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Pathogenesis of emerging avian influenza viruses in mammals and the host innate immune response

November 2008
Immunological Reviews 225(1):68-84

November 2008
225(1):68-84

DOI:10.1111/j.1600-065X.2008.00690.x

Source
PubMed

Authors:

Kristy Szretter

Takeda

Lucy Perrone

University of Washington Seattle

Show all 8 authorsHide

Influenza A viruses of avian origin represent an emerging threat to human health as the progenitors of the next influenza pandemic. In recent years, highly pathogenic avian influenza H5N1 viruses have caused unprecedented epizootics on three continents and rare but highly fatal disease among humans exposed to diseased birds. Avian viruses of the H7 and H9 subtypes have also infected humans but generally resulted in far milder disease, yet they too should be considered as possible pandemic threats. Influenza virus infection elicits a complex network of host immune responses that, in uncomplicated influenza, results in effective control of the virus and the development of long-term memory responses. However, fatal avian H5N1 virus infection in both humans and experimental mammalian models is characterized by a high viral load in the respiratory tract, peripheral leukopenia and lymphopenia, a massive infiltration of macrophages into the lung, and dysregulation of cytokine and chemokine responses. This review focuses on avian influenza viruses as a pandemic threat, their induction of host innate immune responses in mammalian species, and the contribution of these responses to the disease process.

. Determination of proinflammatory cytokine levels in the lungs and brains of BALB/c mice infected intranasally with 1000 MID 50 of avian H5N1 viruses isolated from humans and exhibiting a HP (filled bars) or LP (shaded bars) phenotype . Tissues were collected at the indicated times, and clarified tissue homogenized were assayed for levels of the indicated cytokines or chemokine by ELISA. Bars represent the mean protein concentration (in pg/ml) Æ SD. Constitutive levels of cytokine/chemokine in normal mice are indicated by the dotted lines. Ã P 0.05, HP versus LP

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Pathogenesis of emerging avian

influenza viruses in mammals and

the host innate immune response

Summary: Inﬂuenza A vir uses of avian origin represent an emerging threat

to human health as the progenitors of the next inﬂuenza pandemic. In

recent years, highly pathogenic avian inﬂuenza H5N1 viruses have caused

unprecedented epizootics on three continents and rare but highly fatal

disease among humans exposed to diseased birds. Avian viruses of the H7

and H9 subtypes have also infected humans but generally resulted in far

milder disease, yet they too should be considered as possible pandemic

threats. Inﬂuenza virus infection elicits a complex network of host

immune responses that, in uncomplicated inﬂuenza, results in effective

control of the virus and the development of long-term memory responses.

However, fatal avian H5N1 virus infection in both humans and experi-

mental mammalian models is characterized by a high viral load in the

respiratory tract, peripheral leukopenia and lymphopenia, a massive

inﬁltration of macrophages into the lung, and dysregulation of cytokine

and chemokine responses. This review focuses on avian inﬂuenza viruses

as a pandemic threat, their induction of host innate immune responses in

mammalian species, and the contribution of these responses to the disease

process.

Keywords: avian inﬂuenza virus, pathogenesis, innate immunity

Introduction

Inﬂuenza has long been recognized as a signiﬁcant public

health problem that presents a considerable economic burden

on society due to both epidemics, local or regional outbreaks,

and pandemics, epidemics on a global scale. Seasonal epi-

demics of inﬂuenza cause 4300 000 deaths annually around

the world. In the United States alone, an average of 36 000

inﬂuenza-related deaths and over 200 000 hospitalizations

occur annually due to seasonal inﬂuenza, primarily among

persons aged 65 years and over (1). In the 20th century, three

novel inﬂuenza strains emerged to cause the pandemics of

1918, 1957, and 1968. These emerging viruses differed

substantially in their overall mortality rates and apparent

virulence for humans, the 1918 pandemic being the most

severe with overall estimated numbers of deaths worldwide

ranging from 20 to 100 million (2). Although pandemics

result in excess deaths in people of all ages, a large proportion

Taronna R. Maines

Kristy J. Szretter

Lucy Perrone

Jessica A. Belser

Rick A. Bright

Hui Zeng

Terrence M. Tumpey

Jacqueline M. Katz

Immunological Reviews 2008

Vol. 225: 68–84

r2008 The Authors

Journal compilation r2008 Blackwell Munksgaard

Immunological Reviews

0105-2896

Authors’ address

Taronna R. Maines

, Kristy J. Szretter



, Lucy Perrone

, Jessica A. Belser

Rick A. Bright

w,1

, Hui Zeng

, Terrence M. Tumpey

, Jacqueline M. Katz

Inﬂuenza Division, National Center for Immunization

and Respiratory Diseases, Centers for Disease Control &

Prevention, Atlanta, GA, USA.

Correspondence to:

Jacqueline Katz

1600 Clifton Road, MS-G16

Atlanta, GA 30333, USA

Tel.: 11 404 639 4966

Fax: 11 404 639 2350

e-mail: jkatz@cdc.gov

Acknowledgement

The ﬁndings and conclusions in this report are those of the

authors and do not necessarily represent the views of the

Centers for Disease Control and Prevention.



Present Address: Washington University School of

Medicine St Louis, MO, USA.

Present Address: Vaccine Development Global Program,

PATH Washington, DC, USA.

68 r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008

of deaths in the early pandemic periods occurs among those

o65 years of age (3). The unusually high rate of mortality

among younger, healthy adults in 1918–1919, remains an

enigmatic property of this deadliest of inﬂuenza viruses.

Whether severe disease and death in younger adults due to

emerging inﬂuenza A viruses are a result of inherent virulence

of the virus, a robust or exacerbated host immune response, a

level of protective immunity in older adults, or combination of

these factors remains unknown.

Since late 2003, highly pathogenic avian inﬂuenza (HPAI)

H5N1 viruses have caused unprecedented outbreaks in poultry

in over 60 countries, broadened their host range in birds and

mammals, and as of June 2008, have caused over 380 human

H5N1 virus infections with an overall fatality rate of over 60%

(4). The exceptional virulence of this virus in humans,

together with its ongoing genetic evolution in birds, identiﬁes

this emerging inﬂuenza virus as a signiﬁcant pandemic threat.

Nevertheless, two other avian inﬂuenza subtypes have emerged

to infect humans that also warrant our attention. Avian H9N2

viruses are known to infect swine and humans, are endemic

among poultry in which they may not cause overt disease, and

in some cases, possess altered receptor-binding properties that

may promote their ability to infect and spread in humans (5,

6). Likewise, contemporary North American H7N2 viruses

recently have been shown to possess altered receptor-binding

properties that may enhance their ability to infect and spread

among humans (7). Here we review the emergence of avian

inﬂuenza viruses as a pandemic threat, their induction of host

innate immune responses in mammalian species, and the

contribution of these responses to the disease process.

Inﬂuenza virus overview

Inﬂuenza viruses belong to the Orthomyxoviridae family and are

divided into three genera, inﬂuenzavirus A,inﬂuenzavirus B, and

inﬂuenzavirus C (8); however, only inﬂuenza A and B viruses

cause epidemic human disease. Only inﬂuenza A viruses cause

pandemics due to the availability of a diverse reservoir of virus

subtypes that exist in nature, and as such, they are the focus of

this review. Inﬂuenza A viruses are subtyped based on their two

surface glycoproteins, hemagglutinin (HA) and neuraminidase

(NA), and currently, 16 known types of HA and 9 known types

of NA have been isolated from aquatic birds, the natural

reservoir for all inﬂuenza viruses (9, 10). Various subtypes

infect a number of mammalian species, but stable lineages have

evolved only in humans, pigs, and horses. Mutations accumu-

late in the surface glycoproteins of inﬂuenza viruses that enable

the viruses to evade the host immune response. This process,

known as antigenic drift, is the cause of annual epidemics of

inﬂuenza and is the reason for yearly updating of annual

vaccines (11). Because inﬂuenza viruses have a segmented

genome, they have the unique capability of undergoing

reassortment with other inﬂuenza viruses to generate entirely

novel viruses that are antigenically distinct; this process is

called antigenic shift. If a viable virus with a novel HA is

generated from this process that has the ability to cause disease

and spread efﬁciently among humans that lack immunity to the

novel HA, a pandemic could occur.

Inﬂuenza A virus structure

Inﬂuenza A viruses have a single-stranded, negative-sense RNA

genome made up of eight gene segments encoding 10–11

proteins (12, 13). Virions are enveloped and have two surface

glycoproteins, HA and NA that are responsible for virus

attachment and release from host cells, respectively, and are

the principal targets of the host antibody response. Each gene

segment is associated with three polymerase proteins (PB2,

PB1, and PA) and a nucleoprotein (NP), which together form

the eight viral ribonucleoprotein (vRNP) complexes in each

virion (14). The polymerase complex functions as an RNA-

dependent RNA polymerase and mediates the nuclear transport

of the vRNPs facilitating viral transcription (15). The recently

identiﬁed PB1-F2 protein, encoded in the 11 reading frame of

the PB1 gene segment of a majority of inﬂuenza A viruses,

localizes to the mitochondria and is involved in the induction

of apoptosis within host cells (13). PB1 functions in transcrip-

tion initiation and elongation (16), PB2 is involved in acquir-

ing cap structures from cellular mRNAs (17), and PA has been

recently implicated in virus assembly and release (18, 19). The

NP protein is an integral structural component of vRNPs and is

important for viral RNA transcription and assembly (20). The

M gene encodes the M1 matrix protein, which is abundant in

infected cells and forms a matrix on the interior side of the

virion lipid envelope (21). The M2 protein is derived by

alternative splicing of the M gene mRNA, forms ion channels

within the viral envelope, and functions by acidifying the

virion and facilitating virion uncoating (22). The NS gene

encodes NS1, which has multiple functions including obstruc-

tion of the host antiviral response (23, 24), and NS2, also

known as nuclear export protein (NEP), which is expressed by

alternative mRNA splicing and is important for nuclear export

of vRNPs (25).

