﻿Published Ahead of Print 14 July 2010.
2010, 84(18):9427. DOI: 10.1128/JVI.00373-10.
J. Virol.
Fryer, Jennifer Mosse, Angela R. McLean and Ian G. Barr
Aeron C. Hurt, Siti Sarah Nor'e, James M. McCaw, Helen R.
Model
Ferrets, Using a Competitive-Mixtures
Oseltamivir-Resistant Influenza Viruses in
Assessing the Viral Fitness of
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JOURNAL OF VIROLOGY, Sept. 2010, p. 9427­9438 Vol. 84, No. 18
0022-538X/10/$12.00 doi:10.1128/JVI.00373-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Assessing the Viral Fitness of Oseltamivir-Resistant Influenza Viruses
in Ferrets, Using a Competitive-Mixtures Model
Aeron C. Hurt,1,2
* Siti Sarah Nor'e,1,2
James M. McCaw,3
Helen R. Fryer,4
Jennifer Mosse,2
Angela R. McLean,4
and Ian G. Barr1,2
WHO Collaborating Centre for Reference and Research on Influenza, 10 Wreckyn St., North Melbourne, Victoria 3051, Australia1
;
Monash University, School of Applied Sciences, Churchill, Victoria 3842, Australia2
; Vaccine and Immunisation Research Group,
University of Melbourne, Melbourne School of Population Health and Murdoch Childrens Research Institute,
Royal Childrens Hospital, Melbourne, Victoria 3010, Australia3
; and University of Oxford, Department of Zoology,
Institute for Emergent Infections of Humans, James Martin 21st Century School,
Oxford OX1 3PS, United Kingdom4
Received 18 February 2010/Accepted 2 July 2010
To determine the relative fitness of oseltamivir-resistant strains compared to susceptible wild-type viruses,
we combined mathematical modeling and statistical techniques with a novel in vivo "competitive-mixtures"
experimental model. Ferrets were coinfected with either pure populations (100% susceptible wild-type or 100%
oseltamivir-resistant mutant virus) or mixed populations of wild-type and oseltamivir-resistant influenza
viruses (80%:20%, 50%:50%, and 20%:80%) at equivalent infectivity titers, and the changes in the relative
proportions of those two viruses were monitored over the course of the infection during within-host and over
host-to-host transmission events in a ferret contact model. Coinfection of ferrets with mixtures of an oselta-
mivir-resistant R292K mutant A(H3N2) virus and a R292 oseltamivir-susceptible wild-type virus demonstrated
that the R292K mutant virus was rapidly outgrown by the R292 wild-type virus in artificially infected donor
ferrets and did not transmit to any of the recipient ferrets. The competitive-mixtures model was also used to
investigate the fitness of the seasonal A(H1N1) oseltamivir-resistant H274Y mutant and showed that within
infected ferrets the H274Y mutant virus was marginally outgrown by the wild-type strain but demonstrated
equivalent transmissibility between ferrets. This novel in vivo experimental method and accompanying math-
ematical analysis provide greater insight into the relative fitness, both within the host and between hosts, of
two different influenza virus strains compared to more traditional methods that infect ferrets with only pure
populations of viruses. Our statistical inferences are essential for the development of the next generation of
mathematical models of the emergence and spread of oseltamivir-resistant influenza in human populations.
The neuraminidase (NA) inhibitors are a class of influenza
antiviral drugs that are specifically designed to inhibit the en-
zymatic function of the NA, thereby preventing normal viral
replication. Since 1999, two NA inhibitors (NAIs), oseltamivir
(Tamiflu) and zanamivir (Relenza), have been shown to be
effective for the treatment and prophylaxis of patients infected
with not only seasonal influenza, but also highly pathogenic
A(H5N1) and the newly emerged A(H1N1) pandemic virus.
