Viral Replication Rate Regulates Clinical Outcome and
CD8 T Cell Responses during Highly Pathogenic H5N1
Influenza Virus Infection in Mice
Yasuko Hatta1,2., Karen Hershberger1,2., Kyoko Shinya3, Sean C. Proll4, Richard R. Dubielzig2, Masato
Hatta1,2, Michael G. Katze4, Yoshihiro Kawaoka1,2,5*, M. Suresh1,2*
1Influenza Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States of America, 2Department of Pathobiological Sciences, University of
Wisconsin-Madison, Madison, Wisconsin, United States of America, 3Division of Zoonosis, Department of Microbiology and Infectious Disease, Graduate School of
Medicine, Kobe University, Kusunoki-cho, Chuo-ku, Kobe, Hyogo, Japan, 4Department of Microbiology, School of Medicine, University of Washington, Seattle,
Washington, United States of America, 5Division of Virology, Department of Microbiology and Immunology and International Research Center for Infectious Diseases,
Institute of Medical Science, University of Tokyo, Tokyo, Japan
Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to
occur with a staggering mortality rate of .60%. Although sustained human-to-human transmission has not occurred yet,
there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite
progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system
interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two
human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of
viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was
directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within
48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the
induction of IFN-b or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the
lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology
and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highlylethal H5N1 viruses could
simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However,
therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the
survivalofmicefollowingalethal H5N1infection.Thesefindingssuggest thatsuppression ofearlyH5N1virusreplication iskey
to the programming of an effective host response, which has implications in treatment of this infection in humans.
Citation: Hatta Y, Hershberger K, Shinya K, Proll SC, Dubielzig RR, et al. (2010) Viral Replication Rate Regulates Clinical Outcome and CD8 T Cell Responses during
Highly Pathogenic H5N1 Influenza Virus Infection in Mice. PLoS Pathog 6(10): e1001139. doi:10.1371/journal.ppat.1001139
Editor: Guus F. Rimmelzwaan, Erasmus Medical Center, Netherlands
Received May 25, 2010; Accepted September 8, 2010; Published October 7, 2010
Copyright: ? 2010 Hatta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by ERATO (Japan Science and Technology Agency) and by a Grant-in-Aid for Specially Promoted Research and by a contract
research fund for the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases from the Ministry of Education, Culture, Sports,
Science, and Technology, and by grants-in-aid from the Ministry of Health, Labor, and Welfare of Japan and by National Institute of Allergy and Infectious Disease
Public Health Service research grants to Dr. Yoshihiro Kawaoka. The work was also supported by grants (Systems Biology Contract [HHSN272200800060C] and P51
RR00166) from the National Institutes of Health to Dr. Michael Katze. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (MS); email@example.com (YK)
. These authors contributed equally to this work.
Severe outbreaks of highly pathogenic avian influenza (AI) H5N1
viruses in poultry continue to occur and are often coupled with reports
of direct bird-to-human viral transmission. Between 2003 and 2009,
406 confirmed human cases of AI H5N1 were reported, with a fata-
lity rate of .60% (http://www.who.int/csr/disease/avian_influenza/
country/cases_table_2010_01_28/en/index.html). Although sustain-
ed human-to-human transmission has not yet occurred, there is
increasing concern that these H5N1 AI viruses might acquire the
ability to transmit efficiently between humans and cause a pandemic.
The high virulence of H5N1 viruses in humans can be attributed to
either a delay in development or the ineffectiveness of innate and/or
adaptive immune mechanisms to control the infection in a timely
fashion. However, little information exists on the dynamics of adaptive
immune responses to H5N1 viruses during a primary infection, which
lethal H5N1 infection in humans.
The adaptive immune response to seasonal influenza viruses has
been extensively characterized using a murine model of intranasal
(I/N) infection with mouse-adapted influenza viruses [1,2,3,4,5,6].
Elicitation of a potent CD8 T cell response is of critical importance
in resolving a primary influenza virus infection in mice [1,3,4,7].
However, both CD8 T cells and antibodies might be required to
clear highly pathogenic influenza viruses . Mouse-adapted
influenza viruses elicit robust CD8 T cell responses in the
PLoS Pathogens | www.plospathogens.org1October 2010 | Volume 6 | Issue 10 | e1001139
respiratory tract, which typically peak at day 10 after infection
[5,6]. Effector CD8 T cells control influenza virus replication by
cytolytic mechanisms that require Fas and/or perforin . In
addition to their role in viral clearance, CD8 T cells are also
implicated in mediating immune-mediated lung injury following
influenza virus infection [9,10,11]. Pertaining to primary infection
with H5N1 viruses, we do not yet know whether CD8 T cell
responses are induced in the respiratory tract, or whether virus-
specific CD8 T cells play a protective or immunopathologic role
during a primary H5N1 infection. A high viral load is one of the
hallmarks of a fatal H5N1 infection in humans , but the effect
of high-level H5N1 virus replication on the emergence of CD8 T
cell responses in the respiratory tract has not been studied.
In this study, using two human isolates of H5N1 viruses that
differ in their pathogenicity in mice, we have systematically
examined the following: 1) the relationship between the speed of
H5N1 virus replication and viral pathogenicity on the dynamics of
CD8 T cell responses, 2) whether ineffective control of H5N1 virus
infection is related to the suppression of virus-specific CD8 T cell
responses, 3) the effect of CD8 T cell deficiency on host survival,
and 4) the effect of anti-viral therapy on CD8 T cell responses.
Findings from these studies have provided novel insights into the
virus/immune system interactions during an H5N1 infection from
the standpoint of viral pathogenesis, immune control of viral
replication, and immunopathology.
The pathogenicity of H5N1 viruses in mice is associated
with accelerated viral replication in the lungs
Unlike seasonal strains of influenza viruses, human isolates of
H5N1 viruses readily replicate in other mammals, including mice,
without prior adaptation and induce varying levels of pathogenic-
ity . Experimental infections of mice with the H5N1 viruses
have led to the identification of viral determinants of pathogenicity
[13,14,15,16,17]. Although high cleavability of hemagglutinin is
essential to cause a lethal infection, a single amino acid residue in
the PB2 protein controls the pathogenic potential of these AI
viruses in mice . An extremely low-dose infection of mice with
the A/Hong Kong/483/97 (HK483) virus that has a Lys at
position 627 of the PB2 protein induces a lethal infection (a dose of
virus that kills 50% of infected mice (MLD50) of 1.8 plaque-
forming units [PFU]), whereas the A/Hong Kong/486/97
(HK486) virus that has a Glu at position 627 of PB2 is less
pathogenic (MLD50of 7.66103PFU). To determine whether the
two viruses differ in their rate of viral replication in vivo in the
respiratory tract, we performed a detailed kinetic analysis of viral
titers in the lungs of HK483- and HK486-infected mice
(Figure 1). Although mice were infected with the same dose of
both viruses and reached comparable maximum titers, viral
growth kinetics in the lungs were dramatically different. The
Outbreaks of avian influenza (AI) viruses have continued in
chickens in Southeast Asia, coupled with regular instances
of direct bird to human transmission, with extremely high
case fatality rates. The mechanisms underlying the disease
pathogenesis and high mortality rate in humans are not
well understood. In particular, we lack information on the
development and/or failure of adaptive immune responses
during AI infection. Our studies in mice have linked the
pathogenicity of AI viruses to the virus’ rate of replication
in the lungs. Surprisingly, a strong T cell response was
triggered by the infection, but virus-specific T cells were
ineffective in controlling the rapidly replicating virus. The
extremely high rate of AI virus replication likely outpaces
and overwhelms the developing immune response.