Infection and replication

In aquatic birds, replication of inﬂuenza viruses occurs primar-

ily in the intestinal tract facilitating virus spread among avian

Maines et al Inﬂuenza virus pathogenesis and innate immunity

r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008 69

species via fecal–oral transmission (26). Most infections

in aquatic birds are asymptomatic; however, certain viruses

within the H5 and H7 subtype have been associated with severe

disease and mortality in domestic poultry. Avian inﬂuenza

viruses are considered to be of either high or low pathogenicity

(HPAI or LPAI) based on their intravenous pathogenicity index

(27). Cleavage of the HA is an essential step during infection

but the amino acids around the HA cleavage site determines the

susceptibility of the HA to host proteases and has been

identiﬁed as a determinant of tissue tropism for inﬂuenza

viruses (reviewed in 28). LPAI and human inﬂuenza viruses

have a cleavage site that is cleaved by host trypsin-like proteases

that limit the virus to tissues of the respiratory tract. However,

HPAI viruses possess additional basic amino acids within the

HA cleavage site that make the HA susceptible to a wide

range of proteases and allows the virus to replicate outside

of the respiratory tract. Sporadic infections with avian

inﬂuenza viruses have been reported in several mammalian

species but stable lineages have emerged only in humans,

pigs, horses, and more recently, dogs (29). Furthermore,

only three subtypes have been documented to cause wide-

spread and sustained disease in humans (H1N1, H3N2, and

H2N2) (10).

Inﬂuenza virus infection occurs after the virus attaches to

sialic acid (SA)-terminated glycans on the surface of host cells

via the receptor binding domain of the HA surface

glycoprotein. The binding preference of the HA for particular

SA moieties is an important host range determinant. Human-

adapted inﬂuenza viruses preferentially bind to a terminal SA

linked to galactose by an a2,6 linkage (a2,6 SA), a major

glycan of human respiratory epithelia, whereas avian inﬂuenza

viruses preferentially bind SA in an a2,3 linkage with galactose

(a2,3 SA), which are abundant in the respiratory and intestinal

tracts of aquatic birds (30–33). After attachment, the virus

can enter the cell either by clathrin-mediated or a clathrin- and

caveolin-independent endocytic pathway (34, 35). At low pH

within the endosome, the HA undergoes a conformational

change that results in fusion of the viral and endosomal

lipid membranes releasing the viral RNPs into the cytoplasm

of the host cell (36). Viral RNPs are transported to the nucleus

for vRNA replication and mRNA transcription using cellular

50-methylated cap structures (m

GpppX

), which are more

readily available in the nucleus because NS1 blocks cellular

mRNA transport to the cytoplasm but viral mRNAs are

transported to the cytoplasm for translation (37). M1, NP,

and NS2 are transported back to the nucleus for translocation of

vRNAs to the cytoplasm for virion assembly (38, 39). HA, NA,

and M2 proteins migrate to the apical cell membrane and are

poised for virion budding (40). The NA is required for release

of budding virions by cleaving the SA receptors.

Inﬂuenza pandemics of the 20th century

Although inﬂuenza pandemics have likely occurred at varying

intervals in human history, only the pandemics of the 20th

century have been studied in detail. The pandemic H1N1 virus

of 1918 may have been derived from a virus wholly of avian

origin with the accumulation of multiple adaptive mutation

that allowed it to cause disease and spread among humans,

although the absolute origin and adaptive host remain

unknown (41–43). The H2N2 pandemic in 1957 arose when

a circulating human inﬂuenza virus acquired the H2, N2, and

PB1 genes from an avian inﬂuenza virus, and the H3N2

pandemic in 1968 occurred after a circulating human inﬂuenza

virus acquired the H3 and PB1 genes from an avian inﬂuenza

virus (44). Although the factors allowing an inﬂuenza virus to

acquire pandemic capability are poorly understood, the accu-

mulation of human host-speciﬁc adaptive mutations is critical.

Previous pandemic viruses crossed the species barrier after

acquiring mutations that changed the binding preference of the

HA from avian-like, a2,3 SA, to human-like, a2,6 SA (31, 32,

45, 46). Some subtypes of avian inﬂuenza viruses have caused

limited human infections, but none have acquired the capacity

for efﬁcient and sustained transmission among humans, a key

property of a pandemic virus.

Human and avian inﬂuenza virus infection in humans

Seasonal inﬂuenza virus infections can vary in severity from an

asymptomatic infection to a severe febrile illness characterized

by headache, sore throat, cough, runny nose, muscle aches,

fatigue, and in some cases gastrointestinal symptoms, a more

pronounced symptom of children, lasting a week or more

(47). Complications of inﬂuenza virus infection arise primarily

in the very young or elderly and can involve the exacerbation of

underlying health conditions such as chronic pulmonary and

cardiopulmonary diseases and can progress to viral or, more

commonly, secondary bacterial pneumonia (1, 48). In primary

inﬂuenza virus pneumonia, infection occurs in the alveolar

epithelial lining which undergoes necrosis and desquamation.

Alveolar macrophages are present in large numbers and in early

stages, neutrophils predominate the inﬁltrating leukocytes,

while in later stages, lymphocytes and plasma cells predomi-

nate (49).

Proinﬂammatory cytokines including type I interferon (IFN-

ab), interleukin-1a(IL-1a) and b, IL-6, IL-8, and tumor

necrosis factor-a(TNF-a) are detected in the respiratory tract

in the acute phase of inﬂuenza virus infection in humans at the

Maines et al Inﬂuenza virus pathogenesis and innate immunity

70 r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008

time of peak illness (50). Levels of T-cell regulatory cytokines

IFN-gand IL-10 are also transiently elevated in infected

individuals, while the increased expression of chemokines,

such macrophage inﬂammatory protein-1a(MIP-1a), MIP-

1b, and monocyte chemotactic protein-1 (MCP-1), is

consistent with their role in the recruitment of lymphocytes

and monocytes to the infected respiratory epithelium (51). In

human volunteer challenge studies, increased levels of the IL-6

and IFN-acorrelate with the magnitude and time course of

virus replication and symptoms, particularly fever, in infected

individuals (50, 52). Although these local cytokine responses

in human inﬂuenza have been evaluated experimentally using

challenge strains of modest virulence for obvious ethical

reasons, these studies nevertheless provide insight into the

dynamics of the host innate response in uncomplicated

infections.

Avian inﬂuenza viruses of the H7, H9, and H5 subtypes have

been responsible for human infections (Table 1). Historically,

H7 human infections were largely limited to laboratory and

occupational exposures (53–58) but more recently have

resulted from exposure to infected poultry during outbreak

responses. In 2003 in the Netherlands, an outbreak of highly

pathogenic H7N7 virus resulted in 89 reported infections

among veterinarians and individuals involved in poultry

culling operations and a few family members of cullers who

had no direct exposure to infected poultry (59). The majority

of patients presented with conjunctivitis, a typical clinical sign

of H7 human infection, although respiratory illness was seen in

a few individuals, and in one, the disease progressed to a fatal

pneumonia with acute respiratory distress syndrome (60, 61).

In recent years, repeated outbreaks of both HPAI and LPAI have

occurred in Europe and North America that have been

associated with rare infections among humans exposed to

infected poultry, primarily resulting in conjunctivitis or

relatively mild respiratory illness (59, 61–65). Low

pathogenic H9N2 avian inﬂuenza viruses have caused a small

number of documented human infections in mainland China

and Hong Kong, SAR, primarily in young children who

experience mild inﬂuenza-like illness and fully recovered

(66–68).

HPAI of the H5N1 subtype have caused the most numerous

and severe human infections of any of the avian inﬂuenza

viruses. HPAI H5N1 viruses were ﬁrst recognized to infect and

cause fatal disease in humans in 1997 after Hong Kong live bird

markets experienced widespread outbreaks of disease in

multiple poultry species. The 18 conﬁrmed human cases, six

of them fatal, resulted from exposure to infected birds and/or

the contaminated environment at the live bird markets

(69–71); the outbreak was ended by the slaughter of all

poultry in Hong Kong. Two additional human cases, a father

and son, were reported in Hong Kong, SAR in 2003 (72). The

father eventually succumbed to infection, but the son survived.

Since late 2003, over 380 human infections of H5N1 virus

have been conﬁrmed in 15 countries in Asia, Africa, and

Europe, with about 60% of those being fatal (4). The majority

of human infections have been the result of exposure to H5N1

virus-infected poultry, but occasional family clusters have

provided evidence for limited person-to-person transmission.

The viruses isolated to date from humans are wholly avian in

origin and lack the ability for sustained transmission in

humans. Clusters of H5N1 virus disease among blood relatives

have repeatedly raised the question as to whether there is a host

genetic component that inﬂuences the susceptibility and/or

severity of disease in humans, although in many cases,

epidemiologic evidence for similar environmental exposure

among family members cannot be ruled out (73, 74).

