NS2) and several accessory pro-
teins, such as PB1-F2 and PA-X, which mediate virus-
host interactions, modulate innate immunity, and affect
viral pathogenicity (reviewed in [7]). PB1 (containing
an RNA-dependent RNA polymerase domain), PB2
(containing a cap-binding domain), and PA (contain-
ing an endonuclease domain) form a heterotrimeric
polymerase complex that is essential for viral RNA
synthesis. NP encapsidates vRNA and complementary
RNA (cRNA) and is also required for viral RNA synth-
esis as demonstrated by influenza virus minigenome
assay [8,9]. HA binds to the host receptor and mediates
membrane fusion. NA facilitates the release and spread
of progeny virions by cleaving sialic acid on the cell
surface. M1 mediates viral particle assembly and bud-
ding from the plasma membrane. M2 is an ion channel
protein on the viral membrane that is required for viral
replication and modulation of cellular homoeostasis.
NS1 impairs host antiviral responses via multiple
mechanisms as described below in details. NEP/NS2
Figure 1. Illustration of influenza virion components, genomic organization, and viral genes. (a) Influenza virus is an enveloped RNA
virus, which has 3 envelope proteins (HA, NA, and M2) on the viral membrane, an M1-formed matrix layer, and eight vRnps. (b) Each
vRNP consists of one vRNA segment wrapped with NP and associated with polymerase complex PB2/PB1/PA. (c) Each vRNA segment
encodes 1–3 genes, through alternative splicing (NS2/NEP and M2) and frameshifting (such as PA-X and PB1-F2). Accessory proteins
expressed through frameshifting are shown as filled dark green bars/boxes.
2Y. LIANG

facilitates the export of vRNAs from the nucleus to the
cytoplasm for assembly.
2.2. Influenza virus life cycle
To initiate an infection, the influenza virus first binds
to the cell surface receptor, sialic acid residues of gly-
coproteins or glycolipids, through the receptor-binding
site (RBS) in the HA protein. Viruses are internalized
into early endosomes and trafficked to late endosomes,
where the low pH environment causes conformational
changes in HA to expose a fusion peptide that leads to
the fusion of viral and endosomal membranes. The M2
proton channels on the viral membrane mediate H
+
influx into the viral interior, which lowers the pH to
facilitate the release of vRNPs from M1 into the cell
cytoplasm in a process called uncoating [1,7].
The released vRNPs are then imported into the nucleus
for viral RNA replication and transcription. Viral RNA
synthesis is mediated by the heterotrimeric polymerase
complex PB1/PB2/PA [10]. The polymerase complex
recognizes the terminal promoter sequence of each
vRNA segment and generates complementary positive-
strand RNA (cRNA), which is then used to produce more
copies of vRNA. mRNA is transcribed from vRNA and
initiated by cap structures stolen from cellular pre-
mRNAs in a cap-snatching mechanism, by which PB2
binds to the 5’ m [7]G cap, and the PA endonuclease
cleaves it. The 3’-polyadenylated tail is added to viral
mRNAs through a stuttering mechanism. Thus, influenza
mRNAs are 5-’ capped and 3-polyadenylated. Some viral
transcripts (M and NS) are processed by the host RNA
splicing machinery. Both primary and processed viral
transcripts are exported to the cytoplasm for translation
by the host ribosomes. PB1, PB2, PA, and NP are
imported into the nucleus to increase viral RNA synthesis
and vRNP formation. Viral membrane proteins (HA, NA,
and M2) traffic to the plasma membrane through cellular
secretory pathways. Other non-structural proteins, such
as NS1, PB1-F2, and PA-X, modulate host cell responses.
The newly synthesized vRNPs are exported from the
nucleus with assistance from M1 and NEP/NS2, and
transported to the plasma membrane for assembly and
budding [11]. M1 interacts with both viral membrane
proteins and vRNPs to mediate viral particle formation
and is also a major player in virus budding. Eight vRNPs
are packaged into viral particles in a “1 + 7” configuration,
where one central vRNP is surrounded by 7 vRNPs [12].
The newly formed progeny virions form aggregates at the
cell surface because of the binding of HA to sialic acids on
viral envelope proteins and on the cell surface. NA neur-
aminidase activity is thus required to release new virions
that can spread to infect new target cells.
Three classes of FDA-approved antiviral drugs target
different stages of the viral life cycle [4]. The M2
inhibitors (M2I) – amantadine and rimantadine –
block M2 ion channel activity. Zanamivir and oselta-
mivir are NA inhibitors (NAI) that prevent the release
of viral particles from the cell surface and stop the viral
spread. Baloxavir marboxil (BXM) is an inhibitor of PA
endonuclease activity (PAI) and suppresses viral tran-
scription. Drug-resistant mutations have been identi-
fied in each class of antivirals [4]. Resistance to M2Is
develops rapidly and requires a single or a limited
number of mutations in M2 such as L26F, V27A, and
S31N, which are prevalent in circulating influenza viral
strains [13]. Thus, the M2Is amantadine and rimanta-
dine are no longer recommended to treat influenza
infections. NAI-resistant mutations such as H275Y,
E119G, I223R, R292K, and Q136K, as well as PAI-
resistant mutations such as I38M or I38T, have been
identified from influenza isolates [4,14,15], though their
global frequency was still low [16]. Nevertheless, novel
classes of antiviral drugs and/or host-based therapeutics
should be developed to treat influenza infections.
2.3. Influenza virus pathogenesis
Human influenza viruses are transmitted through the
respiratory route, whereas avian influenza viruses are
spread between birds primarily through direct contact
via faecal-oral, faecal-faecal, and faecal-respiratory
routes [1]. Viruses replicate mainly in the epithelial
cells lining the respiratory or intestinal tract [1,17].
Virus replication peaked at ~48 h after inoculation
and declined slowly thereafter, with little shedding
after 6–8 days [17]. Mild influenza infections generally
involve the upper respiratory tract and trachea, whereas
the most severe and fatal human infections are asso-
ciated with viral infections in the lower respiratory
tract. Influenza virus causes the death of epithelial
cells through various mechanisms. In addition, infected
epithelial cells release cytokines and chemokines to
attract infiltrating inflammatory cells such as neutro-
phils and macrophages and activate adjacent endothe-
lial cells. These activated immune and non-immune
cells produce even more inflammatory cytokines, such
as IL − 6, IL − 1β, TNFα, and CCL − 2, to stimulate
further infiltration, damage the epithelial-endothelial
barrier, and cause more epithelial cell death. Zoonotic
avian H5N1 viruses can cause a highly lethal disease in
humans, which is associated with viral dissemination to
broad tissue tropism, high viral titers in multiple organs
[18,19], and a hyperinflammatory response [18]. Thus,
the pathogenesis of severe influenza disease is caused
not only by direct viral cytopathic effects but also by
VIRULENCE 3

exacerbated host inflammatory responses [18,20].