Prior to 2007, resistance to this class of drugs was considered
relatively uncommon, particularly in comparison with the other
class of influenza antivirals, the adamantanes, which readily
select for viral resistance in treated patients. During early clin-
ical trials, oseltamivir resistance was detected in only 1 to 2%
of adults (14) and 5 to 6% of children (33) under treatment,
although later studies detected resistance in up to 18% of
oseltamivir-treated children (16). In contrast, resistance fol-
lowing zanamivir treatment is rare, with only one reported case
observed in an immunocompromised patient (6). Influenza
viruses that develop resistance to these drugs typically contain
mutations in the NA which, either directly or indirectly, alter
the shape of the NA enzymatic site, thereby reducing the
ability of the drugs to bind to this specific pocket. One of the
most commonly observed mutations in oseltamivir-resistant
A(H3N2) viruses is an arginine-to-lysine mutation at residue
292 (R292K) of the NA, while the predominant NA mutation
in oseltamivir-resistant A(H1N1) viruses is a histidine-to-ty-
rosine mutation at residue 274 (H274Y) (N2 NA amino acid
numbering, equivalent to residue 275 based on N1 numbering).
Both of these mutations have an indirect impact on drug bind-
ing, as they affect the ability of the glutamic acid residue at
position 276 to reorientate, as required for slow binding by
oseltamivir (3). Many mutations that cause NAI resistance also
cause reduced NA enzyme activity and, consequently, can com-
promise viral fitness.
Previous studies have demonstrated that viruses with an
R292K NA mutation demonstrated compromised growth in
vitro (36) and in ferrets were significantly less infectious and
did not transmit (9). The replication and transmission fitness of
the H274Y mutation has also been studied previously. An
H274Y mutant A(H1N1) strain isolated from a patient under
oseltamivir treatment demonstrated compromised growth in
cell culture compared to a wild-type (WT) virus (13), although
a strain carrying the same mutation selected in vitro was found
to replicate as well as the wild type (32). The infectivity and
transmissibility of an H274Y mutant were found to be re-
stricted in ferrets (13), although a second study demonstrated
* Corresponding author. Mailing address: WHO Collaborating Cen-
tre for Reference and Research on Influenza, 10 Wreckyn St., North
Melbourne, Victoria 3051, Australia. Phone: 613 9342 3914. Fax: 613
9342 3939. E-mail: aeron.hurt@influenzacentre.org.

Published ahead of print on 14 July 2010.
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that transmission of the mutant virus between ferrets was pos-
sible, but required a greater viral dose of the mutant compared
to the wild type (10). These results suggest that resistant virus
variants with the same NA mutation may differ in replication
or transmission fitness depending on other viral components.
Nevertheless, based on these data and the viral fitness of other
resistant mutants, it was believed that NAI-resistant viruses
were unlikely to spread throughout the community due to their
compromised viral fitness in the absence of drug selective pres-
sure. This was proven incorrect during the Northern Hemi-
sphere 2007-2008 influenza season, when large numbers of
oseltamivir-resistant seasonal A(H1N1) viruses with an H274Y
mutation were detected in patients who had not been treated
with oseltamivir (4, 24). The mutant strain continued to spread
to the Southern Hemisphere, such that by late 2008 virtually all
circulating seasonal A(H1N1) viruses were oseltamivir resis-
tant (11). The rapid global spread of this strain clearly sug-
gested that the oseltamivir-resistant seasonal A(H1N1) virus
had fitness equivalent to or greater than that of the previous
oseltamivir-sensitive A(H1N1) strain. The reasons for en-
hanced viral fitness in this strain, when previous studies dem-
onstrated that the acquisition of an H274Y mutation led to
reduced viral fitness, remain unclear but probably involve com-
pensatory mutations or reassortment events which may have
improved the hemagglutinin (HA)/NA balance, allowing effi-
cient transmission (5, 26).
Experimental methods have been developed to assess the
relative fitness of NAI-resistant strains compared with respec-
tive wild-type viruses, both in vitro and in vivo. Ferrets have
been considered the most appropriate model animal for influ-
enza research, and fitness studies have assessed variables such
as minimum dose required to achieve infection, duration of
viral shedding, and levels of viral load to allow comparisons
between viruses. The guinea pig model has also been previ-
ously used to assess the viral fitness of influenza viruses, par-
ticularly in comparing the transmissibility of strains via either
the contact or aerosol route (2). As an alternative to these
traditional approaches, we have investigated a methodology
that involves coinfection of ferrets with a mixture of two influ-
enza viruses. Daily monitoring of changes in the relative pro-
portion of those viruses over the course of the infection allows
determination of the relative replication fitness of the viruses.
