Oseltamivir–Resistant Pandemic H1N1/2009 Influenza
Virus Possesses Lower Transmissibility and Fitness in
Susu Duan1,2, David A. Boltz1¤, Patrick Seiler1, Jiang Li3, Karoline Bragstad4, Lars P. Nielsen4, Richard J.
Webby1, Robert G. Webster1,2, Elena A. Govorkova1*
1Division of Virology, Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 2Department of
Pathology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America, 3Hartwell Center for Bioinformatics and Biotechnology, St.
Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 4National Influenza Laboratory, Department of Virology, Statens Serum Institute,
The neuraminidase (NA) inhibitor oseltamivir offers an important immediate option for the control of influenza, and its
clinical use has increased substantially during the recent H1N1 pandemic. In view of the high prevalence of oseltamivir-
resistant seasonal H1N1 influenza viruses in 2007–2008, there is an urgent need to characterize the transmissibility and
fitness of oseltamivir-resistant H1N1/2009 viruses, although resistant variants have been isolated at a low rate. Here we
studied the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, one oseltamivir-sensitive and
one resistant, in the ferret model. The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and
was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses
differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted
efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive
H1N1/2009 virus was efficiently transmitted via both routes (2/2 and 1/2, respectively). The wild-type H1N1/2009 and the
resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We
compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses
and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced
substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These
findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained
efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and
characterization of possible evolving viral proteins during the pandemic.
Citation: Duan S, Boltz DA, Seiler P, Li J, Bragstad K, et al. (2010) Oseltamivir–Resistant Pandemic H1N1/2009 Influenza Virus Possesses Lower Transmissibility and
Fitness in Ferrets. PLoS Pathog 6(7): e1001022. doi:10.1371/journal.ppat.1001022
Editor: Kanta Subbarao, National Institutes of Health, United States of America
Received December 21, 2009; Accepted June 30, 2010; Published July 29, 2010
Copyright: ? 2010 Duan 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 study was supported by Contract No. HHSN266200700005C from the National Institute of Allergy and Infectious Diseases, and the American
Lebanese Syrian Associated Charities (ALSAC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: While the study reported here did not utilize corporate funding, Drs. Elena A. Govorkova and Robert G. Webster are currently performing
a different research study funded by F. Hoffmann-LaRoche, Ltd., Basel, Switzerland. The authors declare no competing financial interests.
* E-mail: email@example.com
¤ Current address: Microbiology & Molecular Biology Division, IIT Research Institute, Chicago, Illinois, United States of America
A novel swine-origin H1N1 influenza virus emerged in Mexico
in April 2009 and rapidly spread worldwide, causing the first
influenza pandemic of the 21st century [1,2]. Most confirmed
human cases of H1N1/2009 influenza have been uncomplicated
and mild , but the increasing number of cases and affected
countries warrant optimal prevention and treatment measures. At
present, two classes of antiviral drugs are approved for specific
management of influenza: M2-ion channel blockers (amantadine
and rimantadine) and neuraminidase (NA) inhibitors (zanamivir
and oseltamivir). However, variants resistant to both classes of
drugs have emerged. During the 2007–2008 season, most
circulating seasonal H3N2 influenza viruses, and H1N1 viruses
in certain geographic areas, were reportedly resistant to M2-
blockers [4,5]; today, almost all of the pandemic H1N1/2009
viruses tested are resistant to M2-blockers . Therefore, only the
NA inhibitors are currently recommended for treatment of
The NA-inhibitor resistance-associated mutations in influenza
viruses are drug-specific and NA subtype-specific . Until 2007,
the clinical data indicated only sporadic, rare emergence of
oseltamivir resistance under drug selection pressure (,1% in
adults and 4%–8% in children) [9–11]. Later reports observed
increased frequency of oseltamivir-resistant variants (18% and
27%) in drug-treated children [11,12]. The situation changed
dramatically during the 2007–2008 season, when seasonal H1N1
influenza viruses with the common oseltamivir-resistance NA
H275Y mutation (275 in N1 numbering, 274 in N2 numbering)
became widespread in first the northern  and then the
PLoS Pathogens | www.plospathogens.org1 July 2010 | Volume 6 | Issue 7 | e1001022
southern  hemispheres. It remains uncertain where these
naturally resistant H1N1 influenza viruses originated and how
they acquired optimal fitness and transmissibility, but the resistant
variants were clearly becoming the dominant strain at the time the
swine-origin pandemic H1N1/2009 virus emerged [15–17].
During the H1N1/2009 influenza pandemic, to date, almost all
tested viruses have remained susceptible to oseltamivir and zanamivir
, but oseltamivir-resistant variants with H275Y NA mutation have
immunocompromised patients  under drug selection pressure.
Oseltamivir-resistant variants also have been isolated from untreated
patients [21,22] and from a few community clusters [23–25],
including two suspected cases of nosocomial transmission among
immunocompromised patients [23,24], although it is uncertain
whether the mutants came from secondary transmission or arose
spontaneously. The isolation of resistant H1N1/2009 viruses with no
link to oseltamivir use raised serious concern that these viruses might
acquire fitness and spread worldwide, as had oseltamivir-resistant
seasonal H1N1 viruses during 2007–2008.