In general, patients infected with H5N1 viruses presented

with fever, respiratory symptoms including cough and

shortness of breath (60, 75–77), gastrointestinal symptoms

including diarrhea (78, 79), and in one pediatric case,

neurological complications. Leukopenia and lymphopenia

have been associated with a poor prognosis (4, 79). Severe

cases were characterized by pneumonia, multi-organ failure,

and in some cases acute respiratory distress syndrome and

death (60, 76, 77). The average incubation period is 2–5 days,

and for the fatal cases, death occurred approximately 9 days

Ta b l e 1 . Avian inﬂuenza virus subtypes that have caused human infections

Subtype Year Location



Cases Fatalities

H7N7 1996, 2003 UK, NL 90 1

H5N1 1997 HK 18 6

H9N2 1999, 2003, 2007 CN, HK 9 0

H7N2 2002, 2003, 2007 US, UK 6 0

H5N1 2003–2008 HK, CN, TH, VN, KH, ID, AZ, DJ, EG, IQ,TR, LA, MM, NG, PK, BD 367 237

H7N3 2004 CA 2 0



Country codes for the United Kingdom, the Netherlands, Hong Kong Special Administrative Region, China, the United States, Thailand, Vietnam,

Cambodia, Indonesia, Azerbaijan, Djibouti, Egypt, Iraq, Turkey, Laos, Myanmar, Nigeria, Pakistan, Bangladesh, and Canada.

Maines et al Inﬂuenza virus pathogenesis and innate immunity

r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008 71

after symptom onset, a more rapid progression to fatal disease

than seen in the ﬁrst human cases in 1997 (4, 71). In contrast

to the older age for fatal disease with seasonal inﬂuenza viruses,

the highest case fatality rate among H5N1-infected patients is

among individuals 10–19 years of age. Although few in

number, post-mortem examinations conducted among H5N1

virus victims have revealed diffuse alveolar damage with

hyaline membrane formation, interstitial lymphocytic

inﬁltrates, bronchiolitis, pulmonary congestion, and

hemorrhage; alveolar damage with macrophage, neutrophil,

and activated lymphocyte involvement was noted in patients

who died acutely, within 2 weeks of symptom onset. Apoptosis

in alveolar cells and inﬁltrating leukocytes and lymphocyte

depletion in lymphoid organs have been detected (80).

Hemophagocytosis, a disorder characterized by excessive

activation of mononuclear phagocytosis of erythrocytes,

platelets, and leukocytes, and one which is thought to be

cytokine-driven, has been observed among the limited H5N1

fatal case studies (72, 81–83). Fatal H5N1 virus disease was

associated with high viral load in the pharynx, more

pronounced cytokine dysregulation, and more frequent

detection of viral RNA in plasma compared with non-fatal

H5N1 cases (83). There are several reports of extrapulmonary

detection of viral RNA in intestine or fecal specimens and

cerebrospinal ﬂuid, but more data are needed to conﬁrm

dissemination of H5N1 viruses in humans. Patients with

severe H5N1 virus infections were found to have elevated

serum IFN-g, sIL-2R, IL-6, IFN-inducible protein-10 (IP-10),

and MCP-1 compared with uncomplicated inﬂuenza virus

infections (72, 82, 83).

Emerging threat

Human infection with avian inﬂuenza viruses remains a rare

occurrence, even for H5N1 viruses. However, the ongoing

endemicity of avian inﬂuenza viruses among some domestic

poultry populations provides increasing opportunities for

human exposure and infection. Together with the continuous

genetic evolution of avian inﬂuenza viruses in the avian host,

the possibility that one of these novel viruses may adapt to the

humans and result in a pandemic strain is ever-present. Figure 1

depicts how avian inﬂuenza virus could acquire pandemic

capabilities by one or more mechanisms. The results of human

Fig. 1. Generation of a pandemic inﬂuenza virus. Pandemic inﬂuenza A viruses may arise by one of two general mechanisms. One possible

mechanism is through a reassortment event in a mammalian host as postulated for the H2N2 and H3N2 pandemics (155). Given that some circulating

avian inﬂuenza viruses are capable of directly infecting multiple mammalian species, including humans, a reassortment event could occur in any of

these mammalian hosts that are simultaneously infected with both a human and an avian inﬂuenza virus. Alternatively, an avian inﬂuenza virus could

acquire pandemic capability by adapting to humans without a reassortment event but with the gradual accumulation of genetic mutations, as is

speculated to have been the case for the 1918 pandemic virus. All previous pandemic strains adapted to humans after acquiring mutations in the HA that

changed the receptor speciﬁcity of the virus from an avian-like to a human-like receptor binding preference. Compensatory changes in the NA are likely

also necessary. The precise mutations required to switch HA receptor speciﬁcity of the previous pandemic viruses is now known, but even with this

information, the mutations that would confer human-like receptor binding properties on currently circulating avian strains remains incomplete (156).

Maines et al Inﬂuenza virus pathogenesis and innate immunity

72 r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008

and avian virus reassortment studies suggest that genetic changes

in addition to those that confer human-like receptor binding are

likely to be required for H5N1 viruses and potentially other

avian strains to acquire pandemic capability (84). Identifying

this constellation of genetic factors as well as a better under-

standing of mechanisms of inﬂuenza pathogenesis and transmis-

sion and identiﬁcation of effective prevention strategies will be

paramount in our preparedness for the next pandemic.

Mammalian models of inﬂuenza pathogenesis

Because there is only limited information on the mechanisms

of inﬂuenza pathogenesis of emerging avian viruses and the

role of host innate immune response in virulence in humans,

animal models are necessary to identify virus and host deter-

minants of disease. Multiple mammalian models have been

used to investigate avian inﬂuenza virus pathogenesis (85–88),

with each animal system contributing valuable information.

Our laboratory has used inbred mice and outbred ferrets as

mammalian models to better understand the virus host rela-

tionship of potential emerging pandemic viruses. Ferrets are

widely recognized to be an excellent small animal model of

inﬂuenza, as they are susceptible to human inﬂuenza and avian

inﬂuenza viruses and exhibit disease signs and a level of disease

severity resembling that seen in humans. Ferrets, like humans,

have a predominance of a2,6 SA receptors on upper respiratory

tract tissue (89, 90). Furthermore, van Riel et al. (91) demon-

strated the H5N1 virus displayed a binding pattern to respira-

tory tract epithelial tissues of ferrets that was most similar to

human tissue, binding primarily to type II pneumocytes,

macrophages, and non-ciliated cuboidal epithelial cells in the

alveoli. Infection of ferrets with seasonal human inﬂuenza

H1N1 or H3N2 viruses causes fever and mild respiratory

illness, with the animals recovering fully in 7–10 days (64,

87, 92, 93). In contrast, infection of this host with some HPAI

H5N1 or H7N7 viruses results in a severe and in some cases

fatal disease (87, 94–96).

We observed a notable difference in the severity and kinetics

of disease in ferrets caused by highly virulent H5N1 viruses

isolated from humans in 1997 and those isolated after 2004

(84, 95). H5N1 viruses isolated from humans in 1997

(A/HK/486/97 and A/HK/483/97) caused substantial

weight loss, lethargy, respiratory signs, neurological signs,

and secondary bacterial infections in some animals typically

during the second week of illness and was lethal in

approximately 35% of ferrets. In contrast, H5N1 viruses

isolated from humans during 2004 (e.g. A/Thailand/16/04,

A/Vietnam/1203/04) caused a disease characterized by

extreme weight loss, more severe lethargy, transient

lymphopenia, and respiratory signs and were lethal in 100%

of ferrets within the ﬁrst week of infection. These results

recapitulated the contracted period of severe illness and

overall higher rate of mortality observed with more recent

human H5N1 virus infections (97) compared with those

documented in 1997 and conﬁrmed that the ferret is an

appropriate model for studies of avian inﬂuenza virus

pathogenicity in mammals. However, a current limitation of

this model is the lack of ferret-speciﬁc immunological reagents

and genomic sequence information, which has hindered the

analysis of the innate immune response in this model. Recently,

a panel of ferret cytokine genes was cloned and sequenced and

a semi-quantitative real time polymerase chain reaction (PCR)

assay was established (98) to identify immune correlates of

disease severity in animals infected with seasonal human

inﬂuenza viruses (93). Cytokine mRNA levels were measured

in nasal washes collected from ferrets during the ﬁrst 4 days

after intranasal infection. A rapid and strong induction of

IFN-a, IFN-g, and TNF-awas observed in nasal washes of

ferrets with mild disease, while ferrets with severe disease,

characterized by increased lung pathology and delayed viral

clearance compared with mild disease, had a reduced level of

IFN-aand expression of IFN-gand TNF-awas delayed. IL-6

was only detected in ferrets exhibiting severe disease, whereas

IL-8 expression was detected in ferrets with mild disease. These

results suggest less virulent inﬂuenza viruses elicit a stronger

innate immune response in ferrets, whereas infection with

more virulent strains led to a weaker host response. As more

ferret gene sequence information and ferret-speciﬁc antibodies

become available (99, 100), additional immunological

questions regarding host response to inﬂuenza virus infection

can be addressed. In the meantime, the inbred mouse remains

our primary tool for such analyses.