Monitoring of recipient ferrets exposed to the infected ferrets
enables the relative transmissibility of the viruses (henceforth,
the relative transmission fitness) to be determined. In this
study, the "competitive-mixtures" methodology was used to
assess the relative replication and transmission fitness of an
oseltamivir-resistant R292K mutant A(H3N2) virus compared
with an oseltamivir-sensitive A(H3N2) wild-type strain and
also to asses the relative replication and transmission fitness of
an oseltamivir-resistant H274Y seasonal A(H1N1) mutant
compared with an oseltamivir-sensitive A(H1N1) wild-type
strain. Quantitative estimates for the replication fitness of mu-
tant viruses were determined using a simple mathematical
model of within-host viral replication and mixed-effects statis-
tical tests. Transmission fitness was evaluated by application of
a graphical technique that demonstrated the relationship be-
tween the proportion of mutant virus in the infectee ferrets as
a function of the proportion of mutant virus in the infector
ferrets.
Inferences drawn from the statistical analyses presented
here are essential for the refinement of existing mathematical
models that simulate the spread of influenza in the human
population and model the deployment of antiviral agents.
These models are designed to assess the likely impact of dif-
ferent antiviral agent deployment strategies to control pan-
demic influenza (18, 21, 35). At present, data on the probability
of emergence of NAI-resistant strains, the relative transmis-
sion fitness of these strains, and the probability of an individ-
ual's infection reverting to an NAI-sensitive strain in the ab-
sence of ongoing selective pressure are severely limited. In
consequence, human population-level models of influenza
spread must make gross assumptions on the likely character-
istics of NAI-resistant strains. Data such as those presented
here will be used to inform new models of drug deployment
and result in improved pandemic policy advice (20, 23).
MATERIALS AND METHODS
Viruses. Two oseltamivir-resistant viruses were paired with respective wild-
type viruses. The first pair of viruses was composed of a wild-type (WT) NAI-
sensitive strain, A/Fukui/20/2004 A(H3N2) (referred to as "R292 WT"), and an
oseltamivir-resistant mutant, A/Okayama/23/2004 A(H3N2), which contained an
R292K NA mutation (referred to as "R292K MUT"). The second pair of viruses
was composed of a wild-type NAI-susceptible seasonal A(H1N1) strain, A/Bris-
bane/59/2007 (referred to as "H274 WT"), and an oseltamivir-resistant seasonal
A(H1N1) strain, A/Sydney/142/2007, which contained an H274Y NA mutation
(N2 numbering) (referred to as "H274Y MUT"). Each of the viruses was isolated
in Madin-Darby canine kidney (MDCK) cells (obtained from ATCC; #CCL-34)
and had undergone at least two further passages in MDCK cells prior to two
rounds of plaque purification in MDCK cells to achieve a homogeneous popu-
lation. The viruses were then expanded once to achieve sufficient stock material
for in vitro and in vivo experiments. The infectivity titers of the R292 WT, R292K
MUT, H274 WT, and H274Y MUT stock viruses were 2.69  106
, 1.58  106
,
3.16  108
, and 3.16  108
50% tissue culture infectious doses (TCID50)/ml,
respectively.