The increasing concern about oseltamivir-resistant H1N1/2009
viruses prompted us to evaluate transmissibility and growth fitness
of one oseltamivir-resistant variant. The infectivity and transmis-
sibility (and thus the clinical relevance) of several NA inhibitor-
resistant influenza viruses have previously been studied in
experimental animal models [26–29]. These studies differed in
the influenza A subtypes studied (H1N1, H3N2, or H5N1), the
NA mutations involved (H275Y, R292K, E119V or I222V), the
animal model used (ferret or guinea pig), and the transmission
routes studied (direct contact and respiratory droplets); in these
studies, the transmissibility of most of the NA inhibitor-resistant
influenza viruses was to some extent less efficient. Here we
characterized in vitro and in a ferret model a pair of pandemic
H1N1/2009 clinical isolates. The pandemic A/Denmark/524/09
viruses were isolated from a small cluster of patients with
H1N1/2009 virus infection . The A/DM/528/09 virus,
carrying the H275Y NA mutation, was isolated from a patient on
oseltamivir prophylaxis, and its ancestor is likely to have been A/
DM/524/09 virus. By recapitulating two natural routes of
influenza virus transmission in ferrets, we found that the
oseltamivir-resistant virus was less transmissible than its sensitive
counterpart through the respiratory droplet route but retained
efficient transmission through direct contact.
Sequencing and phylogenetic analysis of NA genes
Sequence analysis of the NA genes revealed that A/DM/524/
09 virus encoded a conserved H residue at amino acid position
275, whereas A/DM/528/09 virus had an H275Y amino acid
mutation caused by a single T-to-C nucleotide substitution at
codon 275 (Table 1). Pairwise sequence analysis of the full viral
genomes showed that the A/DM/524/09 and A/DM/528/09
viruses had no amino acid differences other than the H275Y NA
mutation and were a highly matched pair. Sequence analysis and
phylogenetic analysis of the two viruses’ NA and HA genes (data
not shown) confirmed that the wild-type A/DM/524/09 and
mutant A/DM/528/09 viruses belonged to the swine-origin 2009
pandemic virus lineage. The alignment of the NA and HA
sequences showed that viruses with H275Y NA substitution have
some amino acid differences from certain wild-type viruses
(without H275Y NA mutation), but these differences also were
observed in other wild-type viruses. Comparison of the NA and
HA amino acid sequences of A/DM/528/09 virus with sequences
of other 24 H275Y mutants and around 2000 wild-type H1N1/
2009 viruses available in Gene Bank did not reveal an increased
frequency of any specific amino acid mutation(s) shared among the
viruses analyzed (data not shown).
NA inhibitor susceptibility and NA enzyme kinetics
To assess the NA inhibitor susceptibility of the two viruses, we
performed NA enzyme inhibition assays with the NA inhibitors
oseltamivir carboxylate (active metabolite of oseltamivir) and
zanamivir. The wild-type A/DM/524/09 virus was susceptible to
oseltamivir carboxylate (mean IC50: 5.0 nM), but the A/DM/528/
09 carrying the H275Y NA mutation had IC50values approxi-
mately 200 times that of the wild-type virus (Table 1). The IC50of
Most of the currently circulating pandemic H1N1/2009
(‘‘swine’’) influenza viruses are susceptible to the anti-
influenza drug oseltamivir. Many countries have stockpiled
oseltamivir for pandemic preparedness, and to date only a
small proportion of the H1N1/2009 viruses isolated have
been oseltamivir-resistant. However, if these viruses can be
readily transmitted, oseltamivir resistance may spread. We
evaluated the transmissibility of a pair of pandemic H1N1/
2009 influenza viruses in ferrets. One virus was oseltamivir-
sensitive and the other carried the oseltamivir resistance-
associated H275Y NA mutation. We also investigated the
viruses’ susceptibility to NA inhibitors (the drug class to
which oseltamivir belongs), their NA enzyme kinetics, and
their replication efficiency in cultured cells. Under identical
conditions, the resistant H1N1/2009 virus was not trans-
mitted by respiratory droplets but was efficiently trans-
mitted by direct contact, while the sensitive H1N1/2009
virus was efficiently transmitted by both routes.
Table 1. Neuraminidase enzymatic properties of the H1N1 influenza viruses.
H1N1 virus Sequence at NA position 275a
NA enzyme inhibition IC50±SDb(nM) Enzyme kineticsc
NucleotideAmino acid Oseltamivir carboxylateZanamivirKm (m mM) Vmax (U/sec)
A/Denmark/524/09CACH 5.060.81.360.15 55.164.2101.667.9
A/Denmark/528/09TACY 9726283*1.060.1380.366.0* 86.865.6*
aThe full genomes of both viruses were sequenced; only differences are shown. In order of segments, the GenBank accession numbers are CY043339–CY043346 for A/
DM/524/09 virus and CY043347–CY043354 for A/DM/528/09 virus genome sequences.
bMean 6 SD from five independent measurements.
cAssayed in parallel with reference A/Fukui/08/02 (H3N2) virus. Km and Vmax values were derived from the Michaelis-Menten plot.