However, administration of anti-viral drugs, only early in
the infection slowed viral replication, enhanced the
number of effector CD8 T cells in the lung, and promoted
survival and recovery from infection. These findings
highlight the role of viral replication rate in pathogenesis
and underscore the importance of controlling viral
replication as an adjunct to immunotherapies in the
treatment of this infection in humans.
Figure 1. Virus replication kinetics in lungs of mice infected with H5N1 viruses HK483 or HK486. At indicated days after I/N infection
with 18 PFU of HK483 or HK486 virus, mice were euthanized and lung virus titers were determined by plaque assay on MDCK cells. The data are the
average titers from 3 mice 6 SD at each time point. Data is representative of 2 independent experiments. * P=0.01; ** P=0.0005; *** P=0.0005; ****
P=0.02; ***** P=0.004; ****** P=0.002.
H5N1 Virus Replication and CD8 T Cell Responses
PLoS Pathogens | www.plospathogens.org2October 2010 | Volume 6 | Issue 10 | e1001139
HK483 virus replicated at a remarkable pace within the first
24 hours, and the coefficient of expansion was calculated to be
,4.3 log PFU/day; peak virus titers of ,107PFU/gram were
attained in the lungs within 48 hours after infection. In striking
contrast, the coefficient of expansion for the less pathogenic
HK486 virus in the first 24 hours was only ,1.3 log PFU/day,
and peak titers in the lungs were not attained until 5 days after
infection. Thus, the speed of early viral replication in the lungs
might be a necessary and distinguishing trait of highly pathogenic
AI viruses to rapidly reach high titers and potentially overwhelm
the host immune responses.
The enhanced replication of the HK483 virus could be related
to the virus’s ability to evade the innate immune mechanism(s),
especially the type I IFN pathway . However, microarray
analysis showed that the induction of IFN-b and interferon-
stimulated genes is greater in the lungs of HK483-infected mice
compared to HK486-infected mice at 48 hours after infection
(Figure S1). To examine whether Type I IFNs play any role in
controlling infection with HK483, we infected groups of wild type
C57BL/6 (n=5) and Type I IFN receptor-deficient (IFNRI2/2)
mice (n=5) with 18 PFU of HK483 virus. Upon infection with the
HK483 virus, all wild-type mice survived at least until day 7 after
infection, but 4 of 5 IFNRI2/2 mice died by day 5 postinfection
(PI), and the remaining IFNRI2/2 mouse died on day 7 PI.
These data suggested that the type I IFN pathway is induced and
functional in HK483-infected mice, which is consistent with a
recently published report .
CD8 T cell responses to H5N1 influenza viruses in mice
To understand the relationship of viral pathogenicity and/or
rapid viral replication rate to the development of adaptive
immunity, we infected BALB/c mice I/N with the HK483 or
HK486 virus and studied the evolution of virus-specific CD8 T
cell responses in the respiratory tract. Sequence comparisons
showed that the Kd-restricted CD8 T cell epitope NP147 of the
PR8 virus was conserved in both HK483 and HK486 viruses. In
mice infected with the less pathogenic HK486 virus, high numbers
of CD8 T cells in the lung airways were not detectable until day 8
PI but rapidly accumulated within the next 24 hours (Figure 2A).
The kinetics of the CD8 T cell response to HK486 were similar to
those of mouse-adapted human influenza viruses [5,6]. Surpris-
ingly, in HK483-infected mice, virus-specific CD8 T cells were
detectable earlier, at day 7 PI, and peak numbers were attained at
day 8 PI. It is noteworthy that the peak numbers of NP147-specific
CD8 T cells in HK483-infected mice attained at day 8 PI were
lower, as compared to those in HK486-infected mice (day 9 PI).
Additionally, CD8 T cells in HK483-infected mice appear to have
initiated contraction between days 8 and 9 PI, when the number of
CD8 T cells continued to increase in the respiratory airways of
HK486-infected mice (Figure 2A). Similar contraction in the
number of CD8 T cells was seen in the lungs of HK483-infected
mice between days 8 and 9 PI in a separate experiment (data not
In order to track the early events of CD8 T cell activation in the
draining lymph nodes (DLNs), we adoptively transferred carboxy-
fluorescein succinimidyl ester (CFSE)-labeled influenza HA518-
specific Clone 4 (CL-4) TCR transgenic CD8 T cells into congenic
BALB/c mice [5,6], which were subsequently infected I/N with
18 PFU of HK483 or HK486 virus. By day 5 PI, .90% of CL-4
CD8 T cells had divided several times in the DLNs of HK483-
infected mice (Figure 2B), and a substantial fraction of these cells
also exhibited markers of activation (LFA-1HI, CD43Hi, and
CD62LLo) (Figure 2C). In contrast, in the DLNs of HK486-
infected mice, only ,50% of CL-4 CD8 T cells had proliferated
by day 5 PI, and these cells did not upregulate expression of LFA-1
or CD43 until day 6 PI. The increased percentage of proliferated
CL-4 CD8 T cells in the lymph nodes of HK483-infected mice
was not linked to reduced trafficking of these cells out of the lymph
nodes into the lungs because, the number of CL-4 CD8 T cells in
the BAL of HK483-infected mice (2.9–4.16103) were higher than
in the BAL of HK486-infected mice (1.0–1.46103) at day 5 PI. In
addition to increased proliferation, a larger percentage of CL-4
CD8 T cells expressed granzyme B in HK483-infected mice at day
6 PI compared to those in HK486-infected mice (Figure 2C).
Thus, CD8 T cells underwent accelerated activation in the DLNs
of mice infected with the HK483 virus compared to those in
HK486-infected mice. The early activation of virus-specific CD8
T cells in lymph nodes of HK483-infected mice corresponds with
the faster replication kinetics of the virus in the respiratory tract.