Full-genome sequence analysis of the viruses demonstrated a high genetic
homology between each pair. The H274 WT and H274Y MUT viruses shared
99.8% amino acid similarity across the entire genome. Comparison of each
segment for the H274 WT and H274Y MUT viruses revealed no amino acid
differences in the HA and M segments (100% similarity) and two changes in the
NA: the H274Y mutation (that confers oseltamivir resistance) and a G354D
mutation (which has previously been shown to be associated with H274Y mutant
viruses [26]). A high genetic similarity in the PB1, PB2, PA, NP, and NS segments
was also observed between the H274Y WT and H274Y MUT strains, with amino
acid homology ranging from 99.7% to 99.9%. The R292 WT and R292K MUT
viruses also showed a high degree of similarity, although slightly lower than that
seen with the H274 WT and H274Y MUT viruses, with 98.8% amino acid
homology across the entire genome and segment-specific homology ranging from
96.6% to 99.6%. Of the few amino acid differences detected between each pair,
none was in a location known to impact on growth, replication, or virulence.
Plaque purification and cell culture. For plaque purification, confluent
MDCK cells in six-well tissue culture plates were prepared a day prior to infec-
tion in MDCK cell culture growth medium containing Dulbecco's modified
Eagle's medium (DMEM)-Coons basal medium (SAFC Biosciences) supple-
mented with 2 mM L-glutamine (SAFC Biosciences), 1% nonessential amino
acids (NE) (SAFC Biosciences), 0.05% sodium bicarbonate (SAFC Biosciences),
1 M HEPES (SAFC Biosciences), 2% penicillin-streptomycin (Sigma-Aldrich),
and inactivated fetal bovine serum (JRH Biosciences). MDCK cells were in-
fected with 0.4 ml of virus per well (from a dilution range of 101
to 108
). The
plates were incubated at 35°C for 30 min in a 5% CO2-gassed incubator. After 30
min of incubation, the inocula were removed using a plastic Pasteur pipette, and
4 ml of overlay containing equal proportions of liquefied 1% agarose and 2
minimal essential medium (MEM) plus NE (MEM/NE) supplemented with 8
g/ml trypsin (SAFC Bioscience) was added to each well. The plates were kept
in the biohazard cabinet until the overlay solidified and then were placed back in
the 5% CO2-gassed incubator. The appearance of foci was examined after 4 days
of incubation at 35°C. The three most-separated and smallest plaques were
harvested by removing a plug with a sterile 100-l micropipette tip. Each of the
9428 HURT ET AL. J. VIROL.
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plugs was placed into 0.5 ml of CaMg-free phosphate-buffered saline (PBS) and
stored at 70°C prior to further plaque purification or viral expansion. Viral
expansion in MDCK cells was done in maintenance medium containing DMEM-
Coons basal medium (SAFC Bioscience), 2 mM L-glutamine (SAFC Bioscience),
1% nonessential amino acids (SAFC Bioscience), 0.05% NaHCO3 (SAFC Bio-
science), 1 M HEPES (SAFC Bioscience), 2% penicillin-streptomycin (Sigma-
Aldrich), 2 g/ml amphotericin (Gibco), and 4 g/ml trypsin (SAFC Bioscience).
RNA extraction, RT-PCR, and sequencing. Total RNA was extracted from the
cell culture supernatant using the robotic MagNA Pure LC instrument and the
MagNA Pure LC total nucleic acid isolation kit (Roche Applied Science).
The RNA extraction procedure was performed according to manufacturer's
instructions, with a total elution volume of 50 l. RNA extracts were stored at
70°C. A 5-l aliquot of RNA was used to amplify the selected influenza virus
gene using specific primers (sequences available by request) and SuperScript III
Platinum one-step reverse transcription (RT)-PCR system (Invitrogen) reagents.
Amplicons were visualized on a 2% agarose gel. PCR products were purified for
use in a sequencing reaction using the QIAquick PCR purification kit (Qiagen).
DNA sequencing was carried out using the ABI Prism dye terminator III cycle
sequencing kit (Applied Biosystems) followed by the removal of excess dye
terminators using a DyeEx spin kit (Qiagen). The sequence was determined
using an automated capillary DNA sequencer (ABI Prism 377 located at the
Institute of Medical and Veterinary Science, Adelaide, Australia). Sequences
were assembled and aligned using the DNAStar Lasergene 8 software.