Therefore, effective therapeutics should aim to reduce
both viral replication and pathogenic airway
inflammation.
3. Mechanism of influenza virus evolution
3.1. History of influenza virus evolution
IAVs have 18 HA-and 11 NA-known subtypes. Except
for H17N10 and H18N11 from bats, all other 16 HA
and 9 NA subtypes were found in aquatic birds (ducks,
geese, and shorebirds) [21]. Avian influenza viruses can
infect poultry (chickens, turkeys, and domestic ducks),
causing major economic damage. A few of these avian-
origin subtypes have established within-host infections
in other species, such as H1N1, H2N2, and H3N2 in
humans and pigs; H7N7 and H3N8 in equines; and
H3N8 and H3N2 in canines.
Human influenza viruses have changed over the
years [4] (Figure 2). Since the 1918 Spanish pandemic
flu, the IAV subtype H1N1 persisted in humans until it
was replaced in 1957 by an H2N2 subtype (Asian pan-
demic). H2N2 circulated in humans until 1968 when it
was replaced by a H3N2 subtype (Hong Kong pan-
demic). The H1N1 virus reappeared in 1977 and was
replaced by a new H1N1 strain in 2009 (swine pan-
demic). Currently, two IAV subtypes, H1N1 and H3N2,
together with two lineages of IBVs (B/Yamagata and B/
Victoria) [22], co-circulate in humans. Therefore,
annual flu vaccines are quadrivalent and contain two
IAV subtypes and two IBV lineages.
Humans are occasionally infected by influenza
viruses from pigs and birds, which sometimes results
in severe disease and death, but has not been estab-
lished in humans. Avian influenza viruses of subtypes
H5, H6, H7, and H9 are known to cause zoonotic
infections. In particular, H5 and H7 have caused
major outbreaks in birds, with significant losses in
the poultry industry and thousands of human infec-
tions with a high case fatality rate (CFR) (reviewed in
[3]). The highly pathogenic avian influenza virus
(HPAIV) H5N1 was first detected in Hong Kong in
1997 and has been circulating among domestic poul-
try and wild migratory birds in Asia, Europe, and
Africa since 2003. The avian H5N1 virus has caused
868 laboratory-confirmed human infections and 457
deaths globally from 2003 to 2022 (CFR:53%) [23].
Novel H7N9 viruses that emerged in China in 2013
have caused a total of 1,568 laboratory-confirmed
human infections, including 616 fatal cases
(CFR:39%) as of January 2023, according to the
WHO avian influenza weekly update [24]. These
avian influenza viruses have yet to establish efficient
human-to-human transmission, but their potential
acquisition of airborne transmissibility through con-
tinuing viral evolution poses a potential pandemic
threat [25]. Since the fall of 2022, an HPAIV H5N1
of clade 2.3.4.4b was found to infect many mamma-
lian species, including foxes, cats, ferrets, seals, griz-
zly bears, and humans, and more alarmingly, spread
among minks in mink farms [26]. Whether the mink
virus can infect humans and cause efficient human-to
-human infections is unknown.
Figure 2. Influenza virus evolution. Almost all IAV subtypes (H1 to H16, N1 to N9) have natural hosts in water birds, of which some
have established infections in other species, such as H1N1, H2N2, and H3N2 in humans and pigs. Human influenza viruses have
changed over the years mainly due to the emergence of four pandemic flu viruses. Currently, circulating influenza viruses include
two IAV subtypes H1N1 and H3N2, and two IBV lineages (B/Yamagata and B/Victoria). Some animal IAVs, particularly bird and swine
flu, can occasionally spill over to cause zoonotic infections in humans. In recent years, avian H5 and H7 viruses caused human
infections with a high case fatality rate.
4Y. LIANG

3.2. Mechanisms of influenza virus evolution –
antigenic drift and shift
The evolution of influenza viruses is driven by two
major mechanisms: antigenic drift and antigenic shift.
Antigenic drift is the accumulation of mutations in the
viral genome during viral replication owing to the lack
of proofreading activity of viral RNA-dependent RNA
polymerase [5]. Mutations located within the antibody
epitopes of the surface envelope proteins, HA and NA,
can reduce the recognition of pre-existing antibodies
elicited against previous viral strains. Antigenic drift
explains the occurrence of seasonal influenza infections
and the need for annual influenza booster vaccines.
Antigenic shift is the result of the reassortment of
viral genomic RNA segments of two different influenza
viruses co-infecting the same cell to generate new
strains and/or subtypes, which have the potential to
cause severe disease and/or spread quickly in
a population with no pre-existing immunity. The
mechanism of influenza virus reassortment has been
reviewed [27]. Antigenic shift or reassortment is
a major driver of the generation of pandemic or zoo-
notic strains. The 1957 pandemic influenza virus
resulted from the reassortment of an H2N2 avian
virus and a human H1N1 virus, while the 1968 pan-
demic virus was the result of reassortment of an H3
avian virus and a human H2N2 virus then. The 2009
H1N1 pandemic virus (2009 H1N1pdm) was generated
after multiple reassortment events in swine. Avian
H5N1 and H7N9 viruses, which cause zoonotic infec-
tions in humans with high mortality, are generated
through reassortment with other subtypes of avian
viruses, especially avian H9N2 viruses.
4. Viral determinants for pathogenicity and
virulence
Influenza replication capacity, disease severity, and
transmissibility are both virus- and host-specific. Low-
pathogenic avian influenza viruses cause mild symp-
toms in wild birds and poultry, with little or no signs of
illness, whereas highly pathogenic avian influenza
viruses cause high mortality in infected poultry.
Compared to seasonal influenza viruses, pandemic
strains cause high morbidity, mortality, and rapid
transmission in the human population. Even among
the pandemic strains, the 1918 virus is unique in its
extreme virulence (estimated 1% mortality) and its dis-
tinct “W-shaped” curve of age-specific death rates, with
peak deaths observed in young adults in addition to the
elderly and infants [2]. Zoonotic infections by HPAIV
H5N1 and H7N9 viruses led to 30% to 50% mortality in
infected patients, but limited human-to-human trans-
mission [3].
Extensive studies have been conducted to identify
and characterize viral determinants associated with
increased pathogenicity, virulence, and transmission.
In particular, the genetic changes required for adaptive
replication and airborne transmissibility in mammalian
hosts [28–32] have been studied extensively (reviewed
in [33]). The major virulence determinants (Table 1)
include changes that affect viral entry, increase viral
polymerase activity, and modulation of host responses.