Viral pathogenicity regulates CD8 T cell responses to
H5N1 influenza viruses
The pathogenicity of influenza viruses in the experimental
mouse model has been defined based on MLD50. Next, we
determined whether the accelerated kinetics of CD8 T cell
contraction is related to the clinical outcome of infection, i.e.,
lethality. Typically, varying the infecting dose alters the disease
process and clinical outcome of influenza viruses, but this
procedure is not feasible with the HK483 virus because of the
extremely low MLD50of 1.8 PFU. Therefore, we examined the
effect of viral dose (based on MLD50) on the kinetics of CD8 T cell
contraction by infecting BALB/c mice with 1 (4.66103PFU) or 10
MLD50(4.66104PFU) of the HK486 virus. As controls, mice
were infected with 10 MLD50of the HK483 virus (18 PFU). As
shown in Figure 3, premature contraction of total and NP147-
specific CD8 T cells occurred in mice infected with 10 MLD50of
the HK483 or HK486 virus, but not in mice infected with 1
MLD50of the HK486 virus. Thus, regardless of the H5N1 virus
strain used, the pathogenicity of H5N1 viruses in mice (which is a
function of infecting dose for the HK486 virus) regulated the
dynamics of CD8 T cell contraction in the respiratory tract
following infection with H5N1 viruses.
Highly pathogenic H5N1 influenza viruses trigger
accelerated apoptosis of CD8 T cells and lung damage by
Next, we determined whether infection with the highly
pathogenic H5N1 virus caused premature contraction of CD8 T
cells by affecting cellular apoptosis. Following infection of BALB/c
mice with 18 PFU of HK483 or HK486 viruses, apoptosis of CD8
T cells in the lung was assessed at days 7, 8, and 9 after infection
by staining for active caspase 3. At all time points, the fraction of
apoptotic CD8 T cells in the lungs of HK483-infected mice was
two- to four-fold higher than in HK486-infected mice (Figures 4A
and 4B). These findings suggested that infection with highly
pathogenic AI viruses induces accelerated apoptosis and prema-
ture contraction of CD8 T cells in the respiratory tract.
Two distinct pathways of caspase-dependent cellular apoptosis
have been described: the intrinsic and extrinsic pathways [20,21].
The intrinsic apoptotic pathway is initiated following activation of
the pro-apoptotic BH3-only proteins, such as Bcl-2-interacting
mediator of death (BIM). On the other hand, interaction between
death receptors and their ligands, such as Fas and Fas ligand,
triggers the extrinsic pathway of cellular apoptosis. To determine
which pathway of cellular apoptosis is triggered in CD8 T cells by
the highly pathogenic AI virus, we infected wild-type C57BL/6
(+/+), Fas-mutant lpr/lpr (Fas KO), and BIM-deficient (BIM KO)
H5N1 Virus Replication and CD8 T Cell Responses
PLoS Pathogens | www.plospathogens.org3 October 2010 | Volume 6 | Issue 10 | e1001139
mice with the HK483 virus. At day 8 PI, we quantified the
number of apoptotic active caspase 3+veCD8 T cells in the BAL of
HK483-infected mice (Figure 4C). As expected, a substantial
fraction of CD8 T cells was apoptotic in the lungs of C57BL/6
mice, and Fas deficiency did not significantly affect apoptosis of
CD8 T cells induced by highly pathogenic HK483 infection.
Notably, the percentage of apoptotic CD8 T cells was reduced by
,90% in BIM KO mice compared to C57BL/6 or Fas KO mice.
These data suggested that the apoptosis of CD8 T cells induced by
highly pathogenic AI viruses is triggered by the intrinsic pathway
of cellular apoptosis. As a consequence of reduced apoptosis in the
absence of BIM activity, the numbers of PA224-specific CD8 T
cells in the BAL of BIM KO mice (5.46104) were higher than in
+/+ mice (3.66104).
Because BIM deficiency protected against CD8 T cell apoptosis,
we next examined whether the loss of BIM would also improve
Figure 2. CD8 T cell responses in the lung airways of mice infected with H5N1 viruses. Panel A, Groups of BALB/C mice were infected I/N
with 18 PFU of HK483 or HK486 virus. At the indicated days after infection, pooled cells from broncoalveolar lavage of 3 mice were collected. Cells
were stained with anti-CD8, anti-LFA-1, and Kd/NP147 MHC I pentamers, and pentamer-binding LFA-1HiCD8 T cells were quantified by flow
cytometry. The data are representative of two independent experiments. Panel B, CFSE-labeled Thy1.1+veTCR transgenic CL-4 CD8 T cells were
adoptively transferred in to congenic Thy1.2/BALB/c mice, and infected I/N with 18 PFU of HK483 or HK486 virus 24 hours later. At day 5 after
infection, cells from deep cervical lymph nodes were stained with anti-CD8, anti-Thy1.1, and Kd/HA518 tetramers. The histograms are gated on CD8+/
Thy1.1+MHC-I tetramer-binding cells, and the numbers are the percentage of divided cells of total gated cells. Data are representative of analysis of
pooled cells from 3 mice/group. Panel C, As in panel B, CL-4 CD8 T cells were adoptively transferred into BALB/c mice, and infected with HK483 or
HK486 virus. At the indicated time points after infection, cells from the deep cervical lymph nodes were stained with anti-CD8, anti-Thy1.1, Kd/HA518
tetramers, anti-LFA-1, anti-CD43, anti-CD62L, or anti-granzyme B. The flow cytometry plots are gated on CD8+/Thy1.1+MHC-I tetramer-binding cells,
and the numbers are the percentage of LFA-1LO/HI, CD43LO/HI, or CD62LO/HI, or granzyme BLO/HIof total gated cells.
H5N1 Virus Replication and CD8 T Cell Responses
PLoS Pathogens | www.plospathogens.org4 October 2010 | Volume 6 | Issue 10 | e1001139
survival of HK483-infected mice. Groups of +/+, Fas KO, and
BIM KO mice were infected with the HK483 virus as above, and
their survival was monitored daily. As shown in Figure 5A, a
majority of +/+ mice infected with HK483 succumbed to infection
by day 12 PI. Likewise, BIM KO and Fas KO mice also
succumbed to HK483 infection, albeit with a slight delay. Neither
BIM nor Fas deficiency significantly affected viral titers in the
lungs (Figure S2). These data suggested that BIM deficiency-
induced enhancement of virus-specific CD8 T cell responses is
insufficient to enhance survival following a highly pathogenic
H5N1 virus infection.
Next, we assessed whether BIM deficiency affected HK483 virus-
induced cell damage in the lungs (Figure 5B). At day 8 after
infection withthe HK483 virus, the lung pathology in +/+ mice was
characterized by extensive cellular necrosis and tissue disruption of
medium-sized blood vessels and bronchioles, which was associated
with infiltration of inflammatory cells composed mostly of
neutrophils. Additionally, apoptotic cells were frequently observed
in the lungs of +/+ mice. In striking contrast, in the lungs of BIM2/
2 mice, cellular necrosis was less pronounced and the tissue
integritywasmore intact,with lessfrequent apoptoticcells.Notably,
the lungs of BIM2/2 mice contained lymphocytic infiltrates in the
connective tissues near medium-sized blood vessels and, more
prominently, adjacent to the bronchioles (Figure 5B). Based on
these findings, we infer that HK483-induced lung pathology is at
least in part mediated by BIM-dependent mechanisms.