Determination of viral infectivity. To determine the infectivity titer of each virus,
confluent MDCK cells were infected with 10-fold serially diluted influenza virus
supernatant to determine the TCID50. Cells (2  105
cells/well) were prepared in
96-well flat-bottom plates in MDCK growth medium 24 h before use. The MDCK
growth medium was removed, and the monolayer washed twice with CaMg-free
PBS. One hundred microliters of each virus diluted in MDCK maintenance medium
was inoculated into the wells in triplicate. The inoculated viruses were incubated at
35°C in 5% CO2 for 2 h, after which virus inocula were removed and 200 l of
maintenance medium supplemented with 4 g/ml of trypsin was added. The plates
were incubated at 35°C in 5% CO2 for 4 days. Cell viability in virus-infected wells was
determined using a neutral red staining method (31). The log10 TCID50/ml was
calculated as described by Reed and Muench (27).
In vitro replication kinetics. In vitro replication kinetics experiments were
performed in conventional MDCK cells and also in MDCK-SIAT1 cells (kindly
provided by Hans-Dieter Klenk, University of Marburg, Marburg, Germany) as
this modified cell line has an increased surface expression of human-like -2,6-
linked sialic acid receptors and may be better suited to analysis of NAI-resistant
viruses (19, 36). The replication efficiency of each virus was investigated using
both a single-step replication cycle, which involved the infection of MDCK or
MDCK-SIAT1 cells with a high virus titer and sampling of the supernatant over
a short time period, and multiple-step replication cycles, which involved the
infection of MDCK or MDCK-SIAT1 cells with a lower virus titer and sampling
of the supernatant over a longer period of time. Both growth studies were
performed in triplicate. For the single-step replication cycle, confluent MDCK or
MDCK-SIAT1 cells in 24-well plates were inoculated with viruses at a multiplic-
ity of infection (MOI) of 5 TCID50/cell. Flasks were incubated at 35°C for 1 h to
allow virus absorption. The cells were washed with 0.9% aqueous NaCl solution
(pH 2.2) to remove any unattached infectious virus particles and then washed
twice with PBS to adjust the pH (36). The cells were then overlaid with 1 ml
maintenance medium. The flasks were incubated at 35°C in 5% CO2, and su-
pernatant was collected 2, 4, 6, 8, and 10 h postinfection with each virus and
stored at 70°C prior to determination of viral infectivity. For the multiple-step
replication cycles, confluent MDCK or MDCK-SIAT1 cells in each T-25 (25-
cm3
) cell culture flask were inoculated with viruses at a multiplicity of infection
(MOI) of 0.01 TCID50/cell. After initial incubation, as described for the single-
step replication cycle, the infected cells were then overlaid with 10 ml mainte-
nance medium and incubated at 35°C with 5% CO2. Three hundred microliters
of supernatant was collected 6, 24, 30, 48, 54, 72, 78, 96, and 102 h postinfection
for each virus and stored at 70°C for titration prior to determination of viral
infectivity. To ensure consistency, viral infectivity assays were performed using
the same cell line (either MDCK or MDCK-SIAT1 cells) from which the repli-
cation kinetics experiments were performed.
Ferret experiments. To prepare the viruses for inoculation into ferrets, each of
the four strains (R292 WT, R292K MUT, H274 WT, and H274Y MUT) was
diluted to a standardized tissue culture infectivity titer of 1  105
TCID50/ml.