They function synergistically to cause efficient infection
and sustained transmission in new hosts [46,68,95]. For
example, a set of five or six substitutions in three viral
genes (HA, PB2, and PB1) are required for an avian
H5N1 virus to acquire airborne transmissibility [28,32].
Identifying viral outbreaks of pandemic potential
through active surveillance of the prevalence and evo-
lution of influenza A viruses in avian, swine, and
human hosts to identify molecular markers of virulence
is an important component of the pandemic prepared-
ness plan.
4.1. HA variants expand tissue tropism and
increase host cell susceptibility
HA is a major determinant of host range because of its
essential role in host receptor binding and membrane
fusion during viral entry. The HA trimer on the viral
membrane consists of two domains: a globular head
containing a receptor-binding site, and a membrane-
proximal stem domain. The receptor-binding site con-
sists of three secondary elements, the 130-loop, 190-
helix, and 220-loop, and includes four highly conserved
residues (Y98, W153, H183, and Y195) [96]. All the HA
positions were based on H3 numbering [97]. HA
mutants allow viruses to expand tissue tropism, switch
receptor specificity, increase host receptor binding, and
optimize membrane fusion at different temperatures or
pH environments.
4.1.1. HA cleavage by host activating proteases
Low-pathogenic avian influenza virus (LPAIV) of H5
and H7 subtypes can mutate to HPAIV, mainly due to
the acquisition of a multi-basic cleavage site [60]. Host
protease-mediated proteolytic cleavage of the HA pre-
cursor molecules HA0 to HA1 and HA2 activates
a conformational change in HA, which is essential for
membrane fusion and viral infectivity [98]. Therefore,
the distribution of activating proteases in the host is
a major determinant of viral tissue tropism. The HA
proteins of LPAIVs and mammalian influenza viruses
contain a single basic residue at the cleavage site and
VIRULENCE 5

Table 1. Molecular markers associated with virulence and pathogenicity of influenza virus. HA substations are based on the H3
numbering system[97].
Genes Subtype Position Molecular markers and functions References
HA H5 83, 128, 197, 496 K83, P128, K197, K496 work in two or more combinations to increase binding
to α2–6 glycans
[34]
H7 104 N104K increases affinity to both α2,3- and α2,6-linked SA [35]
H5 110 H110Y increases thermostability and pH stability, adaptive mutation for airborne
transmission
[32,36,37]
H5 126 S121N enables H5N1 virus binding to both a2–3 and α2–6 glycans [38]
H5, H7 138 L129V/A138V increase H5N1 binding to α2–6 glycans, A138V decreases α2–3
binding; A138-V186-P221 increased H7N9 binding to human-type receptors
[39–42]
H5 143 G143R increases H5N1 virus binding to α2–6 glycans [34]
H5 158 N158D leads to loss of N-glycosylation site, increases human-type receptor
binding, adaptive mutation for airborne transmission
[31,36,38,43]
H5 160 T160A increases overall virus binding to both α2,3-SA and α2,6-SA, adaptive
mutation for airborne transmission
[28,32]
H7 186 A138-V186-P221 increase H7N9 binding to human-type receptors, V186G/
K-K193T-G228S or V186N-N224K-G228S can switch the receptor specificity of
the H7N9 HA from avian- to human-type
[39,44,45]
H9 187, 227 T187P-M227L increase H9N2binding to human-type receptors, enhanced
replication and virulence in mice
[46]
H1 190 D190-D225 determines α − 2,6 binding specificity of H1N1. E190-G225 switched
to α − 2,3-binding
[47–49]
H7 193 V186G/K-K193T-G228S can switch the receptor specificity of the H7N9 HA from
avian- to human-type
[44,45]
H5 196 Q196R/H increases H5N1 virus binding to α2–6 glycans [29,34,50]
H5 214 V214I increases H5N1 virus binding to α2–6 glycans [50]
H7 221 A138-V186-P221 increase H7N9 binding to human-type receptors [39]
H5, H7 224 V186N-N224K-G228S can switch the receptor specificity of the H7N9 HA from
avian- to human-type, N224K increased H5N1 binding to human-type
receptors, adaptive mutation for airborne transmission of H5N1
[31,35,44]
H1 225 D190-D225 determine α − 2,6 binding specificity of H1N1. E190-G225 switched
to α − 2,3-binding
[47,48,51]
H2, H3, H5, H7,
H9, H10,
H15
226 Q226L-G228S change H2 and H3 receptor binding specificity from α2–3 to
α2–6 glycans; Q226L increases HA binding to human-type receptors of
various subtypes
[52,53,5439,55–58]
H5, H9 227 S227N increases H5N1 virus binding to α − 2,6 glycans, T187P-M227L increase
H9N2 binding to human-type receptors
[29,38]
H2, H3, H5, H7,
H9, H10,
H15
228 Q226L-G228S change H2 and H3 receptor binding specificity from α2–3 to α2–6
glycans; G228S increased virus binding to α − 2,6 glycans
[39,52,53,55–58]
H5 318 T318I increases HA stability, adaptive mutation for airborne transmission [28,32,36,37,59]
H5, H7 329 Insertion of multi-basic residues expands tissue tropism in birds and generates
HPAIVs
[60,61]
H5, H7 58 (HA2) K58I increases thermostability and pH stability [35,62,63]
PB2 H5N1 9 D9N enhances viral polymerase activity, inhibits MAVS to suppress IFN-I
production
[64,65]
PB2 H1N1 271 T271A enhances polymerase activity in human cells [66]
PB2 H7N9, H5N1,
H3N2
526 K526R cooperates with E627K to synergistically increase viral polymerase activity [67]
PB2 627 E627K is a mammalian-adaptive mutation, increases avian virus replication in
mammalian cells
[68,69]
PB2 H9N2 526, 543, 627, 655 E627V-E543D-A655V-K526R mutations acted cooperatively to increase viral
polymerase activity in human cells
[70]
PB2 701 D701N is a mammalian-adaptive mutation, increases avian virus replication in
mammalian cells, improved binding of PB2 to importin protein in mammalian
cells
[69,71]
PB2 H7N7 714 S714R is a mouse-adapted mutation, enhances viral polymerase activity in
mammalian cells
[72]
PB1 H5N1 3 D3V enhances viral polymerase activity [73]
PB1 H5N1 99 H99Y increases viral polymerase activity, mammalian-adaptive mutation [28,32]
PB1 H1N1, H5N1,
H7N9
612 K612 SUMOylation is critical for the viral RNA binding activity of PB1, essential
for viral pathogenesis and transmission
[74]
PB1 H5N1 622 D622G enhances viral polymerase activity, virulence in mice [73,75]
PA H7N9 37 S37A increases polymerase activity in human cells [76]
PA H7N7 63 V63I increases viral polymerase activity, viral replication and virulence in
mammalian cells
[77]
PA H1N1 85, 186, 336 85I − 186S −336 M enhances avian virus polymerase activity in mammalian cells [78]
PA H5N1 224, 383 P224-D383 synergistically enhance viral replication in mammalian cells [79]
PA H7N9 383 N383D increases polymerase activity in human cells [76]
PA H5N6 343 A343T cooperates with E627K to synergistically increase viral polymerase activity
in mammalian cells
[40]
PA H9N2 356 K356R cooperates with E627K to synergistically increase viral polymerase activity [67,80]
PA H1N1 552 T552S increases avian virus polymerase activity in mammalian cells [81]
NP H7N9, H9N2 41 V41 enhances viral polymerase activity [82]
NP H7N9, H9N2 210 E210D enhances viral polymerase activity [82]
(Continued )
6Y. LIANG

can only be cleaved by extracellular host proteases,
which restricts viral spread to tissues where specific
proteases are present. HPAIV HA proteins acquire
a multi-basic cleavage site, which allows intracellular
cleavage by ubiquitously present proteases, resulting in
the systemic dissemination of HPAIVs and lethal dis-
ease in poultry.