The murine model has been used extensively to identify

virulence determinants of inﬂuenza viruses. Although most

human inﬂuenza A viruses require prior adaptation in mice

before they will result in a productive infection, avian inﬂuenza

viruses will replicate and cause disease in mice without prior

adaptation. This outcome is most likely due, at least in part, to

the predominance of SAa2,3 (receptor preferred by avian

viruses) present in murine respiratory tissues (101). As is seen

in humans, onset of symptoms, lung pathology, and cytokine

production are temporally related to virus replication

(102–105). Differential pathogenicities of H5 avian inﬂuenza

viruses have been demonstrated in mice. A disease of low

pathogenicity (LP) is generally characterized by a non-lethal

infection that is restricted to the respiratory tract and is cleared

Maines et al Inﬂuenza virus pathogenesis and innate immunity

r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008 73

within a week, whereas a high pathogenicity (HP) phenotype

in mice is characterized by a lethal infection, systemic

replication, and peripheral blood lymphopenia (85, 95,

106–108). Viruses of the HP phenotype were at least 1000-

fold more lethal for mice than their LP counterparts (106, 109).

A similar dichotomy of pathogenicity phenotypes in mice were

observed among viruses of the H7 phenotype (96). Two HPAI

H7N1 viruses isolated from Italy in 2000 were also shown to be

neurovirulent in mice (110). However, H9N2 viruses exhibited

a low virulence phenotype in mice, although they replicated

efﬁciently and to high titers in the lungs of infected mice (111).

A summary of the relative levels of virulence exhibited in mice

and ferrets compared with humans is presented in Ta b l e 2 . Based

on the overall similarities between the relative virulence of

avian inﬂuenza viruses observed in humans and recapitulated in

the mouse model, we felt that the further use of inbred mouse

model for investigation of the role of the innate immune

response in the pathogenesis of these emerging viruses with

pandemic potential was warranted.

Molecular determinants of virulence

Pathogenesis studies in animal models are critical for the

identiﬁcation of virulence determinants of disease to better

understand the disease caused by inﬂuenza viruses observed in

humans. Multiple molecular determinants affecting virus viru-

lence have been identiﬁed. In mammalian models, the viru-

lence of H5N1 viruses is not always dictated by the multi-basic

cleavage site of the HA, indicating that additional virulence

determinants are involved (94, 95).

The polymerase complex (PB2, PB1, and PA) has been

identiﬁed as a virulence determinant of H5N1 viruses,

although phenotypes vary among animal models. The

presence of a Lys at 627 or an Asn at 701 in the PB2 protein of

H5N1 viruses confers a virulent phenotype in inbreed mice but

not in ferrets (94, 95, 112, 113) and does not always correlate

with severe disease in humans (83). Additional studies have

also implicated the polymerase complex in HPAI virus

virulence and virus adaptation to mammalian hosts (114,

115) and identiﬁed efﬁcient polymerase activity as a virulence

marker. The PB1-F2 protein targets alveolar macrophages and is

associated with secondary bacterial infections and virulence in

mice (13, 116–120). The NS1 protein of inﬂuenza viruses acts

as a virulence determinant by disrupting the IFN response of

hosts and preventing an antiviral state in infected cells from

being achieved (23, 24). The NS proteins of 1997 H5N1

viruses were shown to upregulate the expression of

proinﬂammatory cytokines in mice and pigs, directly

contributing to the virulent phenotype observed (121, 122).

HPAI virus virulence is dictated by multiple determinants

encoded on multiple gene segments. Continued characterization

of the molecular basis of inﬂuenza virus pathogenicity and the

identiﬁcation of key host factors in mammals is essential to

identify better ways to prevent disease in humans.

Innate immunity to avian inﬂuenza viruses

Cellular inﬁltration into the H5N1 virus-infected lung

When administered at high infectious dose [100–1000 50%

mouse infectious dose (MID

)] both HP and LP H5N1 viruses

Ta b l e 2 . Comparison of mouse and ferret models and human disease outcomes for avian inﬂuenza virus subtypes

Subtype Avian Pathotype Case, Outcome Human Disease Mice (LD

)



Ferret

(Lethality %) References

H5N1 (1997)

HPAI 13/Male Fatal Respiratory, lymphopenia, ARDS 1.6 33 (71, 95)

H5N1 (1997)

HPAI 5/Female Survived Respiratory, gastrointestinal, lymphocytosis 47 36 (71, 95, 106)

H5N1 (2004)

‰

HPAI 58/Female Fatal Respiratory, Renal, lymphopenia, ARDS 5.5 0 (95)

H5N1 (2004)

HPAI 7/Male Fatal Respiratory, gastrointestinal, lymphpenia, ARDS 1.7 100 (4, 95)

H7N7 (2003)

HPAI 57/Male Fatal ARDS 2.5 67 (59, 61, 96, 157)

H7N7 (2003)



HPAI Adult Survived Conjunctivitis 47 0 (59, 61, 96, 158)

H7N2 (2002-3)

LPAI Adult/Male Survived Respiratory 47 0 (96, 159, 160)

H7N3 (2004)

HPAI/LPAI 45/Male Survived Respiratory 47 0 (7, 62, 63, 96)

H9N2 (1999)

‰‰

LPAI 4/Female Survived Respiratory 48 0 (111, 161)



Expressed as the log

EID

required to give 1 LD

.AnLD

o3 is considered highly pathogenic.

A/HongKong/483/97.

A/HongKong/486/97.

‰

A/Thailand/SP83/04.

A/Thailand/16/04.

A/the Netherlands/219/03.



A/the Netherlands/230/03 and A/the Netherlands/33/03.

A/New York/107/03.

A/Canada/504/04.

‰‰

A/Hong Kong/1073/99.

Maines et al Inﬂuenza virus pathogenesis and innate immunity

74 r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008

replicate extensively in the lungs of infected BALB/c mice.

However, compared with the LP virus, the HP virus elicited a

rapid and massive depletion of peripheral lymphocytes (80%

drop in lymphocyte numbers on day 4 post-infection), deple-

tion of CD4

and CD8

T cell in lung and lymphoid tissue as

well as depletion of CD4CD8 double positive thymocytes on

day 6 post-infection (107). Interestingly, the HP virus repli-

cated to substantial titers in the thymus and thymic involution

was observed (95, 123), suggesting that subsets of T cells may

support replication of HP H5N1 strains in vivo. More recent

studies from this laboratory by Perrone and colleagues (127)

have compared two H5N1 viruses isolated from humans in

2004 (A/Thailand/16/2004 and A/Thailand/SP83/2004),

which exhibited differential virulence in BALB/c mice and

ferrets (95). Using a sub-lethal dose (approximately 1–10

MID

), infection of mice permitted characterization of the

phenotype and extended kinetics of leukocyte inﬁltration into

the infected lung. Using this approach, macrophages and

neutrophils were shown to be the predominant cell types

inﬁltrating the lung early after infection and peaking around

day 7 post-infection; signiﬁcantly higher numbers of these two

cell types were detected in HP virus-infected lungs compared

with LP virus infection, which also correlated in this compar-

ison with the reduced lung viral titers achieved by the LP virus

compared with the HP strain. In contrast, no signiﬁcant

differences in the percentages of dendritic cells (DCs), CD4

and CD8

T cells in the lungs between HP and LP virus

infections were observed.

Proinﬂammatory cytokine and chemokine response in

H5N1 virus-infected mice

Following primary infection of mice with mouse-adapted

strains of inﬂuenza A viruses, multiple proinﬂammatory cyto-

kines are produced in the lungs, including IL-1b, TNF-a, and

IFN-g(102, 103, 105, 124). The differential pathogenicity

phenotypes exhibited by HPAI H5N1 viruses in mice prompted

us to examine further the virus–host interactions in this system.

We ﬁrst addressed the level of proinﬂammatory cytokine and

chemokines in the lungs and brains of mice infected with a

high dose of prototype H5N1 viruses isolated from humans in

1997 that exhibited either a HP or LP phenotype in BALB/c or

C57Bl/6 mice (107, 123). Elevated levels of IL-1b, TNF-a,

IFN-g, IL-6, MIP-1a, MIP-2, and RANTES (regulated upon

activation, normal T-cell expressed, and presumably secreted)

proteins were detected 3–5 days after infection with either HP

or LP virus (107, Szretter et al., unpublished data) (Fig. 2).

However, by day 5–6 post-infection, concentrations

of IL-1b, IFN-g, and MIP-1awere signiﬁcantly lower in HP

virus-infected mice than in mice infected with the LP virus. In

contrast, levels of IL-6 were higher in the lungs of mice

infected with the HP virus, whereas levels of TNF-awere

comparable for the two viruses. Recently, acute lung injury in

H5N1 virus-infected mice was shown to be associated with

elevated IL-6 expression and mediated by TLR4 (Toll-like

receptor 4)-TRIF (TIR-domain-containing adapter-inducing

IFN-b)-TRAF6 (TNF receptor-associated factor)-NF-kB

(nuclear factor-kB) signaling (125). Because HP viruses but

not LP viruses administered intranasally at lethal doses spread

to the brain, we also assessed the inﬂammatory cytokine/

chemokine response in this organ. As expected, there was no

signiﬁcant production of any of the cytokines or chemokines

tested in mice infected with the LP virus. In contrast, mice

infected with HP virus exhibited signiﬁcantly elevated levels of

IL-1b, TNF-a, IFN-g, IL-6, MIP-2, and MIP-1aby day 6 post-

infection, a time point that immediately preceded the death of

the mice. The local synthesis of proinﬂammatory cytokines,

such as TNF-aor IL-1, within the brain can lead to anorexia,

weight loss, and death (126), possibly contributing to H5N1

virus pathogenesis in the murine model.