*P,0.05 compared to value for respective wild-type virus.
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zanamivir was comparable for both viruses and was uniformly low
(mean IC50#1.3 nM) (Table 1). These results showed that the
H275Y NA mutation conferred resistance to oseltamivir carboxyl-
ate but did not alter susceptibility to zanamivir.
To understand the impact of the H275Y mutation on the NA
enzymatic properties of the H1N1/2009 viruses, we determined
the NA enzyme kinetics of both viruses. Km is an estimate of the
dissociation equilibrium for substrate binding to enzyme and the
reciprocal of Km approximates the affinity of substrate binding,
while Vmax reflects the enzyme’s catalytic activity. The NA of
resistant A/DM/528/09 virus had a slightly higher Km and lower
Vmax than the NA of the sensitive A/DM/524/09 virus (Table 1).
The H275Y NA mutation reduced NA affinity for substrate and
NA catalytic activity, although the function of NA was not severely
impaired. This finding in the H1N1 pandemic virus is similar to
that reported by another group, in which NA enzymatic function
was not impaired in some naturally resistant seasonal viruses
isolated during the 2007 season . Our study is the first to show
reduced but not severely impaired NA enzymatic function in a
resistant H1N1/2009 virus with the H275Y mutation.
Plaque morphology and growth kinetics in MDCK and
To determine whether the H275Y NA mutation affects virus
growth in vitro, we characterized virus plaque morphology and
growth kinetics in both MDCK and MDCK-SIAT1 cells. The
latter have increased surface expression of human-like a2,6-linked
terminal sialic acids  and may better assess the growth
capability of human influenza viruses. In MDCK cells, both
pandemic H1N1/2009 viruses formed pinpoint-like (0.3 mm)
plaque phenotype (Figure 1A), differing significantly from some
seasonal H1N1 viruses, such as A/Brisbane/59/2007 (BR/59/07)
virus, which formed large plaques (1.3 mm) (P,0.05) (data not
shown); however, the plaque size did not differ between the
oseltamivir-sensitive and -resistant viruses (Figure 1A), indicating
that the H275Y NA mutation did not alter plaque morphology. In
MDCK-SIAT1 cells, both the pandemic viruses and seasonal BR/
59/07 (data not shown) formed only pinpoint-like plaques
(Figure 1B), consistent with a previous report  that this cell
line did not generate clear plaques for influenza viruses.
To further evaluate the impact of the H275Y NA mutation on
virus growth in vitro, we performed single- and multiple-cycle growth
studies of both viruses in MDCK and MDCK-SIAT1 cells. In single-
cycle growth in the two cell lines, the two viruses reached comparable
levels 6 hours post-infection, but the initial growth of the oseltamivir-
resistant virus was significantly delayed in comparison to its sensitive
counterpart (P,0.05) (Figure 1C): at 4 hours post-infection, the yield
of resistant viruses was at least 1 log10TCID50/ml lower (P,0.05).
Likewise, in multiple-cycle growth, the two viruses reached
comparable yields 24 hours post-infection, but the resistant virus
showed a significant growth delay during the first 12 hours post-
infection (P,0.05); this delay was more conspicuous in MDCK-
SIAT1 cells than in MDCK cells (Figure 1D), probably because
overexpressed a2,6 receptorson cell surfacecouldbetterdifferentiate
NA’s function in support of viral growth. Therefore, final virus yields
of oseltamivir-resistant pandemic virus in the MDCK and MDCK-
SIAT1 cells were not altered, but their growth at the initial infection
stage was significantly delayed.
Transmissibility among ferrets via direct contact and
The transmissibility of pandemic H1N1/2009 viruses was
studied in a ferret model. Two naı ¨ve ferrets were housed at day
2 post-inoculation (p.i.) in the same cage with one inoculated
ferret (direct contact), and two naı ¨ve ferrets were placed in an
adjacent cage separated from the donor’s cage by two layers of
wire mesh (respiratory droplet exposure). Transmission of
H1N1 virus was assessed by detection of infection in recipient
ferrets (nasal wash titers, clinical signs, and seroconversion).
Virus samples in nasal washes at day 4 p.i. or post-contact
(p.c.) were sequenced to detect the presence of the H275Y NA
The donor ferret inoculated with oseltamivir-sensitive A/DM/
524/09 virus shed virus until day 6 p.i. (Figure 2A, Table 2).
Two of 2 direct-contact ferrets and 1 of 2 respiratory droplet-
exposed ferrets were infected through virus transmission, as
indicated by the virus titers and inflammatory cell counts in their
nasal washes (Figure 2) and by seroconversion (Table 3). Virus
shedding and nasal inflammation began earlier in the direct-
contact ferrets, suggesting that transmission through respiratory-
droplets may have a greater lag time. One respiratory droplet-
exposed ferret showed no detectable virus shedding or inflam-
mation, but its post-contact serum had a positive HI titer (320).
Although seroconversion indicated infection in this ferret, the
time of infection could not be determined and therefore we could
not attribute the infection to direct contact with the co-caged
ferret versus respiratory droplet transmission from the adjacent
The donor ferret inoculated with oseltamivir-resistant A/DM/
528/09 virus shed virus until day 8 p.i. (Figure 2), with a peak
virus titer comparable to that of A/DM/524/09 virus (Table 2).