A neuraminidase inhibitor prevents accelerated CD8 T
cell contraction and enhances survival during a highly
pathogenic H5N1 influenza virus infection
The neuraminidase inhibitor, oseltamivir phosphate, is an
effective treatment for influenza A virus infection in humans if
given early in the infection [22,23,24]. Treatment with oseltamivir
reduces viral load and protects mice against a lethal H5N1 virus
infection [25,26]. It was of interest to determine whether a high
rate of virus replication in HK483-infected mice, especially early
in the infection could 1) lead to early activation and contraction of
virus-specific CD8 T cells in the lung airways and 2) outpace and
overwhelm the CD8 T cell response. Additionally, the effects of
oseltamivir treatment on the adaptive immune response to H5N1
infection have not been examined. Therefore, we asked whether
the reduction of virus replication by oseltamivir protected against
the accelerated activation and contraction of CD8 T cells
following infection of mice with the highly pathogenic HK483
virus. Mice that were infected with the HK483 virus were treated
with graded doses of oseltamivir only early in the infection, and
virus-specific CD8 T cells were quantified at days 7, 8, and 9 after
infection. As expected, CD8 T cells in control vehicle-treated mice
Figure 3. Premature contraction of CD8 T cells in mice infected with lethal dose of highly pathogenic H5N1 influenza viruses. BALB/c
mice were I/N inoculated with 1 MLD50or 10 MLD50of HK486 or 10 MLD50of HK483 virus. At indicated days after infection, pooled cells from the BAL
of 3 mice were stained with anti-CD8, anti-LFA-1, and Kd/NP147 pentamers. The numbers of activated LFA-1HiCD8 T cells (Panel A) or NP147-specific
LFA-1HiCD8 T cells (Panel B) were quantified by flow cytometry. Data are from one of two independent experiments.
H5N1 Virus Replication and CD8 T Cell Responses
PLoS Pathogens | www.plospathogens.org5 October 2010 | Volume 6 | Issue 10 | e1001139
underwent contraction between days 8 and 9 PI (Figure 6A), but
oseltamivir treatment at doses of 10 or 20 mg, but not 5 mg,
mitigated contraction and led to a substantive increase in the
number of virus-specific CD8 T cells in the lung airways of
HK483-infected mice between days 8 and 9 PI. Notably,
Figure 6B shows that oseltamivir treatment reduced viral titers,
especially early in the course of the infection, regardless of the dose
administered, but mouse survival was extended or increased only
at doses of 10 and 20 mg, which suggested that suppression of
early viral replication alone might be necessary but not be
sufficient to enhance mouse survival. However, reduced viral titers
coupled with enhanced CD8 T cell responses were associated with
extended or improved survival.
Effect of CD8 T cell deficiency on the survival of mice
following a highly pathogenic H5N1 virus infection
Our studies showed that infection with the highly pathogenic
HK483 virus elicited a readily detectable CD8 T cell response but
Figure 4. Apoptosis of CD8 T cells in mice infected with H5N1 viruses. A and B, BALB/c mice were infected with 18 PFU of HK483 or HK486.
At the indicated days after infection, the percentages of total CD8 T cells (Panel A) and Kd/NP147-specific CD8 T cells (Panel B) expressing active form
of caspase-3 were determined by flow cytometry; cells in the BAL were stained with anti-CD8, Kd/NP147 pentamers, and anti-active caspase-3. Panel
C, C57BL/6 (+/+), Fas-deficient (Fas KO) or BIM-deficient (BIM KO) mice (n=3) were infected with 18 PFU of HK483. On the eighth day after infection,
the percentages of total CD8 T cells and of Db/PA224-specific CD8 T cells expressing active form of caspase-3 in the BAL were determined as in panels
A and B above.
H5N1 Virus Replication and CD8 T Cell Responses
PLoS Pathogens | www.plospathogens.org6October 2010 | Volume 6 | Issue 10 | e1001139
failed to effectively control viral replication. Because there is
evidence supporting a role for CD8 T cells in augmenting lung
pathology following infection with seasonal influenza viruses
[9,10,11], we questioned whether CD8 T cells contribute to the
lethality induced by infection with highly pathogenic H5N1
viruses. Groups of +/+ and CD8-deficient (CD8 KO) mice were
infected with graded doses of the HK483 virus, and mouse survival
was monitored (Figure 7). As shown in Figures 7A and 7B,
there was no difference in survival between HK483-infected +/+
and CD8 KO mice. These data suggested that the loss of a CD8 T
cell response does not provide either a survival advantage or a
disadvantage to mice infected with the highly pathogenic HK483
Until the AI epidemic of 1997, it was assumed that purely AI
viruses could not cause a lethal disease in humans. However, since
1997, recurring instances of direct transmission of AI viruses from
birds to humans have dismissed this assumption [14,27,28]. A
unique feature of these H5N1 viruses is their ability to replicate to
high levels in the lungs of several mammalian species, including
humans, without adaptation [15,29,30]. Although a high viral load
and hypercytokinemia are recognized hallmarks of fatal AI
infections in humans , we still lack crucial information on
the kinetics, magnitude, and nature of the adaptive immune
response to these infections. In this study, we have examined the
relationship of viral replication kinetics in the lungs and viral
pathogenicity to the dynamics of virus-specific CD8 T cell
responses to AI viruses in mice. We found that the extreme
pathogenicity of H5N1 viruses is directly linked to the high viral
replication rate and the consequent production of peak steady-
state viral titers in the lungs within 48 hours after infection.
Interestingly, we found that lethal H5N1 infection in mice
stimulates a robust, virus-specific CD8 T cell response in the
respiratory tract, but these CD8 T cells fail to control viral
replication and undergo early contraction. The prevention of CD8
T cell contraction did not alter the survival of infected mice, but
inhibition of neuraminidase activity and viral replication by
therapeutic intervention mitigated the premature contraction of
CD8 T cells and enhanced mouse survival following a lethal
H5N1 infection. These findings suggest that the ability of H5N1
viruses to overwhelm and/or undercut the sustenance of the anti-
viral CD8 T cell response and cause a lethal pulmonary infection
is linked to a high viral replication rate, especially early in the
infection. These findings further our understanding of the
Figure 5. Survival of C57BL/6, Fas KO, and BIM KO mice after inoculation with highly pathogenic HK483 virus. C57BL/6 (+/+; n=17),
Fas KO (n=12), or BIM KO (n=5) mice were I/N inoculated with 18 PFU of HK483 virus and mouse survival was monitored for 16 days (Panel A). Panel
B, At day 8 after infection, lungs from infected mice were examined for histopathological changes. Arrows indicate apoptotic cells in lung section
from +/+ mouse or lymphocytic infiltrates adjacent to the bronchioles in lung section from BIM2/2 mice.
H5N1 Virus Replication and CD8 T Cell Responses
PLoS Pathogens | www.plospathogens.org7October 2010 | Volume 6 | Issue 10 | e1001139
pathogenesis of H5N1 viruses, which should have implications on
the development of novel therapies and prophylaxis for H5N1
infection in humans.