In studies which have used sublethal intranasal infection of

mice with even more virulent H5N1 strains isolated from

humans in 2004, elevated levels of MCP-1, MIP-1a,KC

(murine IL-8), IL-1a, IL-6, and IFN-gwere detected on day 4

post-infection, which coincided with peak lung cellularity. In a

side-by-side comparison, the HP 2004 H5N1 virus strain

consistently induced levels of proinﬂammatory cytokines and

chemokines that signiﬁcantly surpassed those induced by the

1918 H1N1 pandemic virus, despite a comparable level of

virus replication (127). Contemporary H5N1 viruses are

approximately 50 times more lethal for BALB/c mice than the

1918 pandemic virus (95, 128). Early and elevated production

of TNF-aand type I IFNs in the lungs of mice is also a notable

feature of infection with a highly virulent H7N7 virus (96).

Role of cytokine and chemokine responses in H5N1 virus

pathogenesis in mice

To more clearly deﬁne the role of certain cytokines or

chemokines implicated in recovery from inﬂuenza infection

and/or contribution to severity of illness, we used mice with

targeted gene disruptions to investigate the consequences of

signaling by IL-6, IL-1b, TNF-a, and MIP-1a, because differ-

ential expression of these cytokines and chemokine were

observed in the murine H5N1 virus disease model. Using this

approach, we observed that despite the strong lung IL-6

production in response to H5N1 virus infection among wild-

type mice, the disruption of IL-6 expression did not

Maines et al Inﬂuenza virus pathogenesis and innate immunity

r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008 75

Fig. 2. Determination of proinﬂammatory cytokine levels in the lungs and brains of BALB/c mice infected intranasally with 1000 MID

avian H5N1 viruses isolated from humans and exhibiting a HP (ﬁlled bars) or LP (shaded bars) phenotype. Tissues were collected at the indicated

times, and clariﬁed tissue homogenized were assayed for levels of the indicated cytokines or chemokine by ELISA. Bars represent the mean protein

concentration (in pg/ml) SD. Constitutive levels of cytokine/chemokine in normal mice are indicated by the dotted lines.



P0.05, HP versus LP

group.

Maines et al Inﬂuenza virus pathogenesis and innate immunity

76 r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008

appreciably affect the kinetics of virus replication and outcome

of infection with HP and LP H5N1 viruses that show different

virulence phenotypes in wildtype mice. This may be due, in

part, to the redundancy of the biological effects of IL-6 in

mammalian systems. Nevertheless, these ﬁndings were consis-

tent with those of Kozak et al. (129), who observed no

signiﬁcant difference in weight loss between IL-6-deﬁcient

and wildtype mice infected with mouse-adapted inﬂuenza A

virus. Similarly, no apparent role for MIP-1achemokine

expression in the H5N1 virus-induced disease process was

identiﬁed. Previous studies with mouse-adapted human inﬂu-

enza viruses suggested that MIP-1acontributed to the severity

of the lung inﬂammatory response and pathology, and viral

clearance, and may contribute to recruitment of virus-speciﬁc

CD8

T cells to the infected lung (130, 131). Our results

suggest that MIP-1aexpression is not critical for virus recovery

from LP H5N1 virus infection, but it remains possible that in

the MIP-1a-deﬁcient mouse, other chemokines with over-

lapping functions may act in a compensatory manner. In

studies where signaling through multiple chemokines was

assessed using mice deﬁcient in the chemokine receptor

CCR5 (receptor for MIP-1a, MIP-1ba, and RANTES), an

earlier, robust macrophage inﬁltration into the inﬂuenza

virus-infected lung was associated with increased mortality

(132). In contrast, mice deﬁcient in the chemokine receptor

CCR2 (receptor for MCP-1a) demonstrated delayed macro-

phage recruitment and increased survival (132). These results

highlight the overall consequences for the outcome of inﬂuen-

za infection when the balance of the inﬁltrating monocyte/

macrophage population is altered.

In lethal high dose infections with the HP H5N1 virus in

mice, we observed previously a signiﬁcant reduction in IL-1b

production in the lung at later times during infection.

Following infection with the LP H5N1 virus, IL-1R-deﬁcient

mice lost signiﬁcantly more weight, exhibited delayed

clearance of virus and had a greater proportion of animals

succumb to infection compared with their immunocompetent

counterparts. These results suggest that IL-1 is important for

the induction of effective adaptive immune responses against

H5N1 viruses that aid in viral clearance and that the reduced

IL-1bproduction observed in HP H5N1-infected wildtype

mice may contribute to rapid and lethal disease outcome.

Other studies with mouse-adapted inﬂuenza virus have shown

a more modest role for IL-1bin virus clearance and recovery,

with diminished or altered antigen-speciﬁc T-cell responses

compared with wildtype mice (124, 133).

Both HP and LP H5N1 viruses induced elevated amounts of

TNF-afollowing productive replication of virus in the lungs of

mice. HP H5N1 virus-infected mice that lacked the TNF

receptor1 (TNFR1) exhibited a signiﬁcant delay in weight

loss, a marker of morbidity, but only a modest delay in time to

death and no effect on virus replication or clearance, lung

histopathology or systemic spread. Similar results were

obtained when TNF-awas neutralized by treatment with anti-

TNF-aneutralizing antibody. An interesting ﬁnding in TNFR1

deﬁcient mice infected with HP H5N1 virus was that thymic

involution, observed in wildtype BALB/c and C57Bl/6x129

mice, was substantially diminished, and the loss of total

thymocytes was reduced in TNFR1-deﬁcient mice compared

with wildtype control mice. Because TNF-aand related TNF-

superfamily members including TNF-related apoptosis-

inducing ligand (TRAIL) are known to mediate apoptosis

of T cells, and in particular, thymocytes (134, 135), our

results suggest that the thymic depletion observed in immuno-

competent H5N1 virus-infected mice is primarily due to the

production of increased levels of TNF-ain response to H5N1

infection, rather than a direct cytolytic effect of the virus itself.

In summary, these results suggest that TNF-amay contribute to

early H5N1 virus disease severity, whereas IL-1 may play a role

in viral clearance late in H5N1 virus infection in the murine

model.

Control of H5N1 virus infection in mice by type I interferon

Type I interferons play a critical role in innate resistance to

inﬂuenza virus infection and the induction of adaptive immu-

nity effector responses. Viral RNA products generated during

infection are recognized by Toll-like receptors (TLRs) and RIG-

I like receptors (RLR) to initiate the interferon response.

Airway epithelial cells recognize the double-strand RNA and/

or 50-triphosphate ssRNA via retinoic acid-inducible gene I

(RIG-I), a cytosolic RNA helicase, resulting in production of

type-I IFN through an adapter protein IPS-1 (136). The

interferon signaling can also be initiated by engagement of

TLR3 to dsRNA in airway epithelial cells (137). In plasmacy-

toid DCs, ssRNA is recognized by TLR7 and its adaptor MyD88,

triggering type I interferon response (138). Production of type

I interferon leads to the induction of interferon-stimulated

genes (ISGs) with antiviral activities mediated through the JAK-

STAT signaling pathway. Evasion of the host innate immunity,

including the type I interferon response, has been proposed as

a mechanism of virulence of avian H5N1 viruses in mammals

including humans. Inﬂuenza virus NS1 protein inhibits the

RIG-I induction of interferon-b(139, 140). Avian H5N1

viruses induced elevated amounts of IFN-ab following pro-

ductive replication of the virus in the lungs of inbred mice

(K. J. Szretter et al. unpublished data). Although inbred mouse

Maines et al Inﬂuenza virus pathogenesis and innate immunity

r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008 77

strains lack a functional Mx1 protein, an ISG product which

confers resistance to orthomyxoviruses, other interferon-in-

duced antiviral mediators are expressed which can relay the

effects of the type I interferon response to inﬂuenza infection in

these model systems. IFN-abR-deﬁcient mice infected with

either HP or LP H5N1 virus had a more rapid time-to-death

and increased viral replication in extrapulmonary organs as

compared with immunocompetent mice (Szretter et al., un-

published data). Furthermore, H5N1/1997 and H5N1/2004

viruses were found to be sensitive to the antiviral effects of

interferon in vitro in that replication of either HP or LP virus was

signiﬁcantly reduced in murine lung epithelial cells (LA-4)

pretreated with either IFN-aor IFN-b. Therefore, in the mouse

model in which avian H5N1 viruses exhibit either high or low

virulence, the type I interferon response contributes to early

containment of H5N1 virus in vivo and control of replication in

vitro. These results are consistent with studies using a virulent,

mouse-adapted virus, in which mice that are unable to respond

to IFN-ab experienced greater mortality and virus dissemina-

tion (141). These results further highlight the fact that the

outcome of inﬂuenza infection depends not only on the dose

and relative virulence of the virus, but also on host-dependent

factors.

Cytokine and chemokine expression in human cells in

response to avian inﬂuenza virus infection

Because innate immune responses are inherently host speciﬁc,

it is important to assess the relative induction of cytokine and

chemokines by avian inﬂuenza viruses in human primary cell

cultures and/or cell lines in order to establish correlating

phenotypes related to the virulence of these viruses in vivo.