These virus preparations, containing 1  105
TCID50/ml, were then used for
inoculation of ferrets either as a pure population or for preparation of mixtures
of wild-type and mutant strains. To prepare the different mutant and wild-type
mixtures, the virus preparations (containing 1  105
TCID50/ml) were combined
in three different proportions (80% WT­20% MUT, 50% WT­50% MUT, and
20% WT­80% MUT). For example, the 80% WT­20% MUT mixture was
prepared by combining 8 ml of wild-type virus (at an infectivity titer of 1  105
TCID50/ml) with 2 ml of mutant virus (also at an infectivity titer of 1  105
TCID50/ml). As such, the two pure populations and three mixtures, now referred
to as "virus groups," all had an infectivity titer of 1  105
TCID50/ml. Female
ferrets of approximately 6 months of age and weighing approximately 800 g were
sourced from various ferret breeders in Victoria, Australia, and were micro-
chipped, anesthetized, and pre-bled via the jugular vein to confirm the absence
of preexisting antibodies to seasonal H1/H3/B influenza strains. For each of the
five virus groups, two ferrets, referred to as "donor 1" (D1) and "donor 2" (D2),
were anesthetized intramuscularly with 20 mg/ml Ilium Xylazil-20 (Troy Labo-
ratories, NSW, Australia) and then dosed intranasally with 0.5 ml of virus (5 
104
TCID50) (prepared following the methods detailed above). Donors 1 and 2
from the same virus group were housed together, but in separate HEPA filtered
caging from the donor ferrets in other virus groups. Twenty-four hours postin-
fection (day 1), ferrets were nasal washed by dispensing 1 ml of nasal wash
solution (CaMg-free PBS with 1% [wt/vol] bovine serum albumin [BSA], 100
g/ml streptomycin, and 100 U/ml penicillin) using an Optiva (Medex) 20-gauge
intravenous (i.v.) catheter and 1-ml syringe into the nostril of each ferret and
collecting the returned nasal wash liquid in a sterile tube, which was stored at
70°C. After thorough cleaning of the cages, a naïve uninfected ferret, termed
"recipient 1" (R1), was introduced into each of the five cages containing the
donor ferrets from the different virus groups. Recipient 1 ferrets were nasal
washed daily, until they were found to be influenza positive by a rapid point-of-
care test (BinaxNOW Influenza A & B test kit; Inverness Medical, Waltham,
MA), and then were removed and housed immediately in a new clean cage
together with a naïve uninfected ferret termed "recipient 2" (R2). Nasal washes
were collected either daily or every 2 days from all donor and recipient ferrets
until day 11 (for the H274Y experiment) or day 18 (for the R292K experiment),
after which they were given a terminal bleed.
Genetic analysis of nasal washes. RNA was extracted as described earlier. To
determine the relative proportions of wild-type and mutant influenza strains in
ferret nasal wash samples, a TaqMan probe-based one-step real-time RT-PCR
assay was conducted. The assay used the TaqMan one-step RT-PCR master mix
reagents kit (Applied Biosystems) together with the ABI PRISM 7500 sequence
detection system (Applied Biosystems) following the manufacturer's instruc-
tions. The primers and probes were kindly designed by Michael Tavaria (Applied
Biosystems, Australia) based on NA sequences of the two pairs of viruses and
following the manufacturer's design guidelines and software. For each pair of
viruses, an assay was developed with two specific PCRs: one designed to detect
the wild-type virus and a second to detect the mutant virus (Fig. 1). Following
FIG. 1. Primer and probe designs for R292K and H274Y real-time RT-PCR assays. Specific nucleotides highlighted in gray represent mutations
associated with NAI resistance within the NA gene. The specific nucleotides underlined represent the location where sequence differs between the
wild type and mutant but is not associated with residue mutation or NAI resistance.
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optimization to achieve the highest specificity for both wild-type and mutant
reactions, primers and probes were used in the PCR assays at concentrations of
5 M and 25 M, respectively. RNAs extracted from the five virus groups used
to infect ferrets (e.g., 100:0, 0:100, 80:20, 50:50, and 20:80) were included in each
PCR assay as positive controls.
Calculation of viral proportions in nasal washes. The relative proportions of
wild-type and mutant viruses in each nasal wash or positive control were deter-
mined by calculating the difference between the cycle threshold (CT) value for
the wild-type PCR and the mutant PCR. CT is defined as the cycle at which a
statistical increase in emission intensity of a specific strand crosses a predeter-
mined fluorescence threshold. Based on the principle that a doubling of the
quantity of PCR product occurs every cycle during the exponential phase of the
reaction, it is inferred that a difference of one CT between samples indicates that
there is twice the amount of starting RNA in one of the samples compared to the
other. Therefore, a 50:50 mixture of wild-type and mutant viruses should give
identical CT values for both the wild-type PCR and for the mutant PCR: i.e., no
CT value difference between reactions indicates equal amounts of starting RNA.