The infection of mice with mouse-adapted strains of influenza
viruses elicits strong CD8 T cell responses in the respiratory tract,
and there is ample evidence indicating an important role for CD8
T cells in the viral control of a primary influenza virus infection
[3,4,7]. In contrast to a sublethal infection, inoculation of mice
with high doses of mouse-adapted influenza virus leads to
apoptosis of virus-specific CD8 T cells and lethal pulmonary
injury . Moreover, based on an analysis of gene expression in
the lungs of mice infected with highly pathogenic H5N1 viruses, T
cell activation might be impaired during an H5N1 virus infection
. However, we showed that the infection of mice with a highly
Figure 6. Oseltamivir therapy protects against accelerated activation and contraction of CD8 T cells following infection of mice
with HK483 virus. C57BL/6 mice were treated daily with indicated doses of oseltamivir (mg/Kg body weight) from 21 day to 3 day relative to
infection with 100 PFU of HK483. Panel A, At days 5, 7, and 9 post-infection, BAL samples from 3 mice/dose were pooled and the number of CD8 T
cells specific to CTL epitope PA224 were determined by flow cytometry. Panel B, At days 1, 2, 3, 5, 7, and 9 PI, 3 mice were euthanized at each time
point/dose and lung virus titers were determined; error bars indicate SD. * P=0.03 between 0 mg and 5 mg doses and P=0.06 between 0 mg and
10 mg doses; ** P=0.0003 between 0 mg and 5 mg doses, P=0.08 between 0 mg and 10 mg doses, and P=0.005 between 0 mg and 20 mg doses;
*** P=0.02 between 0 mg and 5 mg doses, P=0.006 between 0 mg and 20 mg doses; **** P=0.12 between 0 mg and 5 mg doses and P=0.07
between 0 mg and 10 mg doses, and P=0.004 between 0 mg and 20 mg doses. Panel C, Effect of oseltamivir treatment on survival of HK483-
infected mice (n=5/group). Data in panels B and C is representative of two independent experiments.
H5N1 Virus Replication and CD8 T Cell Responses
PLoS Pathogens | www.plospathogens.org8October 2010 | Volume 6 | Issue 10 | e1001139
pathogenic H5N1 virus elicits a readily detectable CD8 T cell
response, which suggests that the initial events of T cell priming,
including trafficking of dendritic cells to the DLN and antigen
processing/presentation, are intact in H5N1 virus-infected mice.
While virus-specific CD8 T cells continued to accumulate in the
lung airways of mice until at least day 9 after infection with the less
pathogenic HK486 virus, CD8 T cells in HK483-infected mice
exhibited a decline after day 8 PI due to BIM-dependent
apoptosis. The BIM-dependent intrinsic pathway of apoptosis of
activated CD8 T cells appears to be unique to highly pathogenic
H5N1 AI viruses because a high-dose infection of mice with
mouse-adapted epidemic strains of the influenza virus induced
CD8 T cell apoptosis that was dependent upon Fas/FasL
interactions . Highly pathogenic AI viruses are known to
trigger hyperinduction of TNF-related apoptosis inducing ligand
(TRAIL) in macrophages and cause T cell apoptosis in vitro .
Because TRAIL-induced apoptosis is BIM-dependent [34,35,36],
we propose that apoptosis of activated CD8 T cells in HK483-
infected mice might be triggered by interactions between
macrophage-derived TRAIL and its receptors on CD8 T cells.
It should be noted that our experiments did not test whether BIM
triggered apoptosis of CD8 T cells by T cell intrinsic mechanisms.
It is possible that reduced CD8 T cell apoptosis in BIM-deficient
mice was an indirect effect, possibly linked to increased survival of
dendritic cells . Do differential direct infection of CD8 T cells
by HK483 and HK486 viruses explain differences in CD8 T cell
apoptosis? Studies of apoptosis in the lungs of mice infected with
HK483 show that apoptotic cells are primarily localized to
bronchial epithelial and subepithelial layers, and not to the cells
with lymphocyte morphology . Additionally, apoptotic
HK483-infected cells are primarily found in the germinal centers
of the spleen , where CD8 T cells are not typically present in
significant numbers. Nevertheless, studies are warranted to assess
whether HK483 but not HK486 induces apoptosis of T cells by
direct infection. Tumpey et al have reported that the total number
of CD8 T cells in the lungs and mediastinal lymph nodes of mice
infected with 100 mouse infectious dose 50 (MID50) of HK483 was
lower than those in HK486-infected mice at day 6 PI .
However, in our experiments, contraction in the number of CD8
T cells in the respiratory airways (Figure 2) or lungs (data not
shown) did not occur until after 8 days after HK483 infection (dose
of 18 PFU/mouse or 10 MLD50); the number of CD8 T cells in
the BAL of HK483-infected mice was lower at day 9 PI, when
compared to those in HK486-infected mice. The discrepancy in
the kinetics of the CD8 T cell response between the two studies
might be related to differences in experimental procedures
including preparation of the virus stock, dose of virus used (100
MID50versus 10 MLD50), infection procedures, and methods used
for isolating mononuclear cells from the tissues.
Despite substantial expansion, virus-specific CD8 T cells were
ineffective in controlling HK483 infection, and all mice suc-
cumbed within 10 days after infection. The inability of CD8 T
cells to effectively control HK483 infection is not associated with
functional impairment because virus-specific CD8 T cells in the
lung airways contained high levels of granzyme (Figure 2) and
also produced cytokines, such as IFN-c, upon antigenic stimula-
tion (Figure S3). Additionally, the impaired control of highly
pathogenic H5N1 infection is not linked to premature apoptosis of
CD8 T cells because protection of CD8 T cells against BIM-
dependent apoptosis did not lead to effective viral control or
enhanced mouse survival (Figure 5). Why is the CD8 T cell
response unable to effectively control a lethal H5N1 infection?
Recent work suggests that the effectiveness of a CD8 T cell
response to successfully control viral replication depends upon the
number and concentration of effector CD8 T cells in relationship
to the number of virus-infected cells [39,40]. Therefore, the
inability of effector CD8 T cells to control the rapidly replicating
HK483 virus might be explained by the large number of virus-
infected cells, which leads to higher ratios of effector CD8 T cells
to the number of virus-infected cells. The effector CD8 T cell
response is perhaps neither fast nor large enough (even in BIM
KO mice) to control viruses such as HK483 that are capable of
rapid replication and dissemination. Immunotherapies to inflate
the number of virus-specific CD8 T cells might be able to control
infections with highly pathogenic H5N1 viruses.
The ratio of effector CD8 T cells to virus-infected cells in the
tissues could be altered by increasing the number of effector CD8
T cells and/or by decreasing the number of virus-infected cells.