Identifying signature phenotypes of virulence in in vitro systems

may be a means with which to rapidly assess the potential

virulence of emerging inﬂuenza viruses for humans, as well as

improve our understanding of the contribution of human

genetics to the severity of inﬂuenza disease in general. In

human primary alveolar (type II pneumocytes) and bronchial

epithelial cells (NHBE), H5N1 viruses from 1997 and 2004

induced higher levels of IP-10, IFN-b, RANTES, and IL-6

mRNA expression compared with a human H1N1 virus

(142), although all viruses replicated to comparable levels in

these two cell types. Production of IP-10, IL-6, and RANTES

protein was also signiﬁcantly higher in H5N1 virus-infected

cultures versus human H1N1 virus-infected cultures, but

production of IFN-bwas below detectable limits for all viruses

up to 24 h post-infection. Interestingly, the 2004 H5N1 viruses

were more potent inducers of IP-10 than the1997 H5N1 virus;

high levels of IP-10, a macrophage chemoattractant, were also

detected in humans with severe or fatal H5N1 virus disease

(72, 83). Because primary human cell cultures may vary

considerably in terms of SA expression and support for virus

replication, our laboratory has used polarized human bronchial

epithelial (Calu-3) cell cultures, which express SA receptors

recognized by both human and avian viruses, for the relative

comparison of cytokine induction by human and avian inﬂu-

enza viruses. In studies focusing on the type I interferon

response, a highly virulent 2004 H5N1 virus was found to

trigger a delayed and weaker IFN-bresponse, weaker JAK-STAT

pathway activation, and ISGs induction compared with a

contemporary human H3N2 inﬂuenza virus (143). In particu-

lar, comparison of H5N1 viruses which exhibited HP and LP

pathogenicity phenotypes in mice and ferrets also displayed

differential induction of IFN-b, with the HP strain exhibiting a

stronger dampening of the early IFN-bresposne in Calu-3 cells

but higher levels of TNF-aexpression (143, H. Zeng et al.,

unpublished data). Therefore, an H5N1 virus which exhibits

heightened virulence in mammalian models demonstrated a

better ability to attenuate the host IFN response in human

respiratory epithelial cells, compared with less virulent avian or

human viruses.

In ongoing studies, we are evaluating cytokine expression in

Calu-3 cells in response to HPAI H7 subtype viruses, to

determine whether features of human cytokine induction are

common for highly virulent avian viruses in respiratory

epithelial cultures that support their replication. Interestingly,

we found that HPAI H7 viruses exhibited a profoundly reduced

IFN-bresponse in Calu-3 cells, a response even lower than levels

induced by an HPAI H5N1 virus (Belser et al., unpublished data).

This delayed and/or weakened type I interferon response in

respiratory epithelial cells infected with highly virulent H5 and

H7 viruses suggests that one mechanism of severe disease may

be due to suppression of the early innate immune response in

cells in which primary virus replication takes place. The

pandemic 1918 virus was recently reported to cause a similar

downregulation of the type I interferon response in infected

non-human primates (144). Studies are underway to determine

whether the NS1 protein, a known viral antagonist of the host

interferon response, contributes to this human response to

highly pathogenic avian strains.

Because macrophage inﬁltration into infected lungs has been

implicated in the immunopathology of H5N1 virus infection

in both humans and a murine model, investigators have used

cultures of primary human monocyte-derived macrophages to

better understand their ability to express cytokines and

chemokines in response to emerging avian inﬂuenza viruses.

Cheung et al. (145) demonstrated that H5N1 viruses from

Maines et al Inﬂuenza virus pathogenesis and innate immunity

78 r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008

1997 were more potent inducers of IFN-band TNF-agene

expression than were human H3N2 or H1N1 inﬂuenza viruses.

In particular, monocyte-derived macrophages infected with a

1997 H5N1 virus produced TNF-ain excess of that stimulated

by LPS treatment. High levels of TNF-awere also induced by a

LPAI H9N2 virus which possessed essentially similar internal

genes to the 1997 H5N1 viruses, including the non-structural

gene which was implicated in contributing to the abundant

TNF-aproduction. Furthermore, infection of monocyte-

derived macrophages with 1997 H5N1 viruses or the LPAI

H9N2 virus, induced high levels of another TNF family

member, TRAIL which contributed to the apoptosis of a co-

cultured Jurkat T cell line (146). Induction of TRAIL by the

avian H7 inﬂuenza subtype has also been implicated in efﬁcient

replication of the virus in epithelial cells (147). Human

peripheral blood mononuclear cell-derived macrophages

supported productive and efﬁcient replication of a 2004 HP

H5N1 virus which induced signiﬁcantly higher levels

of cytokines and chemokines including IL-1b, IL-6, IL-12,

TNF-a, MCP-1, and MIP-1bcompared with a LP strain (127).

High levels of IFN-awere also detected following infection of

human monocyte-derived macrphages with 1997 H5N1

strain, but in comparison, highly pathogenic viruses of the H7

subtype, exhibit reduced and/or suppressed production of

IFN-ain this cell type (Belser et al., unpublished data). In

addition to macrophages, human monocyte-derived and

myeloid DCs are susceptible to infection with H5N1 viruses

(127, 148).

Immunomodulators as interventions for H5N1 virus

infection

Human infections with currently circulating H5N1 viruses are

frequently fatal and intervention strategies are few. Suscept-

ibility to one of two classes of inﬂuenza antivirals, the

adamantane M2 channel blockers, varies widely among the

genetically diverse clades and subclades of H5N1 viruses in

circulation (97) and emergence of resistant strains during

treatment with the NA inhibitor, oseltamivir, has been

reported (149). Late initiation of antiviral therapy, due to

delayed presentation of patients or diagnosis of H5N1 virus

infection, further limits efﬁcacy. The general understanding

that H5N1 viruses induce hypercytokinemia in severely ill

patients has raised the question as to whether the use of

modulators of proinﬂammatory cytokine responses may repre-

sent a beneﬁcial therapeutic strategy. However, early clinical use

of corticosteroids to reduce inﬂammatory responses in H5N1

virus-infected patients proved to be detrimental for disease

outcome (97). Treatment of mice with a gluticocorticoid

cytokine inhibitors failed to reduce the lethality in mice

infected with a highly virulent 2004 H5N1 (150). In contrast,

gemﬁbrozil, a commonly prescribed lipid-lowering drug,

which has also been shown to inhibit proinﬂammatory cyto-

kines, was shown to signiﬁcantly increase survival in mice

infected with an H2N2 virus, similar to that which caused the

1957 pandemic, but of more modest virulence than the avian

H5N1 viruses (151). Cholesterol-lowering statins, which ex-

hibit non-speciﬁc anti-inﬂammatory activities, have been pro-

posed as another possible class of drugs which could be

efﬁcacious in the treatment for human H5N1 virus infection

(152). Preliminary studies conducted in this laboratory with

two FDA-approved statin drugs have shown no beneﬁt to

survival or have seen no effect, and found only modest delay

in morbidity (as measured by weight loss), and no effect on

survival, viral replication or spread in our murine model of

H5N1 virus disease (K. J. Szretter et al., unpublished data).

Although we observed no improvement in the outcome of

disease, we also noted no enhancement of disease caused by

global suppression of the immune response during H5N1 virus

infection. A recent report suggested that a combination therapy

which included a NA inhibitor, celecoxib, a non-steroidal anti-

inﬂammatory, and mesalazine, an anti-inﬂammatory aminosa-

licylate, signiﬁcantly improved survival in mice infected with

highly virulent H5N1 virus (153). While these studies have

taken a rather non-speciﬁc approach to moderating the cyto-

kine response to H5N1 viruses, a more targeted approach,

focusing on the type I interferon response, has yielded

promising results. Prophylactic treatment of mice expressing

the Mx1 gene with human IFN-abefore infection with a highly

lethal dose of H5N1 virus signiﬁcantly reduced viral titers in

the lung, prevented viral spread and protected mice from fatal

disease (154). It will be interesting to determine whether

therapeutic treatment with interferon is also beneﬁcial in this

model system. Furthermore, these results suggest that addi-

tional knowledge of the contribution of cytokines in the

immunopathology of fatal H5N1 virus disease will provide a

more rational and targeted approach in the development of

intervention strategies that can enhance patient survival.

Concluding remarks

Another inﬂuenza pandemic will certainly occur. However,

answers to the critical questions of when, where, and what

virus will emerge continue to evade us. Since 2005, avian

H5N1 viruses have evolved into multiple phylogenetically and

largely geographically distinct groups, but only some of these

Maines et al Inﬂuenza virus pathogenesis and innate immunity

r2008 The Authors Journal compilation r2008 Blackwell Munksgaard Immunological Reviews 225/2008 79

have caused human disease (4). This massive geographic,

genetic, and antigenic diversity among avian H5N1 viruses

has heightened concern that these viruses may be poised to

cause the next pandemic and have complicated international

public health efforts to develop effective prevention and

control strategies. Avian viruses of the H7 and H9 subtypes

have also demonstrated changes in receptor binding that

potentially move them one step closer to a pandemic pheno-

type. Concerns that the next pandemic may be a return to the

H2 subtype are also warranted, because approximately two-

thirds of the today’s global population were born after 1967,

the last time an H2N2 virus circulated in humans, and have no

speciﬁc B cell immunity to the H2 HA. A better understanding

of the complex interplay between the virus and the host and the

consequences for disease outcome, will help us develop

improved methods to identify rapidly the potential for heigh-

tened virulence of an emerging inﬂuenza virus, identify a

possible genetic basis for disease, and develop appropriate

intervention strategies.