4.1.2. HA-dependent host adaptation
Except for bat influenza viruses (HA17 and HA18), HA
proteins (HA1–16) recognize terminal sialic acids (SA)
of cell surface glycoconjugates as receptors. Avian and
equine influenza viruses preferentially bind SA linked
to galactose via an α − 2,3 linkage (α2,3-SA), whereas
human influenza viruses prefer α − 2,6-linked SA and
swine influenza viruses bind to both efficiently [99].
Differential receptor-binding affinity is an important
determinant of the influenza virus host range and
viral pathogenicity. α2,3-SA is prevalent in the intest-
inal epithelial cells of birds, allowing the efficient repli-
cation and shedding of avian influenza viruses. In
humans, α2,6-SA is more abundant in the upper
respiratory tract (URT) than in the lower respiratory
tract (LRT), whereas α2,3-SA is more abundant in the
lungs. Human influenza viruses primarily replicate in
URT, causing mild disease, but efficient transmission
among humans. Avian influenza virus infection in
humans (zoonotic) replicates better in LRT than in
URT, resulting in a more severe disease but limited
ability for transmission [99]. Therefore, avian influenza
viruses must acquire human receptor-binding specifi-
city for efficient infection and airborne transmission in
humans. Pigs have both α2,3-SA and α2,6-SA in the
respiratory tract and can act as mixing vessels for avian
and human influenza viruses [100].
Amino acids that determine receptor-binding speci-
ficity have been defined in many HA subtypes, which
generally require two residue changes within the recep-
tor-binding pocket, such as positions 190 and 225 for
H1N1, 226 and 228 for H2N2 and H3N2 viruses [96].
For H1N1 viruses, D190 and D225 are associated with
α − 2,6 binding, whereas E190 and G225 are associated
with α − 2,3 binding [47,48]. The 1918 H1N1 pandemic
HA protein, containing D190 and D225 residues, binds
α − 2,6 receptors exclusively. D225G substitution led to
mixed α − 2,6 and α − 2,3 binding, whereas D190E sub-
stitution alone resulted in α − 2,3 binding. Consistent
with the α − 2,3 receptor-binding specificity, the recom-
binant 1918 pandemic virus with D190E/D225G muta-
tions lost respiratory transmission between ferrets [49].
The recombinant 2009 H1N1pdm virus with D225G
had dual receptor-binding specificity for α − 2,6 and α
− 2,3-SAs and displayed increased binding to human
LRT, but still retained aerosol transmission in mamma-
lian hosts [51], which helps explain the severe disease
associated with D225G during the 2009 pandemic. For
H2N2 and H3N2 viruses, L226 and S228 are associated
with α − 2,6 binding, whereas Q226 and G228 are asso-
ciated with α − 2,3 binding [52]. Q226L and G228S
substitutions switched receptor binding from α − 2,3
to α − 2,6, which contributed to the emergence of the
1957 H2N2 and 1968 H3N2 pandemic infections.
Q226L and G228S have also been shown to increase
the α − 2,6 binding specificity of other avian HA sub-
types (H5, H7, H9, H10, and H15) . However, switch-
ing these HA proteins to human receptor preference
generally requires additional mutations [33,57]
(Table 1).
Apart from substitutions to switch receptor-binding
specificity, other mutations that increase HA stability
and/or overall receptor binding are required for host
adaptation. Site-directed mutagenesis and experimental
adaptation of an avian H5N1 virus have shown that
airborne transmissibility in ferrets requires only three
to four substitutions in the H5 protein, apart from
substitutions in polymerase genes [28,32]. Among the
H5 substitutions, Q226L or G228S increased mamma-
lian receptor-binding specificity, H110Y altered the
acid and temperature stability of HA, and T160A
increased overall receptor binding [32,36,37]. Another
Table 1. (Continued).
Genes Subtype Position Molecular markers and functions References
NP H7N7 319 N319K is mouse-adapted mutation, improves binding of NP to importin protein
in mammalian cells
[72,83]
NP H1N1 357 Q357K enhances viral replication, mammalian-adaptive mutation [84]
M1 H5N1 30, 215 N30D-T215A increases viral replication and viral virulence in mice [114]
M1 H9N1 37 T37A abolishes the phosphorylation site to stabilize the M1 protein, increases
viral replication in mammalian species
[116]
NS1 H5N1 42 P42S increases viral virulence in mice by preventing the dsRNA-mediated
activation of the NF-κB and IRF − 3 pathways
[119]
NS1 H7N9 106 I106M increases viral virulence in mice by enhanced CPSF30 binding [120,121]
PA-X host- and strain-specific role in viral virulence and pathogenicity [118]
PB1-F2 host- and strain-specific role in viral virulence and pathogenicity [85,131]
PB1-F2 H5N1 66 N66S increases viral pathogenicity in mice [94]
VIRULENCE 7

experimental adaptation study [31] showed that
a laboratory-generated reassortant virus requires four
substitutions (N158D, N224K, Q226L, and T318I)
within the H5 protein to be transmitted among ferrets
through aerosol or respiratory droplets. N158D,
N224K, and Q226L allow the switch to human receptor
binding [31,36], while T318I increases the heat stability
and decreases the pH threshold of HA during mem-
brane fusion [37,59]. As the human-receptor-binding
mutations in H5 reduce the HA heat stability and
increase the pH threshold for membrane fusion, com-
pensatory mutations such as T318I that reverse the
effects can facilitate avian virus respiratory transmis-
sion in humans [101].
The emergence of H7N9 avian influenza viruses in
China causing zoonotic infections with high CFR
since 2013 has attracted considerable attention
because of their continuing evolution and potential
pandemic risk. Most zoonotic H7N9 isolates bind to
both the human and avian receptors [102,103].