Our studies show that oseltamivir treatment can achieve this
objective. Oseltamivir therapy at certain doses not only suppressed
H5N1 viral titers in the lungs but also enhanced the number of
effector CD8 T cells in the lung airways, which in turn led to
improved survival. The mechanism(s) underlying the enhancement
in CD8 T cell responses by oseltamivir is purely conjecture at this
point. One possibility is that oseltamivir reduces viral load, which
in turn leads to inhibition of TRAIL induction and BIM-
dependent apoptosis of effector CD8 T cells. A second theory is
that the diminished viral load in oseltamivir-treated mice would be
expected to reduce the amount of HA and HA-triggered cellular
apoptosis . A third theory is that CD8 T cell contraction is
Figure 7. Effect of CD8 T cell deficiency on survival of mice infected with H5N1 viruses. C57BL/6 (+/+) or CD8-deficient (CD8 KO) mice
were infected intranasally with the indicated doses of HK483 virus (Panels A and B). Three to six mice were infected at each dose and mouse survival
was observed for 21 days.
H5N1 Virus Replication and CD8 T Cell Responses
PLoS Pathogens | www.plospathogens.org9October 2010 | Volume 6 | Issue 10 | e1001139
triggered by extrapulmonary dissemination of the HK483 virus,
which elicits a systemic response, and oseltamivir treatment limits
viral replication to the lungs. A fourth possibility is that reduced
viral load induced by oseltamivir lowered/delayed antigenic
stimulation of T cells by DCs, especially early in the infection,
which in turn prevented accelerated activation and contraction of
CD8 T cells in HK483-infected mice.
It should also be noted that oseltamivir treatment only affected
virus titers early in the infection, and viral load in the lungs at the
time of T cell contraction (8–9 days PI) was similar in the
untreated group as well as in treated groups, regardless of the dose
of oseltamivir. These data suggested that viral titers early in the
infection might control the contraction kinetics of the anti-viral
CD8 T cell response. It has been reported that the early
inflammatory response triggered by an infecting organism
programs the contraction of CD8 T cell responses . Therefore,
the hyperinflammatory response induced by high viral titers early
in the H5N1 infection  could also be involved in accelerating
the kinetics of CD8 T cell activation and contraction in the lungs.
Consequently, lower HK483 viral titers induced by oseltamivir
treatment would be expected to blunt the inflammatory response
thereby delaying the onset of CD8 T cell contraction.
Interestingly, treatment of mice with 10 mg or 20 mg of
oseltamivir reduced viral load in the lungs and modulated CD8 T
cell responses to a largely similar extent. However, only treatment
with 20 mg of oselatmivir led to substantial improvement in
survival of HK483-infected mice. In addition to the well-
characterized anti-viral effects, the increased survival of mice that
received 20 mg of oseltamivir might be explained by at least two
non-mutually exclusive mechanisms. First, only treatment with
20 mg or more of oseltamivir can restrict viral replication to the
lungs and prevent viral dissemination into tissues like the brain,
thereby averting a fatal infection. Second, oseltimivir at this dose
might effectively attenuate the host inflammatory response and
limit tissue damage by inhibiting pro-inflammatory responses of
Although cytolytic influenza virus replication alone can cause
significant cell death, CD8 T cells are implicated in accentuating
tissue damage by immunopathologic mechanisms. We first showed
that CD8 T cell deficiency had minimal effects on the survival of
mice infected with the highly pathogenic HK483 virus. It is
conceivable that in infections with highly lethal viruses, such as
HK483, the extremely high rate of viral replication potentially
outpaces the innate and adaptive immune responses, and
overwhelming tissue damage caused by cytolysis of infected cells
is sufficient to cause a lethal infection. A 100% mortality in +/+
mice and the delayed death in CD8 KO mice imply that viral
replication is not controlled in +/+ mice, despite the development
of a CD8 T cell response.
In summary, in this study, we have defined mechanistic links
among the rate of viral replication, viral pathogenicity, the CD8 T
cell response, and the clinical outcome of a lethal H5N1 infection
in mice. These studies show that the extreme pathogenicity of
H5N1 viruses is directly linked to the ability of virus to replicate
rapidly and attain high steady-state viral titers in the lungs early in
the infection and not due to the lack of a CD8 T cell response.
Perhaps, the rapidly replicating virus simply overwhelms and
outpaces the most potent CD8 T cell response. Therefore,
restraining H5N1 virus replication to levels under a certain
threshold early in the infection not only limits direct virus-induced
cytopathicity but also allows the development of a CD8 T cell
response that can now effectively clear the non-overwhelming
infection. These findings have furthered our understanding of the
pathogenesis of H5N1 infections and are expected to have
significant implications on the development of effective therapies
to treat H5N1 infection in humans.
Materials and Methods
6-week-old BALB/c, C57BL/6, BIM KO , Fas KO (lpr/
lpr) , CD8 KO , and Clone-4 mice  were purchased
from Jackson Laboratory (Bar Harbor, ME). The Type I IFNR2/
2 mice were provided by Dr. Murali-Krishna (University of
Washington, Seattle, WA) . All mice were used at 6–8 weeks of
age according to the protocol approved by the University of
Wisconsin School of Veterinary Medicine Institutional Animal
Care and Use Committee (IACUC). The animal committee
mandates that institutions and individuals using animals for
research, teaching, and/or testing must acknowledge and accept
both legal and ethical responsibility for the animals under their
care, as specified in the Animal Welfare Act (AWA) and associated
Animal Welfare Regulations (AWRs) and Public Health Service
(PHS) Policy. Animal experimentation was done as per the PHS
Policy on Humane Care and Use of Laboratory Animals as
described in the Guide for the Care and Use of Laboratory
HK483 and HK486 viruses that were isolated from patients
during the Hong Kong outbreak of 1997 were derived by reverse
genetics and titered as described before . Mice were infected
I/N with different doses of HK483 or HK486 virus in a volume of
50 ml. Viral titers in tissues were quantified by a plaque assay using
MDCK cells. All experiments with these H5N1 viruses were
performed in a biosafety level 3 containment laboratory approved
for such use by the CDC and United States Department of
Adoptive transfer of CL-4 TCR transgenic CD8 T cells
Thy1.1/CL-4 CD8 T cells were labeled with CFSE and
adoptively transferred into congenic Thy1.2/BALB/c mice by tail
vein injection as described before . Twenty-four hours after cell
transfer, mice were infected I/N with the HK483 or the HK486
Kd/NP147 pentamers were purchased from Proimmune Inc.
(FL USA). The Db/PA224 tetramers were kindly provided by the
NIH Tetramer Facility (Emory University, Atlanta, GA). All
antibodies were purchased from BD-Pharmingen unless stated
otherwise. Mononuclear cells isolated from BAL or lymph nodes
were stained with anti-CD8, anti-LFA-1, anti-CD62L, anti-CD43,
and MHC tetramers/pentamers for 1 hr at 4C. For intracellular
staining, cells were stained for cell surface molecules as above, and
subsequently permeabilized and stained with anti-granzyme
(Invitrogen) or anti-caspase 3 antibodies using the Cytofix/
Cytoperm kit (BD-Pharmingen). Following staining, cells were
fixed with 2% paraformaldehyde and analyzed using a FACSCa-
libur flow cytometer (Becton Dickinson). Flow cytometry data
were analyzed using Flowjo software.