The human and animal studies reviewed here suggest that that

severe and fatal H5N1 virus disease is a consequence of highly

efﬁcient replication and preferred tropism, at least in humans, for

type II alveolar epithelial cells and alveolar macrophages in the

lower airways, followed by a dysregulated host inﬂammatory

response. Whether the high efﬁciency of replication by H5N1

viruses observed in vivo and in vitro is due to molecular

determinants in polymerase gene(s) that enhance inherent

replication efﬁciency of the virus, or those in other genes that

may allow the virus to overcome or delay early host control via

the type I interferon response, or a combination of both, requires

further study. Efﬁcient and rapid viral replication in respiratory

epithelial cells and alveolar macrophages would enhance

expression of chemokines and cytokines, in a virus-dependent

manner, and promote cellular inﬁltration of macrophages,

neutrophils and lymphocytes and their activation in the infected

lung. A second wave of cytokines released from inﬁltrating,

macrophages undergoing virus-induced apoptosis would further

amplify the inﬂammatory response, leading to high

concentrations of cytokines in the circulation which may have

destructive, multi-organ consequences. Several lines of evidence

suggestthatlymphocytes,particularlyTcells,aredepletedfrom

respiratory tissues, and peripheral blood in H5N1 virus infection,

possibly due to apoptosis induced by macrophage-derived TNF

family proteins. The role of CD8

T cells in release of cytokines

and immunopathology, bears further attention, but their lack of

accumulation at least in the murine H5N1 virus-infected lung, is

consistent with the observed uncontrolled replication of HP

H5N1 viruses. Although multiple studies suggest that

proinﬂammatory cytokine responses correlate with disease

severity, the use of gene knockout mice failed to place any single

cytokine in a key role in fatal H5N1 virus disease. Strikingly, the

HP H5N1 viruses exhibited greater virulence in mice, and

produced signiﬁcantly higher levels of cytokines and

chemokines in mouse lungs and human macrophages than the

reconstructed 1918 virus, the so-called ‘mother of all pandemics’

(42). Further studies in mice deﬁcient in multiple cytokines or

cytokine receptors may overcome the redundancies in the

cytokine network and identify more pronounced effects on

disease outcome. Such studies should provide further insight

and a basis for the rational design of intervention strategies that

target speciﬁc host responses for the amelioration of severe

inﬂuenza disease caused by emerging or remerging viruses.

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Influenza A infection dynamics in an ex vivo organ culture of pig trachea

Thesis

Full-text available

Feb 2011

Filipe Nunes

Ex vivo organ cultures (EVOC) of tracheal explants with an air interface system have been successfully developed and used in the study of both human and animal respiratory pathogens. Such systems reproduce, to a great extent, the physiological conditions and the cellular complexity of the respiratory mucosa of the host in a highly controlled setting. In this study, we developed an EVOC system using pig trachea to quantify the infection dynamics of influenza A viruses. Pig tracheal explants maintained the structure and function present in the living host preserving the intricate complexity of the respiratory mucosa during the first four days of the organ culture. Inoculation with the swine virus A/swine/England/435/06 (H1N1) resulted in productive viral infection in a dose-dependent fashion. Infected explants showed histopathology of influenza infection. Viral titres peaked at day 2 post-inoculation and ciliary activity was abrogated shortly after. Explants collected from different sections of the trachea showed significant differences in the production of infectious virus. The host innate immunity induction was evaluated by quantifying the mRNA levels of the cytokines IFN-β, IFN-α, TNF-α, IL-1α, IL-6 and the chemokine IL-8 during influenza infection of tracheal explant. In uninfected explants, IFN-β IFN-α and TNF-α mRNA expression levels did not change while cytokines associated with the inflammatory response - IL-1α, IL-6 and IL-8 - were significantly elevated throughout the organ culture. In influenza-infected explants the elevation in IFN-β expression appeared to be the predominant early response to influenza infection. IFN-α and TNF-α levels were not significantly altered whereas IL-1α, IL-6 and IL-8 expression showed a marked decrease in expression when the viral genome copy numbers peaked. Productive infection was also observed in explants infected with the 2009 influenza pandemic virus A/England/195/09 (H1N1) but not with laboratory-adapted strain A/Puerto Rico/8/34 (H1N1) and not related with the induction of the innate immune system in tracheal explants.

The role of mammals in Avian Influenza: a review

Article

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Mar 2024

Avian influenza (AI) is an infectious viral disease of birds, including domestic poultry, which has been causing outbreaks worldwide, leading to several millions of dead wild birds and culled poultry. AI is mainly found in birds, but recently, there was an increase in reported infections in mammals, ranging from no symptoms to mass mortality events and some human cases. Epidemiologically of great concern, evidence of mammalian adaptations have been found, but the transmission routes and pathogenesis in mammals are still to be defined. Hence, it is paramount to address all facets of AI viruses epidemiology, including investigating taxa not customarily thought to be involved in the transmission and/or trafficking of AI, such as wild mammals. The scope of this report was to assess the role of mammals in AI epidemiology, virology and pathology, i.e. AI maintenance, reservoir role, immunity, role of mammals in a potential pandemic. To do so, we performed an all-encompassing review of the literature on the topic with a two-fold approach: a systematic review of the published AI cases in wild mammals and a narrative approach to provide an expert opinion on the role of mammals in AI spread. The final number of peer-reviewed papers included in the systematic literature review was 76, resulting in 120 unique infection records with AI in wild mammal species. The most represented taxa were included in the order Carnivora. The risk of infection was identified mainly as predation (or feeding) upon infected birds or contact with avian species. Evidence of mammal-to-mammal transmission in the wild is only circumstantial and yet to be confirmed. Cases of AI from the systematic review of experimental findings were discussed concerning epidemiology, pathology and virology. Knowledge gaps and potential pandemic drivers were identified. In summary, although a greater number of infections in wild mammals have been reported, there is no hard evidence for sustained mammal-to-mammal transmission in the wild. The factors contributing to the increased number of infections found in wild carnivores are not clear yet, but the unprecedented global spread of highly pathogenic avian influenza (HPAI) viruses creates ample opportunities for intense, mostly alimentary, contact between infected wild birds and carnivores. Close surveillance of circulating strains and continued assessment of new epidemiological situations are crucial to quickly identify strains with enhanced mammalian fitness.

CHAPTER -8 EPIZOOTIOLOGY

Chapter

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Sanjoy Samanta

Alpha-lipoic acid ameliorates influenza A virus caused acute pneumonia though enhancement of anti-viral T cell immunity and suppression of macrophage activation by inhibiting ERK1/2 signaling pathway

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Aging shapes infection profiles of influenza A virus and SARS-CoV-2 in human lung slices

Preprint

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The recent coronavirus disease 2019 (COVID-19) outbreak revealed the susceptibility of elderly patients to respiratory virus infections, showing cell senescence or subclinical persistent inflammatory profiles and favouring the development of severe pneumonia. In our study, we evaluated the potential influence of lung aging on the efficiency of replication of influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2), as well as determined the pro-inflammatory and antiviral responses of the distal lung tissue. Using precision-cut lung slices (PCLS) from donors of different ages, we found that pandemic H1N1 and avian H5N1 IAV replicated in the lung parenchyma with high efficacy. In contrast to these IAV strains, SARS-CoV-2 early isolate and Delta variant of concern (VOC) replicated less efficiently in PCLS. Interestingly, both viruses showed reduced replication in PCLS from older compared to younger donors, suggesting that aged lung tissue represents a sub optimal environment for viral replication. Regardless of the age-dependent viral loads, PCLS responded to infection with both viruses by an induction of IL-6 and IP-10/CXCL10 mRNAs, being highest for H5N1. Finally, while SARS-CoV-2 infection was not causing detectable cell death, IAV infection caused significant cytotoxicity and induced significant early interferon responses. In summary, our findings suggest that aged lung tissue might not favour viral dissemination, pointing to a determinant role of dysregulated immune mechanisms in the development of severe disease.

Diverse roles of lung macrophages in the immune response to influenza A virus

Article

Full-text available

Sep 2023

Influenza viruses are one of the major causes of human respiratory infections and the newly emerging and re-emerging strains of influenza virus are the cause of seasonal epidemics and occasional pandemics, resulting in a huge threat to global public health systems. As one of the early immune cells can rapidly recognize and respond to influenza viruses in the respiratory, lung macrophages play an important role in controlling the severity of influenza disease by limiting viral replication, modulating the local inflammatory response, and initiating subsequent adaptive immune responses. However, influenza virus reproduction in macrophages is both strain- and macrophage type-dependent, and ineffective replication of some viral strains in mouse macrophages has been observed. This review discusses the function of lung macrophages in influenza virus infection in order to better understand the pathogenesis of the influenza virus.

Understanding Neutrophil Dynamics during COVID-19 Infection

Article

Full-text available

Feb 2023

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results in varied clinical outcomes, with virus-induced chronic inflammation and tissue injury being associated with enhanced disease pathogenesis. To determine the role of tissue damage on immune populations recruitment and function, a mathematical model of innate immunity following SARS-CoV-2 infection has been proposed. The model was fitted to published longitudinal immune marker data from patients with mild and severe COVID-19 disease and key parameters were estimated for each clinical outcome. Analytical, bifurcation, and numerical investigations were conducted to determine the effect of parameters and initial conditions on long-term dynamics. The results were used to suggest changes needed to achieve immune resolution.