Two of 2 direct-contact ferrets were infected through transmis-
sion (Figure 2), but neither respiratory droplet-exposed ferret was
infected, as confirmed by the absence of seroconversion (Table 3).
These results showed that the oseltamivir-resistant H275Y
mutant A/DM/528/09 virus was transmitted efficiently only by
direct contact. Virus shedding in two direct-contact ferrets was
lower and peaked after a longer interval in this group than in the
oseltamivir-sensitive A/DM/524/09 group (Figure 2A), although
the severity and course of disease were similar (Figure 2B,
We verified the sequence stability of the NA at position 275
in each virus after replication and transmission in ferrets.
Direct sequencing of the NA genes from nasal wash samples
revealed no sequence change at this position in either virus
(data not shown). Therefore, no spontaneous H275Y NA
mutation emerged in the wild-type virus and the H275Y
mutation remained stable in the mutant after transmission to a
Co-inoculation with oseltamivir-sensitive and -resistant
Because both the oseltamivir-sensitive and the oseltamivir-
resistant H1N1/2009 viruses were efficiently transmitted by
direct contact, hosts could potentially be exposed to both types
of virus. To compare the relative growth capability and
transmissibility of the sensitive and resistant H1N1/2009
viruses within the host, we co-inoculated a ferret with a 1:1
ratio of the sensitive A/DM/524/09 and resistant A/DM/528/
09 viruses. The pattern of virus shedding and the clinical signs
were similar to those in ferrets inoculated with either A/DM/
524/09 or A/DM/528/09 virus (Figure 3A). By using a relative
quantification of single nucleotide polymorphism (SNP) method
to detect the NA genotype at codon 275 (CAC or TAC), we
found that the virus population in the co-inoculated ferret’s
nasal washes remained mixed but was predominantly a wild-
type (oseltamivir-sensitive) population (Figure 3B). The pro-
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portion of wild-type virus in the nasal wash increased
progressively, from 75% on day 1 p.i., to almost 100% on
day 6 p.i. (Figure 3B). Two of 2 ferrets placed in direct contact
with the co-inoculated ferret were infected through transmis-
sion (Figure 3A). SNP analysis of their nasal wash samples
showed only wild-type virus (Figure 3B). In summary, the
oseltamivir-sensitive A/DM/524/09 virus possessed greater
growth capability in the upper respiratory tract than did
resistant A/DM/528/09 virus and thus had an advantage in
Figure 1. Plaque morphology and replication kinetics of two H1N1/2009 influenza viruses in MDCK and MDCK-SIAT1 cells. The
diameters of 20 randomly selected value plaques were measured in MDCK cells (A) and MDCK-SIAT1 cells (B). Values are mean (6 SD) plaque
diameter (mm). Single-cycle (C, D left panel) and multiple-cycle (C, D right panel) growth curves were obtained by using an MOI of ,2 and
,0.001 PFU/cell, respectively. Virus in the supernatant was titrated in MDCK or MDCK-SIAT1 cells and expressed as log10TCID50/ml at the
indicated time post-infection. Each point represents the mean log10TCID50/ml 6 SD from three experiments. * P,0.05 compared to value for
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This study is the first, to our knowledge, to demonstrate the
inefficient respiratory droplet transmission of an oseltamivir-
resistant H275Y mutant of H1N1/2009 in ferrets, which are an
established animal model of the pathogenesis and transmission of
human influenza viruses. The oseltamivir-resistant mutant virus
retained efficient transmission only by direct contact, whereas the
oseltamivir-sensitive pandemic virus was efficiently transmitted by
both routes. These results show that the transmissibility of the
oseltamivir-resistant H1N1/2009 influenza virus had been altered.
We suggest that the lower fitness of oseltamivir-resistant variant
Figure 2. Transmissibility of the two H1N1/2009 influenza viruses among ferrets. The virus titer (A, B left panel) and total number of
inflammatory cells (A, B right panel) in the nasal wash samples from each donor ferret, direct-contact (DC contact) ferret, and respiratory droplet-
contact (RD contact ) ferret. The arrow indicates the first day of exposure of contact ferrets.
Table 2. Clinical signs, virus replication, and seroconversion in inoculated donor ferrets.
H1N1 virus Inoculated donor ferretsa
Clinical signs Virus replication
(observed day of onset)
Last day of
Peak virus titer
Last day of
Serum HI titerf
A/Denmark/524/095.0 (2)3 12 7.3 (2)6 1280
A/Denmark/528/096.2 (2)7 12 6.9 (2)8 640
5.9 (4)7 12 7.7 (2)6 640
an=1 for each virus group.
bThe maximum percent weight loss during the 21 days p.i. Numbers in parentheses indicate the day of maximum weight loss.
cUpper respiratory tract inflammation was defined as a total inflammatory cell count $10 times the baseline count.
dVirus titers in nasal washes (log10TCID50/ml).
eThe first day of observation on which virus was not detected.
fHemagglutination inhibition (HI) antibody titers to homologous virus 21 days p.i.
gCo-inoculation of ferret with A/DM/524/09 and A/DM/528/09 viruses at 1:1 ratio.