Following an adjustment based on the wild-type versus mutant CT values for the
control viruses, the difference between the CT value for the wild-type reaction
and that for the mutant reaction was calculated for each virus sample. The fold
difference in RNA quantity between the WT and mutant viruses is therefore
given by 2CT
, where CT is the CT difference. The final estimated proportion
(percentage) of WT to mutant virus in each sample was then calculated from the
fold difference value. A CT value of greater than 40 cycles was considered to be
a negative result. Assays were considered valid if the difference between the
calculated WT/mutant proportion and the known proportions for any of the five
mixture controls did not exceed 10 percentage points. For example, if calcula-
tions for the 80% WT­20% mutant control sample produced predicted propor-
tions of 69% WT­31% mutant, then the assay was considered invalid and all
samples from that assay would be retested.
Mathematical and statistical analysis. To assess the within-host replication
fitness of the R292K and H274Y mutant strains, we utilized an established
technique previously used to asses the fitness of cytotoxic T-lymphocyte (CTL)
escape mutants in chronic HIV infection (1). Briefly, we assumed both the wild
type and mutant were recognized similarly by the immune system (both innate
and adaptive components) and so the rate of removal, b, of the viruses was the
same. The mutant has a different replication rate, a, compared to that for the
wild type, a. We let a, a, and b vary over time to allow for the increase and then
decrease in viral load, but made the assumption that a  a is constant in time.
It follows that the proportion of the mutant in the viral population at any point
in time is given by the equation
pt 
1
get
 1
(1)
where   a  a is a measure of replication fitness cost of the mutant virus and
g is the ratio of wild type to mutant at the time of inoculation. Given this
functional form for the changing mutant proportion over time, we made a
model-dependent estimate of the replication fitness cost, , for the mutant virus
in each ferret. This measure is the difference between two viral growth rates, a
and a, not a ratio of viral growth rates. We applied the Wilcoxon rank-sum test
for difference between mutation types (R292K versus H274Y). Furthermore, to
make inferences across strain type while accounting for the expected individual
variation among ferrets, we performed a standard mixed-effects statistical anal-
ysis. A transformation of equation 1 yields a linear-model, ln {[1  p(t)]/gp(t)} 
t, where a positive slope, , indicates a compromised mutant virus. We tested
for evidence of a replication fitness cost for the R292K and H274Y mutants
separately and for a difference in replication fitness cost between the two mu-
tants, allowing for the random variation among individual ferrets in our analysis.
Statistical analysis was performed in STATA/IC 10.1 2009. To assess transmis-
sion fitness costs--that is differences in the transmissibility of the mutant com-
pared to that of the wild type--we used a graphical technique presented in the
Results.
Nucleotide sequence accession number. Sequences for each virus segment
have been deposited in GenBank under the following accession numbers:
A/Fukui/20/2004 (R292 WT), CY064955 to CY064962; A/Okayama/23/2004
(R292K MUT), CY064963 to CY064970; A/Brisbane/59/2007 (H274 WT),
CY064971 to CY064978; and A/Sydney/142/2007 (H274Y MUT), CY064979 to
CY064986.
RESULTS
In vitro replication kinetics. The replication kinetics of wild-
type and mutant viruses in single- and multiple-step replication
cycles were determined in MDCK and MDCK-SIAT1 cells. In
the single-step growth studies, the growth rate of the oselta-
mivir-resistant R292K MUT virus was found to be significantly
slower than that of the R292 WT virus (P  0.05) in experi-
ments conducted in both MDCK and MDCK-SIAT1 cells (Fig.
2A), although in the multiple-step growth experiment, there
was no significant difference between the growth curves of the
two strains (Fig. 2B). Comparison of the in vitro replication
kinetics for the H274 WT and H274Y MUT strains revealed no
significant difference in either the single- or multiple-step ex-
periments regardless of the cell line used (Fig. 2C and D). In
both the MDCK and MDCK-SIAT1 multiple-step experi-
ments, the H274 WT and H274Y MUT viruses achieved higher
viral titers between the 20 and 120 h postinfection than those
observed for the R292 WT and R292K MUT viruses during the
same period.