Mice were euthanized, and tissues were collected and fixed in
10% phosphate-buffered formalin. They were then dehydrated,
embedded in paraffin, and cut into 5-mm-thick sections that were
stained with standard hematoxylin-and-eosin.
H5N1 Virus Replication and CD8 T Cell Responses
PLoS Pathogens | www.plospathogens.org10October 2010 | Volume 6 | Issue 10 | e1001139
Oseltamivir phosphate (Tamiflu, Roche Laboratories Inc.,
Basel, Switzerland) dissolved in 50% Ora-Plus Suspending agent
(Paddock Laboratories, Inc., Minneapolis, MN, USA) in water and
administered to mice once daily by oral gavage in a volume of
200 mL at 21 to 3 days relative to infection with HK483 virus.
lungs of HK483-infected mice. BALB/c mice were infected I/N
with HK483 or HK486 virus. At day 2 PI, total RNA extracted
from lungs were subjected to microarray analyses to determine
gene expression profiles using Agilent oligo-nucleotide arrays.
Data was analyzed using Rosetta’s resolver and SpotFire decision
site for functional genomics. Data represent fold increase in gene
expression, as compared to uninfected controls.
Found at: doi:10.1371/journal.ppat.1001139.s001 (0.23 MB TIF)
Induction of IFN-b and IFN-stimulated genes in
mice. Groups of mice were infected with 18 PFU of HK483 virus,
and viral titers in the lungs were determined at the indicated days
after infection. The data for days 1 and 2 PI are from 2–3 mice/
Viral titers in C57BL/6, FAS KO, and BIM KO
group/time point. Viral titers at days 8 and 9 PI are from 3–12
Found at: doi:10.1371/journal.ppat.1001139.s002 (0.12 MB TIF)
T cells in the BAL of mice infected with HK483 virus. Groups of
BALB/c mice were infected with the indicated doses of HK483 or
HK486 virus. Pooled cells from BAL were stimulated for 5 hours
with the NP147 peptide, and IFNc production by CD8 T cells was
assessed by intracellular cytokine staining. The FACS plots are
gated on total CD8 T cells, and the numbers are the percentages
of IFNc-producing cells of CD8 T cells.
Found at: doi:10.1371/journal.ppat.1001139.s003 (0.68 MB TIF)
Interferon gamma production by virus-specific CD8
We wish to thank Dr. Yumi Nakayama, Erin Plisch, and Martha
McGregor for all their help.
Conceived and designed the experiments: YH KH YK MS. Performed the
experiments: YH KH KS SCP MH. Analyzed the data: YH KH KS SCP
RRD MH MGK YK MS. Contributed reagents/materials/analysis tools:
MGK. Wrote the paper: YH YK MS.
1. Graham MB, Braciale TJ (1997) Resistance to and recovery from lethal
influenza virus infection in B lymphocyte-deficient mice. J Exp Med 186:
2. Topham DJ, Tripp RA, Doherty PC (1997) CD8+ T cells clear influenza virus
by perforin or Fas-dependent processes. J Immunol 159: 5197–5200.
3. Woodland DL (2003) Cell-mediated immunity to respiratory virus infections.
Curr Opin Immunol 15: 430–435.
4. Epstein SL, Lo CY, Misplon JA, Bennink JR (1998) Mechanism of protective
immunity against influenza virus infection in mice without antibodies. J Immunol
5. Lawrence CW, Braciale TJ (2004) Activation, differentiation, and migration of
naive virus-specific CD8+ T cells during pulmonary influenza virus infection.
J Immunol 173: 1209–1218.
6. Lawrence CW, Ream RM, Braciale TJ (2005) Frequency, specificity, and sites of
expansion of CD8+ T cells during primary pulmonary influenza virus infection.
J Immunol 174: 5332–5340.
7. Bender BS, Croghan T, Zhang L, Small PA, Jr. (1992) Transgenic mice lacking
class I major histocompatibility complex-restricted T cells have delayed viral
clearance and increased mortality after influenza virus challenge. J Exp Med
8. Lee BO, Rangel-Moreno J, Moyron-Quiroz JE, Hartson L, Makris M, et al.
(2005) CD4 T cell-independent antibody response promotes resolution of
primary influenza infection and helps to prevent reinfection. J Immunol 175:
9. La Gruta NL, Kedzierska K, Stambas J, Doherty PC (2007) A question of self-
preservation: immunopathology in influenza virus infection. Immunol Cell Biol
10. Moskophidis D, Kioussis D (1998) Contribution of virus-specific CD8+ cytotoxic
T cells to virus clearance or pathologic manifestations of influenza virus infection
in a T cell receptor transgenic mouse model. J Exp Med 188: 223–232.
11. Enelow RI, Mohammed AZ, Stoler MH, Liu AN, Young JS, et al. (1998)
Structural and functional consequences of alveolar cell recognition by CD8(+) T
lymphocytes in experimental lung disease. J Clin Invest 102: 1653–1661.
12. de Jong MD, Simmons CP, Thanh TT, Hien VM, Smith GJ, et al. (2006) Fatal
outcome of human influenza A (H5N1) is associated with high viral load and
hypercytokinemia. Nat Med 12: 1203–1207.
13. Hatta M, Gao P, Halfmann P, Kawaoka Y (2001) Molecular basis for high
virulence of Hong Kong H5N1 influenza A viruses. Science 293: 1840–1842.
14. Subbarao K, Klimov A, Katz J, Regnery H, Lim W, et al. (1998)
Characterization of an avian influenza A (H5N1) virus isolated from a child
with a fatal respiratory illness. Science 279: 393–396.
15. Lu X, Tumpey TM, Morken T, Zaki SR, Cox NJ, et al. (1999) A mouse model
for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses
isolated from humans. J Virol 73: 5903–5911.
16. Katz JM, Lu X, Tumpey TM, Smith CB, Shaw MW, et al. (2000) Molecular
correlates of influenza A H5N1 virus pathogenesis in mice. J Virol 74:
17. Jackson D, Hossain MJ, Hickman D, Perez DR, Lamb RA (2008) A new
influenza virus virulence determinant: the NS1 protein four C-terminal residues
modulate pathogenicity. Proc Natl Acad Sci U S A 105: 4381–4386.
18. Seo SH, Hoffmann E, Webster RG (2002) Lethal H5N1 influenza viruses escape
host anti-viral cytokine responses. Nat Med 8: 950–954.
19. Szretter KJ, Gangappa S, Belser JA, Zeng H, Chen H, et al. (2009) Early control
of H5N1 influenza virus replication by the type I interferon response in mice.
J Virol 83: 5825–5834.
20. Opferman JT, Korsmeyer SJ (2003) Apoptosis in the development and
maintenance of the immune system. Nat Immunol 4: 410–415.