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within the host along with its reduced NA enzyme efficiency and
delayed growth of the H275Y mutant virus in vitro may at least in
part explain its impaired transmission among ferrets.
There are limited experimental data about the routes of
transmission of oseltamivir-resistant influenza viruses. The two
natural routes of influenza virus transmission, direct contact with
Table 3. Clinical signs, virus replication, and seroconversion in contact ferrets.
Direct contact Respiratory droplets exposure
Clinical signsVirus detectionClinical signs Virus detection
Last day of
Last day of
A/Denmark/524/092/2 (3.5) 1/2 (7)2/2 (8.3)8, 8 1280, 640 1/2 (6.0) 1/2 (7) 1/2 (7.2)10 1280,320
A/Denmark/528/092/2 (3.3)2/2 (5,7) 2/2 (7.0)8, 10 1280,1280 0/20/2 0/2 NA
2/2 (6.0)1/2 (2) 2/2 (7.1)10, 10 1280,1280NA NA NANA NA
aNumber of animals with weight change/total number (maximum percent weight loss during the 21 days p.c.).
bNumber of animals sneezing/total number during the 21 days p.c. (day of observed onset).
cNumber of virus-shedding animals/total number. Numbers in parentheses indicate mean peak virus titer (log10TCID50/ml) in nasal wash samples).
dThe first day of observation on which virus was not detected.
eHemagglutination inhibition (HI) antibody titers to homologous virus in ferret serum on day 21 p.c.
fDonor ferret was co-inoculated with A/DM/524/09 and A/DM/528/09 viruses at a 1:1 ratio.
Figure 3. Co-infection in a ferret with oseltamivir-sensitive and -resistant H1N1/2009 influenza viruses. Virus titers and inflammatory
cell counts in the nasal wash specimens of ferrets co-inoculated with oseltamivir-sensitive and -resistant H1N1/2009 viruses (A). The arrow indicates
the first day of exposure of contact ferrets. The proportion of wild-type virus (C in SNP sequence) in the mixed virus population (C+T in SNP sequence)
in nasal wash samples from the donor ferret and two direct-contact ferrets (B). Values are the mean 6 SD from three independent measurements.
* P,0.05 compared to value for day 0 p.i.
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fomites and respiratory droplets (aerosol and larger droplets ),
are not mutually exclusive. Therefore, the transmissibility of
influenza virus via both routes must be investigated if the results
are to be clinically relevant. In the earliest studies, oseltamivir-
resistant H3N2 (R292K NA mutant) and H1N1 (H275Y NA
mutant) variants exhibited severely compromised replication and
virulence both in vitro and in vivo [34,35] and were therefore
thought unlikely to be of clinical consequence. In a subsequent
study, an R292K mutant of H3N2 virus was not transmitted by
direct contact among ferrets . Under similar conditions, the
transmission of an E119V mutant of H3N2 virus and an H275Y
mutant of A/New Caledonia/20/99-like (H1N1) virus by direct
contact required a higher dose of inoculum than transmission of
the wild-type viruses, and it occurred more slowly . However,
none of these studies assessed both routes of transmission. The
only study to date that has evaluated both routes of transmission of
oseltamivir-resistant virus showed that recombinant resistant
H3N2 viruses with either the E119V or the E119V+I222V NA
mutation were transmitted efficiently by direct contact but not by
respiratory droplets among guinea pigs . Our study is a latest
addition to the previous data by comparing a highly matched pair
of H1N1/2009 viruses and by assessing the transmissibility of
resistant viruses via two routes in ferrets.
The reduced transmissibility of the oseltamivir-resistant H1N1
viruses could be explained by a number of factors [33,36,37]. First,
host physical exposure to virus is directly affected by the quantity
of virus shed into the environment. In our study, inoculated donor
ferrets shed comparable quantities of both viruses, which indicated
potential comparable environmental contamination in the restrict-
ed space of cages; therefore, it is unlikely that transmission was
affected by the level of donor viral shedding. Other host variables
such as the extent of inflammation could affect the amount and
size of upper respiratory secretions thus the release of infectious
respiratory droplets. For example, sneezing, a common host
symptom believed to mediate viral transmission, was observed
only at later stages in the ferret inoculated with resistant virus,
when inflammation was more severe but virus shedding had
declined greatly. Second, efficient transmission to a naive host
requires not only viral exposure but also successful viral invasion,
effective replication and simultaneous evasion of the first line of
host innate immunity . Our results showed a significant initial
growth delay in two cell lines of the oseltamivir-resistant virus.
This growth delay could be caused by delayed release of progeny
virions from the host cell surface due to reduced NA enzyme
efficiency observed in the resistant virus. Such a delay would not
affect the final virus yield in cell lines, but in the respiratory tract of
ferrets it could allow the host’s first-line innate immune defense
(e.g., macrophages or neutrophils) sufficient time to clear the virus.