Enhanced viral infectivity and reduced interferon production are associated with high pathogenicity for influenza viruses

Article

Full-text available

Feb 2023
PLOS COMPUT BIOL

Epidemiological and clinical evidence indicates that humans infected with the 1918 pandemic H1N1 influenza virus and highly pathogenic avian H5N1 influenza viruses often displayed severe lung pathology. High viral load and extensive infiltration of macrophages are the hallmarks of highly pathogenic (HP) influenza viral infections. However, it remains unclear what biological mechanisms primarily determine the observed difference in the kinetics of viral load and macrophages between HP and low pathogenic (LP) viral infections, and how the mechanistic differences are associated with viral pathogenicity. In this study, we develop a mathematical model of viral dynamics that includes the dynamics of different macrophage populations and interferon. We fit the model to in vivo kinetic data of viral load and macrophage level from BALB/c mice infected with an HP or LP strain of H1N1/H5N1 virus using Bayesian inference. Our primary finding is that HP viruses have a higher viral infection rate, a lower interferon production rate and a lower macrophage recruitment rate compared to LP viruses, which are strongly associated with more severe tissue damage (quantified by a higher percentage of epithelial cell loss). We also quantify the relative contribution of macrophages to viral clearance and find that macrophages do not play a dominant role in the direct clearance of free viruses although their role in mediating immune responses such as interferon production is crucial. Our work provides new insight into the mechanisms that convey the observed difference in viral and macrophage kinetics between HP and LP infections and establishes an improved model-fitting framework to enhance the analysis of new data on viral pathogenicity.

International Journal of Molecular Sciences PGE2 Produced by Exogenous MSCs Promotes Immunoregulation in ARDS Induced by Highly Pathogenic Influenza A through Activation of the Wnt-β-Catenin Signaling Pathway

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Full-text available

Apr 2023
INT J MOL SCI

Acute respiratory distress syndrome is an acute respiratory failure caused by cytokine storms; highly pathogenic influenza A virus infection can induce cytokine storms. The innate immune response is vital in this cytokine storm, acting by activating the transcription factor NF-κB. Tissue injury releases a danger-associated molecular pattern that provides positive feedback for NF-κB activation. Exogenous mesenchymal stem cells can also modulate immune responses by producing potent immunosuppressive substances, such as prostaglandin E2. Prostaglandin E2 is a critical mediator that regulates various physiological and pathological processes through autocrine or paracrine mechanisms. Activation of prostaglandin E2 results in the accumulation of unphosphory-lated β-catenin in the cytoplasm, which subsequently reaches the nucleus to inhibit the transcription factor NF-κB. The inhibition of NF-κB by β-catenin is a mechanism that reduces inflammation.

A target-cell limited model can reproduce influenza infection dynamics in hosts with differing immune responses

Article

Apr 2023
J THEOR BIOL

We consider a hierarchy of ordinary differential equation models that describe the within-host viral kinetics of influenza infections: the IR model explicitly accounts for an immune response to the virus, while the simpler, target-cell limited TEIV and TV models do not. We show that when the IR model is fitted to pooled experimental murine data of the viral load, fraction of dead cells, and immune response levels, its parameters values can be determined. However, if, as is common, only viral load data are available, we can estimate parameters of the TEIV and TV models but not the IR model. These results are substantiated by a structural and practical identifiability analysis. We then use the IR model to generate synthetic data representing infections in hosts whose immune responses differ. We fit the TV model to these synthetic datasets and show that it can reproduce the characteristic exponential increase and decay of viral load generated by the IR model. Furthermore, the values of the fitted parameters of the TV model can be mapped from the immune response parameters in the IR model. We conclude that, if only viral load data are available, a simple target-cell limited model can reproduce influenza infection dynamics and distinguish between hosts with differing immune responses.

1918 Influenza: the Mother of All Pandemics

Article

Full-text available

Jan 2006
EMERG INFECT DIS

Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia

Article

Full-text available

Nov 2006

Avian influenza A (H5N1) viruses cause severe disease in humans, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity. To assess the relevance of these findings for human disease, we performed virological and immunological studies in 18 individuals with H5N1 and 8 individuals infected with human influenza virus subtypes. Influenza H5N1 infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H5N1 cases and was associated with higher pharyngeal viral loads. We observed low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels in H5N1-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H5N1 viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. Our observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment.

A Mouse Model for the Evaluation of Pathogenesis and Immunity to Influenza A (H5N1) Viruses Isolated from Humans

Article

Jul 1999

During 1997 in Hong Kong, 18 human cases of respiratory illness, including 6 fatalities, were caused by highly pathogenic avian influenza A (H5N1) viruses. Since H5 viruses had previously been isolated only from avian species, the outbreak raised questions about the ability of these viruses to cause severe disease and death in humans. To better understand the pathogenesis and immunity to these viruses, we have used the BALB/c mouse model. Four H5N1 viruses replicated equally well in the lungs of mice without prior adaptation but differed in lethality for mice. H5N1 viruses that were highly lethal for mice were detected in multiple organs, including the brain. This is the first demonstration of an influenza A virus that replicates systemically in a mammalian species and is neurotropic without prior adaptation. The mouse model was also used to evaluate a strategy of vaccination against the highly pathogenic avian H5N1 viruses, using an inactivated vaccine prepared from nonpathogenic A/Duck/Singapore-Q/F119-3/97 (H5N3) virus that was antigenically related to the human H5N1 viruses. Mice administered vaccine intramuscularly, with or without alum, were completely protected from lethal challenge with H5N1 virus. Protection from infection was also observed in 70% of animals administered vaccine alone and 100% of mice administered vaccine with alum. The protective effect of vaccination correlated with the level of virus-specific serum antibody. These results suggests a strategy of vaccine preparedness for rapid intervention in future influenza pandemics that uses antigenically related nonpathogenic viruses as vaccine candidates.

Molecular correlates of influenza A H5N1 virus pathogenesis in mice

Article

Oct 2001
Int Congr

Background: In 1997, highly pathogenic avian influenza A H5N1 viruses caused 18 human cases of respiratory illness and six deaths in Hong Kong. The genes of the H5N1 viruses isolated from humans were derived from avian influenza viruses, with no evidence of reassortment with human influenza viruses. The molecular determinants of human pathogenesis were not evident. Methods: The BALB/c mouse was used as a mammalian model to evaluate the biological and molecular basis of human H5N1 virus pathogenesis. Molecular correlates of H5N1 virus pathogenicity for mice were sought by analyzing the nucleotide sequence of human H5N1 viruses. Results: Based on high and low lethality for mice, the pathogenicity phenotype of 15 human H5N1 viruses was characterized; nine viruses displayed a high, five a low, and one an intermediate pathogenicity phenotype. H5N1 viruses with a high pathogenicity phenotype replicated in multiple solid organs and caused depletion of peripheral blood leukocytes. Sequence analysis determined that five specific amino acids in four proteins correlated with pathogenicity in mice. Conclusions: Alone or in combination, these specific residues are the likely determinants of virulence of human H5N1 influenza viruses in this mammalian model.

Avian influenza A/(H7N2) outbreak in the United Kingdom

Article

May 2007

Collective Editorial team

Several cases of influenza-like-illness (ILI) and/or conjunctivitis in humans have been linked to an outbreak of avian influenza in poultry at a smallholding near Corwen in northern Wales in the United Kingdom (UK).

Orthomyxoviridae: The viruses and their replication

Article

Jan 1996

Update: Influenza activity - United States and worldwide, 2006-07 season, and composition of the 2007-08 influenza vaccine

Article

Aug 2007

Sickness behavior in mice deficient in interleukin-6 during turpentine abscess and influenza pneumonitis

Article

Jan 1997

METHODS Animak. Breeder mice deficient in the IL- 6 gene (IL- 6 -/-) were obtained from Dr. Valeria Poli (Istituto di Ricerche di Biologia Molecolare IRBM P. Angeletti, Rome, Italy) and were bred in our barrier facility. The generation of IL- 6 -deficient mice by gene targeting ...

INFLUENZA-VIRUS M2 PROTEIN ENCODES ION CHANNEL ACTIVITY

Article

May 1992

The influenza virus M2 protein was expressed in Xenopus laevis oocytes and shown to have an associated ion channel activity selective for monovalent ions. The anti-influenza virus drug amantadine hydrochloride significantly attenuated the inward current induced by hyperpolarization of oocyte membranes. Mutations in the M2 membrane-spanning domain that confer viral resistance to amantadine produced currents that were resistant to the drug. Analysis of the currents of these altered M2 proteins suggests that the channel pore is formed by the transmembrane domain of the M2 protein. The wild-type M2 channel was found to be regulated by pH. The wild-type M2 ion channel activity is proposed to have a pivotal role in the biology of influenza virus infection.

Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics

Article

Nov 1989

We determined the origin and evolutionary pathways of the PB1 genes of influenza A viruses responsible for the 1957 and 1968 human pandemics and obtained information on the variable or conserved region of the PB1 protein. The evolutionary tree constructed from nucleotide sequences suggested the following: (i) the PB1 gene of the 1957 human pandemic strain, A/Singapore/1/57 (H2N2), was probably introduced from avian species and was maintained in humans until 1968; (ii) in the 1968 pandemic strain, A/NT/60/68 (H3N2), the PB1 gene was not derived from the previously circulating virus in humans but probably from another avian virus; and (iii) a current human H3N2 virus inherited the PB1 gene from an A/NT/60/68-like virus. Nucleotide sequence analysis also showed that the avian PB1 gene was introduced into pigs. Hence, transmission of the PB1 gene from avian to mammalian species is a relatively frequent event. Comparative analysis of deduced amino acid sequences disclosed highly conserved regions in PB1 proteins, which may be key structures required for PB1 activities.

Pathogenesis of emerging avian influenza viruses in mammals and the host innate immune response

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