Q226L and G186V substitutions accounted for the
increased α − 2,6 SA binding [39,56] but were not
sufficient for the switch in receptor specificity.
A combination of three substitutions, V186G/
K-K193T-G228S or V186N-N224K-G228S, is suffi-
cient to switch the receptor specificity of H7N9
from avian to human type [44], of which, G228S is
a known determinant for human receptor binding,
V186G/K/N loses the α − 2,3 binding and increases α
− 2,6 binding, and N224K increases overall receptor
binding [31,35,44].
4.2. Substitutions in the internal genes increase
viral replication in target cells
Influenza virus internal genes, including three poly-
merase proteins (PB2, PB1, and PA), NP, and M1,
contain important virulence markers that increase
viral replication and progeny production.
Polymerase genes play a critical role in the adaptive
replication of avian influenza viruses [72]. These
adaptive mutations in polymerase proteins generally
increase the polymerase activity in mammalian cells.
The major virulence determinant and mammalian
adaptive marker is PB2 E627K [68,69]. Avian influ-
enza viruses generally have E627, while all human
viruses and many zoonotic influenza viruses have
K627, except for the 2009 H1N1pdm virus. Another
mammalian-adaptive substitution is PB2 D701N,
which has been shown to expand the host range of
the avian H5N1 virus to mice and humans and to
increase viral transmission in guinea pigs [69,71].
Surprisingly, 2009 H1N1pdm had neither E627K
nor D701N; the introduction of K627 and N701 did
not enhance virulence or transmission in ferrets or
mice [104].
Studies have shown that polymerase adaptive mutations,
especially PB2 E627K, are caused by species-specific differ-
ences in ANP32 proteins, which are essential host factors
for viral RNA synthesis [105–107]. Compared to avian
ANP32A, human ANP32A lacks a 33-amino acid insertion
in the C-terminal disordered domain, which allows the
binding of PB2 K627, but not PB2 E627 [108,109]. Both
human ANP32A and ANP32B homologs can support viral
RNA synthesis by human influenza viral polymerases or
avian viral polymerases with the PB2 E627K adaptive muta-
tion [110,111]. Other polymerase mutations, such as PB2
K562R, PA A343T, and K356R, can cooperate with PB2
E627K to synergistically increase viral polymerase activity
[40,67,80]. Another mechanism of adaptation has been
shown for host importin-α isoforms, which mediate the
nuclear import of vRNPs and viral proteins for viral RNA
synthesis [112]. The avian PB2 and NP depend on impor-
tin-α3, whereas the mammalian PB2 and NP primarily use
importin-α7 [112]. The PB2 D701N adaptive mutation
enhances the interaction between PB2 and importin-α7 to
promote vRNP nuclear import in mammalian cells
[112,113]. Multiple virulence determinants or adaptive
mutations have been identified in PB2, PB1, PA, and NP
proteins, which function to increase avian viral polymerase
activity in mammalian cells (Table 1), though their
mechanisms of adaptation have not been well
characterized.
As the major driver of viral budding and assembly, M1
also contributes to virulence by increasing viral replication.
A previous study found that D30 and A215 contribute to
the high pathogenicity of avian H5N1 viruses in mice and
that the D30N/A215T double mutations significantly atte-
nuated multiple H5N1 viruses in mice [114]. The M1
A215T substitution eliminates the SUMOylation of M1,
leading to a significant reduction in M1 stability, vRNP
nuclear export, viral replication, and viral progeny produc-
tion [115]. The D30N substitution changes the shape of
H5N1 viral particles from filamentous to spherical,
although the mechanism by which the virion shape affects
viral pathogenicity is unknown [115]. M1 T37A of the
H9N2 virus abolishes the phosphorylation site to stabilize
the M1 protein and increases viral replication in mice and
human cells [116].
4.3. Viral antagonism of host antiviral immunity
Like all other viruses, influenza viruses must overcome
host antiviral immunity for efficient replication. The
viral NS1 protein is a key virulence factor known to
impair host antiviral responses through various
8Y. LIANG

mechanisms (reviewed in [117]). NS1 has been shown
to interact with many viral and host factors through its
functional domains, including an N-terminal RNA-
binding domain (RBD) (aa 1–73), a linker region (aa
74–88), an effector domain (ED) (aa 89–202), and
a C-terminal tail of variable length [118]. Many of the
NS1 functions are strain-specific owing to the sequence
variations of different NS1 proteins. A single substitu-
tion of P42S in the NS1 RBD prevented the dsRNA-
mediated activation of the NF-κB and IRF − 3 pathways
and drastically increased the pathogenicity of an H5N1
virus in mice [119]. The NS1 I106M substitution in an
H7N9 virus increased its binding to CPSF30 and
blocked host gene expression, leading to a more viru-
lent infection in vivo [120]. Two NS1 mutations,
D189N and V194I, present in circulating human
H3N2 viruses impair their ability to inhibit host gene
expression and attenuate viral virulence in mice, while
NS1 V194I affects the thermosensitivity of viral replica-
tion and further attenuated viral virulence [120,121].
Recombinant influenza viruses expressing human NS1
(from human H1N1 or H3N2) induced higher levels of
type I and III interferons (IFN-I/III) than those expres-
sing avian NS1 (from avian H5N1, H7N9, and H7N2)
in dendritic cells (DCs), suggesting that avian NS1 has
an increased ability to antagonize IFN-I/III produc-
tion [122].
PA-X protein is expressed from PA mRNAs via
ribosomal frameshifting [123]. It has the same
N-terminal 191 residues as PA, containing the endo-
nuclease domain and a unique C-terminal domain,
which is 61 aa long for most viral isolates, including
the 1918 H1N1pdm and avian viruses, but is shorter
(41 aa) in the 2009 H1N1pdm virus [123]. PA-X
selectively targets cellular mRNAs, but not viral
mRNAs for degradation, leading to host protein
shutoff and inhibition of cellular antiviral responses
[118,123]. PA-X also modulates host responses such
as inflammation, immune responses, and apoptosis.
The functional mechanism of PA-X in innate
immune suppression is reviewed in detail [118].
Studies on PA-X-deficient influenza viruses have sug-
gested that the biological roles of PA-X in viral repli-
cation and pathogenicity are host- and strain-specific
[118]. PA-X was shown to decrease pathogenicity in
mice by modulating host responses following infec-
tion with the 1918 H1N1pdm virus, the 2009
H1N1pdm strain (A/Beijing/16/2009 (BJ/09), HPAI
H5N1 viruses, and circulating H1N1 strains [123–
125]. Other studies, however, showed a pro-
virulence role of PA-X in the 2009 H1N1pdm strain
A/California/04/09, avian H9N2, and A/PR8 in mice
[126–129].