The NA enzyme also facilitates virus binding, entry, and spread
within the host by removing terminal sialic-acid residues from
mucus and preventing virion self-aggregation , and therefore
the NA mutation could have affected viral penetration into the
host respiratory tract. The slightly reduced (not severely impaired)
NA enzyme function and delayed viral growth of the H275Y
mutant may have been more crucial in recipient ferrets that
acquired virus from environment via natural routes than in donor
ferrets inoculated with a high dose of virus, as we observed delayed
viral shedding or inefficient transmission in the recipient ferrets,
but not in the inoculated donor ferret.
Although the transmissibility of the oseltamivir-resistant
H1N1/2009 virus was reduced by the H275Y NA mutation,
the severity and course of disease was similar to that caused by
oseltamivir-sensitive H1N1/2009 virus in both inoculated and
direct-contact ferrets, with no apparent attenuation of clinical
signs. In inoculated ferrets, the viruses showed comparable
replication in the upper respiratory tract and caused comparable
clinical signs, including weight loss and inflammation. However,
one caveat to ferret model has been noticed that high inoculation
dose may mask the differential viral replication and clinical signs
for different viruses . In the direct-contact ferrets, which
acquired virus though natural routes, the shedding of resistant
virus peaked later than the shedding of susceptible virus, but the
duration of shedding and the severity of disease was not
compromised when compared with sensitive virus. Therefore,
the H275Y mutant of pandemic H1N1/2009 virus is likely to be
of clinical consequence in humans.
The fitness of a virus describes its relative ability to produce
infectious progeny in a host . Competitive growth assay by co-
infection is a method of evaluating the growth fitness of two viruses
[41,42]. In the present study, we inoculated a ferret with equal
doses of oseltamivir-sensitive and -resistant H1N1/2009 viruses to
compare their relative growth fitness within the host. The mixed
virus population in the nasal wash was analyzed at different days
p.i. to determine which viral genotype predominated. To bypass
the time- and labor-intensive process of cloning the desired genes
from the mixed populations and choosing an arbitrary number of
clones for genotypic analysis, we used a new method, relative
quantification of SNP, to determine the ratio of wild-type to
mutant populations. This method showed high reproducibility in
genotyping HIV protease gene . Our study is the first to use
this method to genotypically analyze influenza viruses. For the
H1N1/2009 influenza viruses, we designed a specific probe to
detect the first nucleotide of codon 275 of the NA gene, where a
single C-to-T substitution causes an H-to-Y amino acid substitu-
tion. Our results showed that the oseltamivir-resistant mutant
H1N1/2009 virus possessed less growth fitness than the sensitive
H1N1/2009 virus in the ferret upper respiratory tract. At least
partly for that reason, only wild-type H1N1/2009 virus was
transmitted to the direct-contact ferrets. The competitive trans-
mission advantage of wild-type H1N1/2009 virus should be
confirmed by other types of experiments.
In summary, our study determined the comparative transmis-
sibility of a pair of naturally circulating oseltamivir-sensitive and
oseltamivir-resistant H1N1/2009 viruses. This information from
this study could be useful in assessing the clinical relevance of
contemporary pandemic viruses, considering the extensive use of
oseltamivir during this pandemic. The H275Y mutant of H1N1/
2009 used in this study was the first oseltamivir-resistant H1N1/
2009 isolate from a patient on oseltamivir prophylaxis. As this
study was undertaken, additional H275Y mutants of H1N1/2009
viruses have emerged in the absence of oseltamivir use [21–25].
The emergence of these viruses should raise concerns as to
whether resistant H1N1/2009 viruses will acquire significantly
greater fitness and spread worldwide as did the naturally resistant
H1N1 viruses during the 2007–2008 season. Further studies of
these newly isolated H275Y mutants of H1N1/2009 viruses are
warranted to determine whether they have acquired additional
Materials and Methods
All animal experiments with H1N1 influenza viruses were
performed in biosafety level 3+ facilities at St. Jude Children’s
Research Hospital (St. Jude; Memphis, TN, USA), were approved
by the St. Jude Animal Care and Use Committee, and complied
with the policies of the National Institutes of Health and the
Animal Welfare Act.
Transmissibility of NAI-Resistant H1N1 Viruses
PLoS Pathogens | www.plospathogens.org7 July 2010 | Volume 6 | Issue 7 | e1001022
Viruses and cells
A/Denmark/524/09 (H1N1) influenza virus (A/DM/524/09)
and an oseltamivir-resistant A/Denmark/528/09 (H1N1) virus (A/
DM/528/09) were provided by Statens Serum Institute, Copenha-
gen, Denmark. The resistant virus was isolated from the tthroat
swab of a patient who had influenza-like symptoms and received
post-exposure oseltamivir prophylaxis (75 mg once daily) . A/
Brisbane/59/07 (H1N1) influenza virus (A/BR/59/07) was
provided by U.S. Centers for Disease Control and Prevention.
Stocks of H1N1 viruses were prepared in Madin-Darby canine
kidney (MDCK) cells (ATCC, Manassas, VA) and grown in
minimal essential medium (MEM) supplemented with 5% fetal
bovine serum, 5 mM L-glutamine, 0.2% sodium bicarbonate,
100 U/ml penicillin, 100 mg/ml streptomycin sulfate, and 100 mg/
ml kanamycin sulfate in a humidified atmosphere of 5% CO2. All
strains of virus underwent a limited number of passages in MDCK
cells to maintain their original properties. MDCK cells transfected
with cDNA encoding human 2,6-sialyltransferase (MDCK-SIAT1
cells) were maintained as described previously .