21. Marsden VS, Strasser A (2003) Control of apoptosis in the immune system: Bcl-
2, BH3-only proteins and more. Annu Rev Immunol 21: 71–105.
22. Aoki FY, Macleod MD, Paggiaro P, Carewicz O, El Sawy A, et al. (2003) Early
administration of oral oseltamivir increases the benefits of influenza treatment.
J Antimicrob Chemother 51: 123–129.
23. Gillissen A, Hoffken G (2002) Early therapy with the neuraminidase inhibitor
oseltamivir maximizes its efficacy in influenza treatment. Med Microbiol
Immunol 191: 165–168.
24. Schirmer P, Holodniy M (2009) Oseltamivir for treatment and prophylaxis of
influenza infection. Expert Opin Drug Saf 8: 357–371.
25. Yen HL,MontoAS,WebsterRG,GovorkovaEA(2005)Virulencemay determine
the necessary duration and dosage of oseltamivir treatment for highly pathogenic
A/Vietnam/1203/04 influenza virus in mice. J Infect Dis 192: 665–672.
26. Kiso M, Takahashi K, Sakai-Tagawa Y, Shinya K, Sakabe S, et al. T-705
(favipiravir) activity against lethal H5N1 influenza A viruses. Proc Natl Acad
Sci U S A 107: 882–887.
27. Claas EC, Osterhaus AD, van Beek R, De Jong JC, Rimmelzwaan GF, et al.
(1998) Human influenza A H5N1 virus related to a highly pathogenic avian
influenza virus. Lancet 351: 472–477.
28. Cox NJ, Subbarao K (2000) Global epidemiology of influenza: past and present.
Annu Rev Med 51: 407–421.
29. Cilloniz C, Shinya K, Peng X, Korth MJ, Proll SC, et al. (2009) Lethal influenza
virus infection in macaques is associated with early dysregulation of
inflammatory related genes. PLoS Pathog 5: e1000604.
30. Neumann G, Chen H, Gao GF, Shu Y, Kawaoka Y H5N1 influenza viruses:
outbreaks and biological properties. Cell Res 20: 51–61.
31. Legge KL, Braciale TJ (2005) Lymph node dendritic cells control CD8+ T cell
responses through regulated FasL expression. Immunity 23: 649–659.
32. Fornek JL, Gillim-Ross L, Santos C, Carter V, Ward JM, et al. (2009) A single-
amino-acid substitution in a polymerase protein of an H5N1 influenza virus is
associated with systemic infection and impaired T-cell activation in mice. J Virol
33. Zhou J, Law HK, Cheung CY, Ng IH, Peiris JS, et al. (2006) Functional tumor
necrosis factor-related apoptosis-inducing ligand production by avian influenza
virus-infected macrophages. J Infect Dis 193: 945–953.
34. Meng XW, Lee SH, Dai H, Loegering D, Yu C, et al. (2007) Mcl-1 as a buffer
for proapoptotic Bcl-2 family members during TRAIL-induced apoptosis: a
mechanistic basis for sorafenib (Bay 43-9006)-induced TRAIL sensitization.
J Biol Chem 282: 29831–29846.
35. Werneburg NW, Guicciardi ME, Bronk SF, Kaufmann SH, Gores GJ (2007)
Tumor necrosis factor-related apoptosis-inducing ligand activates a lysosomal
pathway of apoptosis that is regulated by Bcl-2 proteins. J Biol Chem 282:
H5N1 Virus Replication and CD8 T Cell Responses
PLoS Pathogens | www.plospathogens.org11October 2010 | Volume 6 | Issue 10 | e1001139
36. Han J, Goldstein LA, Gastman BR, Rabinowich H (2006) Interrelated roles for Download full-text
Mcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apoptosis. J Biol
Chem 281: 10153–10163.
37. McGill J, Van Rooijen N, Legge KL (2008) Protective influenza-specific CD8 T
cell responses require interactions with dendritic cells in the lungs. J Exp Med
38. Tumpey TM, Lu X, Morken T, Zaki SR, Katz JM (2000) Depletion of
lymphocytes and diminished cytokine production in mice infected with a highly
virulent influenza A (H5N1) virus isolated from humans. J Virol 74: 6105–6116.
39. Li Q, Skinner PJ, Ha SJ, Duan L, Mattila TL, et al. (2009) Visualizing antigen-
specific and infected cells in situ predicts outcomes in early viral infection.
Science 323: 1726–1729.
40. Budhu S, Loike JD, Pandolfi A, Han S, Catalano G, et al. CD8+ T cell
concentration determines their efficiency in killing cognate antigen-expressing
syngeneic mammalian cells in vitro and in mouse tissues. J Exp Med 207:
41. Daidoji T, Koma T, Du A, Yang CS, Ueda M, et al. (2008) H5N1 avian
influenza virus induces apoptotic cell death in mammalian airway epithelial cells.
J Virol 82: 11294–11307.
42. Badovinac VP, Porter BB, Harty JT (2004) CD8+ T cell contraction is
controlled by early inflammation. Nat Immunol 5: 809–817.
43. Cilloniz C, Pantin-Jackwood MJ, Ni C, Goodman AG, Peng X, et al. Lethal
dissemination of H5N1 influenza virus is associated with dysregulation of
inflammation and lipoxin signaling in a mouse model of infection. J Virol 84:
44. Kacergius T, Ambrozaitis A, Deng Y, Gravenstein S (2006) Neuraminidase
inhibitors reduce nitric oxide production in influenza virus-infected and gamma
interferon-activated RAW 264.7 macrophages. Pharmacol Rep 58: 924–930.
45. Bouillet P, Metcalf D, Huang DC, Tarlinton DM, Kay TW, et al. (1999)
Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses,
leukocyte homeostasis, and to preclude autoimmunity. Science 286: 1735–1738.
46. Andrews BS, Eisenberg RA, Theofilopoulos AN, Izui S, Wilson CB, et al. (1978)
Spontaneous murine lupus-like syndromes. Clinical and immunopathological
manifestations in several strains. J Exp Med 148: 1198–1215.
47. Fung-Leung WP, Schilham MW, Rahemtulla A, Kundig TM, Vollenweider M,
et al. (1991) CD8 is needed for development of cytotoxic T cells but not helper T
cells. Cell 65: 443–449.
48. Martinez X, Kreuwel HT, Redmond WL, Trenney R, Hunter K, et al. (2005)
CD8+ T cell tolerance in nonobese diabetic mice is restored by insulin-
dependent diabetes resistance alleles. J Immunol 175: 1677–1685.
49. Kolumam GA, Thomas S, Thompson LJ, Sprent J, Murali-Krishna K (2005)
Type I interferons act directly on CD8 T cells to allow clonal expansion and
memory formation in response to viral infection. J Exp Med 202: 637–650.
H5N1 Virus Replication and CD8 T Cell Responses
PLoS Pathogens | www.plospathogens.org12October 2010 | Volume 6 | Issue 10 | e1001139