PB1-F2 is a small protein expressed in the PB1 gene
through the + 1 reading frame shift [130] and is
a major virulence factor that modulates host innate
immune responses (reviewed in [131]). PB1-F2 expres-
sion is detected in many, but not all, influenza
A strains and is not detected in influenza B viruses
[130]. PB1-F2 proteins vary in length and sequence. As
such, the role of PB1-F2 in viral virulence is strain-
and host-specific [131]. Different PB1-F2 proteins can
differentially modify IFN-I responses, inflammatory
responses, and immune cell death, contributing to
viral virulence and pathogenicity [131]. A single muta-
tion N66S in the PB1-F2 protein is associated with
high pathogenicity of the 1918 H1N1pdm and
HPAIV H5N1 viruses [94]. PB1-F2 of the 1918
H1N1pdm virus, but not of the A/PR8 virus, binds
to DDX3 and causes its degradation [132], which may
explain the severe pathogenicity of the 1918 pandemic
virus. PB1-F2 of the highly pathogenic H7N9 virus,
but not of the A/WSN virus, is a potent inhibitor of
MAVS, an essential mediator of antiviral signalling, by
forming aggregates in the mitochondria to prevent
MAVS aggregation and activation, resulting in sup-
pression of IFN-I production and MAVS-mediated
NLRP3 activation [133]. H7N9 PB1-F2, however,
does not affect extracellular NLRP3 inflammasome
maturation, in sharp contrast to PB1-F2 of the A/
WSN virus, which effectively suppresses IL − 1β pro-
cessing and secretion by all stimuli [134]. Thus, the
differential function of PB1-F2 may account for the
highly elevated cytokine storm observed in the H7N9
infection but in not the WSN infection [134].
5. Host immune responses: immune protection
or immunopathogenesis
The host immune response against influenza viral
infections plays an important role in determining the
disease pathogenesis [135]. A potent and fine-tuned
immune response can effectively control and eliminate
viral infection without significant damage, whereas an
excessive and/or prolonged inflammatory response has
been shown to cause tissue damage and disease exacer-
bation. Elucidating the mechanisms of immune protec-
tion and immunopathogenesis is crucial for the
development of effective therapeutics against influenza.
5.1. Early activation of innate immunity correlates
with protection
Upon infection, influenza viruses are recognized by the
innate immune system through different classes of pat-
tern recognition receptors (PRRs), membrane-bound
VIRULENCE 9

Toll-like receptors (TLR3, TLR7, and TLR8), cytosolic
receptor RIG-I, and NOD-like receptor family NLRP3
[136]. Activation of the TLR and RIG-I signalling path-
ways leads to the upregulation of IFN-I/III and proin-
flammatory cytokines. IFNs act through paracrine and
autocrine signalling to induce the expression of inter-
feron-stimulating genes (ISGs), many of which exert
antiviral activities to restrict viral replication.
Proinflammatory cytokines activate phagocytic cells,
recruit additional immune cells to the infection site,
and contribute to the initiation of adaptive immunity.
The NLRP3 inflammasome triggers the proteolytic
cleavage and release of cytokines IL − 1β and IL − 18
and elicits pyroptosis in infected cells [136]. The pro-
tective roles of these innate immune pathways against
influenza virus infection have been demonstrated in
various knockout animal models. Some ISGs have
demonstrated strong antiviral activity. Mx is a well-
known restriction factor for the influenza virus.
IFITMs block virus-host membrane fusion. The 2’−5’-
oligoadenylate synthase (OAS) and RNase L degrade
viral RNA. PKR binds to dsRNA and blocks protein
translation by phosphorylating eukaryotic translation
initiation factor 2α (EIF2α). Other antiviral factors
have been identified that block influenza virus replica-
tion through various mechanisms (reviewed in [136]).
Early activation of robust innate immunity correlates
with host protection against influenza viral infections in
animal models. A highly pathogenic H7N9 virus caused
complete lethality in mice with a high lung virus titer,
whereas the H9N2 virus caused a mild and self-resolved
infection. Compared to H7N9, H9N2 infection resulted
in early and transient induction of innate immunity, as
evidenced by the upregulation of IFN-Is, ISGs, immune
cell markers, and proinflammatory genes [137,138].
Macrophages were recruited to the lungs at a much
earlier time point (as early as 6 h) for H9N2 infection
than for H7N9 infection (day 3) [137]. Co-infection
with H9N2 resulted in effective protection against
both H7N9 and PR8 (H1N1) infections, supporting
the protective role of early innate immunity [137].
5.2. Pathogenic effect of accelerated activation of
proinflammatory responses
Studies on severe influenza viral diseases in both
humans and animals have demonstrated the pathologi-
cal effect of hyperactivation of immune responses
against elevated viral replication. The 1918 H1N1pdm
virus is highly virulent and causes an abnormally high
death rate in young adults, the elderly, and infants [2].
Mice and non-human primates infected with the recon-
structed 1918 H1N1pdm virus have shown marked and
prolonged activation of proinflammatory and cell death
pathways, suggesting that accelerated activation of the
host immune response contributes to severe pulmonary
pathology [95,139]. Virological and immunological stu-
dies of human fatal cases of avian H5N1 and 2009
H1N1pdm infections suggest that a high viral load
and the resulting intense inflammatory responses are
central to the disease pathogenesis [18,140–142]. The
avian H7N9 virus caused a more severe phenotype in
animals than H9N2 and seasonal H3N2, and the dis-
ease severity correlated with increased infectivity and
elevated induction of proinflammatory cytokines [143–
146]. Excessive levels of proinflammatory cytokines,
such as IL − 6, IL − 8, IP10, G-CSF, MCP − 1, and
MIP − 1α, are associated with pulmonary inflammation
and tissue damage in acute lung injury caused by influ-
enza virus and SARS-CoV −2 infections, as well as in
asthma and chronic obstructive pulmonary disease
[95,145,147,148]. The immunopathogenic roles of cyto-
kine storms and innate immune cells have been
reviewed [149].
5.3. Antiviral and immunopathological roles of
macrophages
Macrophages are important innate immune cells that
protect against viral infections by secreting cytokines,
initiating adaptive immunity, and clearing debris and
dead cells [150]. Alveolar macrophages are tissue-
resident macrophages that play an essential role in the
protection from influenza virus-induced morbidity and
mortality [86]. However, the excessive activation of
cytokines in macrophages contributes to lung injury
and disease severity [141,151]. Thus, the controlled
activation of macrophages is critical for their protective
role following influenza viral infections. Recent studies
have shown that cellular Wnt/β-catenin/HIF −1α sig-
nalling and the transcriptional factor PPAR-γ function
to promote the inflammatory activity of alveolar
macrophages [90,152], and that downregulation of
PPAR-γ following IFN-I antiviral signalling is critical
for the suppression of exaggerated inflammatory
response [152].