The NA inhibitors oseltamivir carboxylate ([3R, 4R, 5S]-4-
ic acid) and zanamivir (4-guanidino-Neu5Ac2en) were provided by
Hoffmann-La Roche, Ltd. (Basel, Switzerland). The compounds
were dissolved in distilled water and aliquots were stored at 220uC
until the time of use.
Infectivity of influenza viruses
The 50% tissue culture infectious dose (TCID50) was deter-
mined in MDCK cells. The cells were infected with serial log
dilutions of the stock viruses, incubated for 1 h at 37uC, washed,
and overlaid with infection medium (MEM with 0.3% BSA and
1 mg/ml TPCK-trypsin). Infection of cells was determined by
hemagglutination assay (HI) after incubation for 3 d at 37uC, and
TCID50was calculated by the Reed-Muench method .
Single-step growth curves were generated for influenza viruses
in MDCK cells or MDCK-SIAT1 cells. Confluent cell monolayers
were infected with viruses at a multiplicity of infection (MOI) of
,2.0 PFU/cell. After incubation, the cells were washed with 0.9%
aqueous NaCl solution (pH 2.2) to remove free infectious virus
particles and then were washed twice with phosphate-buffered
saline (PBS) to adjust the pH. Supernatants were collected 2, 4, 6,
8, 10 and 12 h p.i. and stored at 270uC for titration. To generate
multi-step growth curves, MDCK cells or MDCK-SIAT1 cells
were infected with viruses at a MOI of 0.001 PFU/cell.
Supernatants were collected 12, 24, 36, 48, 60 and 72 h p.i. and
stored at 270uC for titration in the same cell line.
Plaque assay in MDCK and MDCK-SIAT cells
Confluent MDCK or MDCK-SIAT cells were incubated for
1 h at 37uC with 10-fold serial dilutions of virus in 1 ml of
infection medium. The cells were then washed and overlaid with
freshly prepared MEM containing 0.3% BSA, 0.9% bacto-agar,
and 1 mg/ml TPCK trypsin. The plaques were visualized after
incubation at 37uC for 3 d by staining with 0.1% crystal violet
solution containing 10% formaldehyde.
Virus susceptibility to NA inhibitors in vitro
A modified fluorometric assay using the fluorogenic substrate 29-
(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA)
(Sigma-Aldrich) was used to determine viral NA activity . The
fluorescence of the released 4-methylumbelliferonewasmeasured in
a Synergy 2 multi-mode microplate reader(BioTek) usingexcitation
and emission wavelengths of 360 and 460 nm, respectively. The
drug concentration required to inhibit 50% of the NA enzymatic
activity (IC50) was determined by plotting the percent inhibition of
NA activity as a function of compound concentration calculated in
the GraphPad Prism 4 software from the inhibitor-response curve.
The NA inhibitor–sensitive A/Fukui/20/04 (H3N2) influenza virus
was included in every plate for comparison.
NA enzyme kinetics
All H1N1 viruses were standardized to an equivalent dose of
106.0PFU/ml. We measured NA enzyme kinetics at pH 6.5 with
33 mM 2-(N-Morpholino) ethanesulfonic acid hydrate (MES;
Sigma-Aldrich), 4 mM CaCl2, and MUNANA with a final
substrate concentration of 0 to 400 mM. The reaction was
conducted at 37uC in a total volume of 50 ml, and the fluorescence
of released 4-methylumbelliferone was measured every 60 sec for
60 min in a Synergy 2 multi-mode microplate reader (BioTek)
using excitation and emission wavelengths of 360 and 460 nm,
respectively. The Km and Vmax were calculated by fitting the
data to the appropriate Michaelis-Menten equations by using
nonlinear regression in the GraphPad Prism 4 software. The A/
PR/8/34 (H1N1) influenza virus was included for comparison in
Transmission experiments in ferrets
Young adult ferrets (4–5 months of age) were obtained from the
ferret breeding program at St. Jude Children’s Research Hospital.
All ferrets were seronegative for influenza A H1N1 and H3N2
viruses and for influenza B viruses. Ferrets were housed in the
isolators in ABSL3+ facilities and monitored for 3–5 days to
establish baseline body temperature and overall health. Donor
ferrets were initially housed separately from contact ferrets. The
donor ferrets were lightly anesthetized with isoflurane and
inoculated with 106TCID50of A/DM/524/09, A/DM/528/09
virus in 1.0 ml sterile PBS. One donor ferret was inoculated with
106TCID50of a mixture of A/DM/524/09 and A/DM/528/09
viruses (1:1 infectivity ratio). After the donor ferrets were
confirmed to shed virus on day 2 p.i. by the Directigen Flu A+B
quick test (BD, Franklin Lakes, NJ), each was then housed in the
same cage with 2 naı ¨ve direct-contact ferrets. Two additional
recipient ferrets were placed in an adjacent cage isolated from the
donor’s cage by a two layers of wire mesh (,5 cm apart) that
prevented physical contact but allowed the passage of respiratory
droplets. A Borazine gun (Zero Toys, Concord, MA ) was used to
ensure non-directional air flow inside the isolator. The donor and
recipient ferrets were housed together since day 2 p.i until day 21
p.i. Ferret weight and temperature were recorded daily for 21
days. Body temperature was measured by subcutaneous implant-
able temperature transponders (Bio Medic Data Systems Inc,
Collection and titration of nasal wash samples
Nasal washes were collected from donors and recipients on days
1, 2, 4, 6, 8, 10, 12, and 14 p.i. by flushing both nostrils with
1.0 ml PBS, and TCID50titers were determined in MDCK cells.
Inflammatory cell counts were determined as described previously
. Briefly, the nasal washes were centrifuged at 2000 rpm for
5 min. The pellet was resuspended in PBS, and the total cell
number was counted in a hemacytometer under light microscopy.
Inflammation was defined as a cell count $10 times the baseline
count determined before the inoculation or exposure.
Transmissibility of NAI-Resistant H1N1 Viruses
PLoS Pathogens | www.plospathogens.org8July 2010 | Volume 6 | Issue 7 | e1001022
Serum samples were collected from ferrets 3 weeks after virus
inoculation, treated with receptor-destroying enzyme, heat-inacti-
vated at 56uC for 30 min, and tested by HI assay with 0.5% packed
chicken red blood cells (CRBC) as described previously .
Virus sequence analysis
Viral RNA was isolated from ferret nasal washes by using the
RNeasy Mini kit (Qiagen, Valencia, CA). Samples were reverse-
transcribed and analyzed by PCR using primers specific for the
NA gene segment, as described previously . Sequencing was
performed by the Hartwell Center for Bioinformatics and
Biotechnology at St. Jude Children’s Research Hospital. The
DNA template was sequenced by using rhodamine or dRhoda-
mine dye terminator cycle-sequencing Ready Reaction kits with
AmpliTaq DNA polymerase FS (Perkin-Elmer, Applied Biosys-
tems, Inc., Foster City, CA) and synthetic oligonucleotides.
Samples were analyzed in a Perkin-Elmer Applied Biosystems
DNA sequencer (model 373 or 377). DNA sequences were
completed and edited by using the Lasergene sequence analysis
software package (DNASTAR, Madison, WI). The alignment of
NA and HA for multiple sequences was conducted by BioEdit
software (Tom Hall Ibis Therapeutics, Carlsbad, CA).
Relative quantification of single nucleotide
The relative quantification of SNP assay was performed as
described previously , with slight modification. Briefly, an NA
fragment (nucleotide 673 to 1034) containing the codon at NA 275
position was amplified by RT-PCR. Primers were 59-AGAACA-
CAAGAGTCTGAATGTG-39 and 59-CCATTTGCTCCATTA-
GACGATACT-39. Single nucleotide primer extension was per-
formed using a SNaPshot kit (ABI) per the manufacturer’s protocol.
The reaction consisted of 2.5 ml SNaPshot Reaction Mix, 3 ml RT-
PCR product, and 0.2 mmol/L of the extension primer in a 5 ml final
reaction volume. The extension primer, 59-CAGTCGAAAT-
GAATGCCCCTAATTAT-39, was synthesized by IDTDNA and
used to detect the first nucleotide of NA 275 codon. After the
SNaPshot reaction, a unit of shrimp alkaline phosphatase (USB) was
added to remove 59 phosphoryl groups of unincorporated dideox-
ynucleotide substrates as directed by manufacturer’s protocol. One ml
of the SNaPshot products was mixed with deionized formamide and
LIZ120 (ABI) size standard and was injected into the ABI 3730xl
capillary electrophoresis instrument (ABI) per the manufacturer’s
protocol. Data were analyzed by using ABI GeneMapper software.
Serially diluted DNA template (35 ng/ml to 0.02 ng/ml) from each
genotype was used for signal standardization. Spike-in samples were
generated by using 11 different ratios of wild-type and mutant DNA
fragments, e.g. 100% wild-type, 90% wild-type, etc. A good
correlation was achieved between the spike-in ratios and ratios of
fluorescence intensity values (R2=0.9877) (data not shown).
The unpaired t-test or analysis of variance (ANOVA) was used
for all comparisons.
We thank Sharon Naron for editorial assistance and Klo Spelshouse for
illustrations. We thank Christy Brockwell and Mariette F. Ducatez for
advice on phylogenetic analysis. We also thank David S. Carey and Sharon
Lokey for the assistance with animal work in the ABSL3+ laboratory. The
NA inhibitors oseltamivir carboxylate and zanamivir were provided by
Hoffmann-La Roche, Ltd (Basel, Switzerland).
Conceived and designed the experiments: SD RJW RGW EAG.
Performed the experiments: SD EAG. Analyzed the data: SD RJW
RGW EAG. Wrote the paper: SD RJW RGW EAG. Designed the viral
SNP assay and conducted experiments with ferrets: DAB. Conducted
experiments with ferrets: PS. Designed and conducted the viral SNP assay:
JL. Sequenced the H1N1/2009 viruses: KB. Provided the pandemic
H1N1/2009 viruses and advice in experimental design: LPN.
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