Macrophages are susceptible to influenza viral infec-
tion; however, most infections are abortive, and only
a small number of viruses can produce infectious par-
ticles by overcoming cellular blocks [87,88,153].
Viruses that productively infect primary murine alveo-
lar macrophages include a subset of highly pathogenic
H5N1 viruses and A/WSN viruses. Even though pro-
ductive replication in macrophages does not solely
determine viral virulence in vivo, it decreases the
10 Y. LIANG

phagocytic function of macrophages and thus may con-
tribute to disease development [88].
Influenza virus strains may also differentially mod-
ulate macrophage polarization, leading to either anti-
viral or pathogenic state [89]. Macrophages are highly
heterogeneous and include two major subclasses:
proinflammatory M1 and anti-inflammatory M2
macrophages. M1 macrophages are activated early in
infection to secrete inflammatory cytokines, such as IL
− 1, IL − 6, and TNF-α, and trigger antiviral responses.
M2 macrophages are activated later during infection to
terminate inflammation, repair tissue damage, and pro-
duce TGF-β and IL − 10 [154–156]. Recent studies have
suggested that M2 macrophages are beneficial for influ-
enza infection, while M1 polarization is associated with
acute lung injury in severe influenza disease
[155,157,158]. Thus, switching macrophage polariza-
tion from M1 to M2 May be a novel therapy for
influenza diseases [159,160].
5.4. Antiviral and immunopathogenic roles of
T cells
Both CD4 and CD8 T cells are important components
of adaptive immunity to clear viral infections and pro-
tect the host from severe disease [161,162]. Pre-existing
influenza-specific CD4 T cells are associated with lower
viral shedding and less severe disease in healthy volun-
teers following influenza viral infection [91]. The adop-
tive transfer of memory CD4 T cells protects unprimed
mice [92]. Memory CD8 T cells have also been shown
to provide cross-reactive protection [93,163].
A comparison of host immunity in hospitalized
patients after zoonotic H7N9 infection with diverse
disease outcomes revealed an important protective
role of memory CD8 T cells against severe influenza
disease [164,165]. Patients with early recovery showed
an early robust H7N9-specific CD8 T cell response,
while those with prolonged hospitalization showed
late recruitment of CD4 and CD8 T cells. In contrast,
deceased patients showed minimal influenza-specific
immunity and little T-cell activation [164]. These stu-
dies suggest that an early strong memory T cell
response can help reduce disease severity. In contrast,
overactive T cells can be harmful because they cause
tissue damage and/or trigger inflammatory responses
[162,166].
Tissue-resident memory T cells (T
RM
) play an
important role in the first-line defence against re-
infection [167]. Lung CD8 T
RM
cells mediate protection
against respiratory infections by producing immediate
effector responses at the site of pathogen entry; how-
ever, heightened responses may also cause
inflammation and tissue damage [168]. Lung
CD8 + T
RM
cells have been shown to adopt exhausted-
like phenotypes to avoid post-infection inflammation
and fibrotic sequelae after influenza viral infection
[169]. In aged hosts, however, lung CD8 T
RM
cells
exhibit malfunction in antiviral response and support
chronic lung inflammation and fibrotic sequelae [170],
which helps explain severe pneumonia in the elderly
after influenza viral infection.
6. Host factors and cell signalling contribute to
viral replication and pathogenesis
6.1. Pro-viral host factors
Like all other viral infections, influenza viruses rely on
host factors and cellular machinery to reproduce
(Figure 3). Hundreds of pro-viral host factors have
been identified through genome-wide gene knockdown
or knockout by CRISPR or RNAi, and through proteo-
mic analysis of viral protein-associated cellular proteins
[171]. Validating the functional roles of each candidate
in influenza virus infection and characterizing their
molecular mechanisms require tremendous effort and
resources. Limited by space, only some proviral host
factors are briefly summarized here.
Viral entry includes attachment, uptake, and fusion,
and each step involves multiple host factors. Viral
attachment requires sialic acids of cell surface glyco-
proteins or glycolipids. Viral uptake by endocytosis
occurs through coordinated actions of cellular endocy-
tic proteins. Host factors that promote influenza virus
internalization include free fatty acid receptor 2
(FFAR2) and transmembrane protein immunoglobulin
superfamily DCC subclass member 4 (IGDCC4)
[172,173]. Once internalized, viral particles move from
early to late endosomes, where fusion and uncoating
occur to release the vRNPs. Prolidase (PEPD) is impor-
tant for the early endosomal trafficking of viral particles
[174]. The cysteine protease cathepsin W (CtsW) and
lysosomal acid phosphatase 2 (ACP2) are required for
viral fusion [175,176]. Cellular factor epidermal growth
factor receptor pathway substrate 8 (EPS8) has been
shown to promote influenza viral uncoating [177].
Influenza virus components must travel between the
cytoplasm and the nucleus, as viral RNA synthesis
occurs in the nucleus, while viral protein synthesis
and viral particle assembly occur in the cytoplasm.
The influenza virus utilizes the cellular nucleocytoplas-
mic trafficking machinery for the nuclear import and
export of vRNPs, viral mRNAs, and viral proteins
[178]. Nuclear importation depends on cellular impor-
tin proteins [179] and is enhanced by G protein
VIRULENCE 11

subunit β1 (GNB1), which increases the interaction
between PB2 and importin proteins [180], and by
BinCARD1, an isoform of Bcl10-interacting protein
with CARD (BinCARD), which increased the binding
of NP with importin α7 [181]. The nuclear export of
viral mRNAs utilizes the host mRNA export machin-
ery to translocate through the nuclear pore complex
for translation in the cytoplasm [182]. Many cellular
factors are involved in different steps of viral RNA
synthesis and have been reviewed [183]. Newly synthe-
sized vRNPs must accumulate at the plasma mem-
brane for assembly. Cytoplasmic trafficking of
progeny vRNPs depends on their association with the
cellular Rab11 GTPase isoform RAB11A [184] in recy-
cling endosomes along the microtubule network, alter-
native vesicles, or liquid-phase organelles (reviewed
in [185]).
The viral matrix proteins M1 and M2 play a central
role in viral assembly and budding. M1-interacting host
factors have been shown to modulate their functions.
GNB1 binds to M1 via intracellular trafficking to the
plasma membrane and promotes viral release [186].
PSMD12, a 26S proteasome regulatory subunit, med-
iates K63-linked ubiquitination of M1 at K102 to pro-
mote viral budding [187]. M1 phosphorylation at the
T108 site increases multimerization at the cell mem-
brane and controls its binding affinity to the cellular
striatin-interacting phosphatase and kinase (STRIPAK)
complex, which is important for M1 polymerization
and viral replication [188].
6.2. Pro-viral host signalling pathways
In addition to antiviral responses, various cellular path-
ways are activated by influenza virus infections and
exploited to support viral replication. Receptor-
tyrosine kinases (RTKs) and their downstream signal-
ling pathways (Raf/MEK/ERK, phosphatidylinositol −
3-kinase (PI3K)/Akt, JAK/STAT, PLC-γ1, NF-κB)
have been found to facilitate viral replication through
different mechanisms [189]. Influenza virus infection
activates many RTKs, such as epidermal growth factor
Figure 3. Pro-viral host factors and host signalling pathways important for influenza viral replication. Host factors and cellular
signalling pathways are hijacked by the influenza virus to promote viral replication at different steps of the viral life cycle. Host
factors are listed inside light green boxes next to the specific step of viral replication. Receptor tyrosine kinases (EGFR, c-Met, TrkA)
on plasma membranes are activated by influenza viral infection and function to promote viral replication. EGFR, c-Met, and PLC-γ1
enhance viral uptake. TrkA signalling is important for several steps of the viral life cycle: viral RNA synthesis, vRNP nuclear export,
and viral budding and release. NF-κB signalling enhances viral RNA synthesis and induces the expression of proinflammatory genes.
PI3K/Akt and Raf/MEK/ERK pathways also strongly increase viral replication.
12 Y. LIANG

receptor (EGFR), c-Met, and tropomyosin receptor
kinase A (TrkA), possibly through clustering of lipid
rafts induced by multivalent virus binding [190,191].
Activated EGFR and c-Met promote efficient viral
uptake [190]. EGFR inhibition by small compounds
or the protein antagonist SOCS5 reduces influenza
virus replication [192,193]. In addition to directly pro-
moting viral entry, EGFR activation has been shown to
suppress IFN-I/III production and antiviral response
[194,195], further increasing viral production.
However, the role of EGFR signalling in influenza dis-
ease could be complex. EGFR has been predicted to be
a key regulator of viral pathogenicity by network-based
analyses of transcriptomic and proteomic data from
mice infected with six influenza virus strains with dif-
ferential disease severity [196]. Treatment of virus-
infected mice with the EGFR inhibitor gefitinib caused
more body weight loss after non-lethal infections, but
not after lethal infections [196], suggesting an antiviral
role of EGFR during non-lethal infections.
TrkA is another RTK activated by influenza virus
infection but has post-entry functional roles in the viral
replication cycle [191]. TrkA and its high-affinity
ligand, nerve growth factor (NGF), are essential for
neuron cell development and functions [197]. TrkA is
also expressed in a wide variety of non-neuronal tissues
and cell types, including human lung epithelial and
endothelial cells [198], and plays an important role in
allergic airway inflammation [199,200]. TrkA inhibitors
block influenza virus replication at multiple steps after
viral entry, viral RNA synthesis, vRNP nuclear export,
and viral budding and release [201,202]. Using a TrkA-
F592A
knock-in mouse model to specifically control
TrkA kinase activity [203], Verma et al. showed that
TrkA not only promotes influenza virus replication in
airway epithelial cells but also contributes to airway
inflammation and lung pathology by activating the
expression of proinflammatory cytokines/chemokines
in infected cells [191]. Thus, targeting TrkA could be
an effective treatment against influenza viral diseases by
blocking both viral replication and airway
inflammation.
NF-κB is a major mediator of proinflammatory cyto-
kines, which can exert antiviral activities [204,205] or med-
iate pathogenic inflammatory responses [206,207].
Interestingly, many studies have demonstrated a pro-viral
role for NF-κB in influenza virus replication [208–212],
specifically in promoting viral RNA replication
[208,209,213,214], suggesting that influenza viruses can
convert this antiviral and proinflammatory pathway for
viral replication. Similar findings were observed for PI3K,
whose activation by influenza viral infection [215–217] was
shown to promote IFN-I production through RIG-I
signalling [217]. PI3K inhibitors, however, have been
found to strongly suppress influenza virus replication at
multiple steps of the viral life cycle [218–220], demonstrat-
ing a pro-viral role of PI3K signalling. In addition, inhibi-
tors of the Raf/MEK/ERK pathway have been shown to
strongly inhibit IAVs and IBVs in vitro and in vivo [221–
224]. Phospholipase C gamma 1 (PLC-γ1) signalling is
virus- and cell-type-specific. Activated PLC-γ1 has an
important pro-viral role in the cellular uptake of H1N1,
but not H3N2 virus, in epithelial cells [225], while it exerts
an antiviral and inflammatory role in H1N1-infected
macrophages by activating NF-κB to express inflammatory
cytokines in a positive feedback loop [226].
Viral infection is expected to activate the host sig-
nalling network, leading to various cellular responses,
such as stress, inflammation, and cell death, which can
restrict viral infection (antiviral) and cause disease
symptoms (proinflammatory). Influenza viruses not
only antagonize antiviral responses but also hijack
the activated signalling network to efficiently accom-
plish different steps of the viral life cycle (pro-viral
role). Thus, targeting the pro-vial and/or proinflam-
matory pathways in the influenza virus-infected cells
can be developed as effective anti-influenza therapeu-
tics [191,210–212,227], which, unlike inhibitors of sin-
gle viral or host targets, generally has a high barrier to
resistance [193,201,210]. The roles of host signalling
pathways in viral replication and disease severity,
however, can be virus- and cell-type-specific and
need to be carefully evaluated for therapeutic
development.
Summary
The continuing evolution of influenza viruses, driven by
antigenic drift (mutation) and shift (reassortment), is
a major barrier to the development of effective vaccines
and antiviral therapeutics. Severe influenza is caused by
direct viral cytopathic effects and immunopathogenesis.
This review summarizes viral determinants and host com-
ponents that affect influenza virus pathogenicity and viru-
lence. Further studies are needed to characterize the
molecular mechanisms of viral virulence determinants
and pro-viral host factors, elucidate the complex roles of
host immunity and cellular pathways in viral replication
and disease development, and identify the control mechan-
isms of the protective and pathogenic roles of host signal-
ling and immune responses. This knowledge will facilitate
the development of new classes of antiviral drugs targeting
different viral and host components as well as novel ther-
apeutics that exploit antiviral roles and dampen the patho-
genic effects of host signalling and immune responses.
VIRULENCE 13

Data sharing statement
Data sharing does not apply to this article, as no new data
were created or analysed in this study.
Disclosure statement
No potential conflict of interest was reported by the author.
Funding
This work was supported in part by the National Institute of
Food and Agriculture – The University of Minnesota Hatch
Formula Fund (MIN-63-074).
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