Validation of the real-time RT-PCR assays for estimating
viral proportions. Prior to testing the nasal wash samples, the
ability of the R292K and H274Y real-time RT-PCR assays to
correctly estimate the wild-type and mutant viral proportions
in the control mixtures 80%:20%, 50%:50%, and 20%:80%
was determined. Analysis of the R292K control mixtures in
seven separate R292K real-time RT-PCR assays revealed that
the estimated mixture proportion, as determined by the assay,
differed from the actual proportions by a minimum of 0.2 and
a maximum of 5.1 percentage points, with an absolute mean (
1 standard deviation) difference of 2.2 ( 1.5) percentage
points. Similarly, analysis of the H274Y control mixtures across
seven H274Y real-time RT-PCR assays demonstrated that the
estimated proportions differed from the actual proportions by
a minimum of 0.1 and a maximum of 7.2 percentage points,
with an absolute mean ( 1 standard deviation) difference of
2.7 ( 2.2) percentage points. To determine if the accuracy of
estimating the viral proportions differed when analyzing mix-
tures with lower viral titers, the three R292K control mixtures
(80%:20%, 50%:50%, and 20%:80%) were diluted (101
to
105
) and tested in the R292K real-time RT-PCR assay. The
absolute mean differences between the estimated and actual
proportions for the three control mixtures were 1.8, 2.1, 2.4,
and 2.0 percentage points for the undiluted and 101
-, 102
-,
and 103
-diluted controls, respectively. The control mixtures
diluted to 104
or 105
could not be detected by the PCR
assay. These results indicated the ability of the PCR assay to
accurately estimate viral proportions, irrespective of viral load,
within the limit of detection of the assay.
Analysis of viral proportions in ferret nasal washes. Figures
3 and 4 summarize the relative proportions of wild-type and
mutant virus in each ferret over time for the R292K and
H274Y experiments, respectively. We first present within-host
viral replication data in the donor and recipient ferrets (repli-
cation fitness), before examining the characteristics of trans-
mission of viral mixtures from donor ferrets to recipient ferrets
(transmission fitness).
Summary for the R292K experiment. For the R292K exper-
iment, we observed that in all donor ferrets the mutant virus
(black) is outgrown by the wild-type virus (white) (Fig. 3). Both
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donor ferrets (D1 and D2) infected with 100% R292 WT virus
shed virus until 8 or 10 days postinfection, with no detection of
any mutant virus in any nasal washes during this period (Fig.
3). Recipient 1 (R1) became infected by day 6 of the experi-
ment (5 days after being housed with the donor ferrets) and
continued to shed detectable virus for another 4 days. Virus
was detected in recipient 2 (R2) on the second day it was
housed with R1 (day 8); R2 continued to shed virus until day
14 (8 days postexposure). The two donor ferrets infected with
100% R292K MUT virus shed detectable virus for 6 or 8 days,
although the proportions of R292K MUT in the viral popula-
tion, which started at 100%, were significantly reduced over the
course of the infection to only 61% and 1% in D1 and D2,
respectively, being replaced by R292 WT virus (Fig. 3). None
of the R292K MUT virus was detected in the R1 ferret, and the
R292 WT virus was found to persist for 6 days before R2
became infected, again with only the R292 WT virus (100%).
A similar outcome was observed with the ferrets infected with
the mixtures of mutant and wild-type virus. In each case, the
proportion of the R292K MUT virus decreased over time in
the donor ferrets and R292K MUT was not detectable in any
of the R1 ferrets (Fig. 3).
Summary for the H274Y experiment. Donor ferrets infected
with a pure population of H274 WT virus demonstrated de-
FIG. 2. In vitro replication kinetics of wild-type and mutant viruses in MDCK and MDCK-SIAT1 cells. Replication kinetics of the A/Fukui/
20/2004 (R292 WT) (E) and A/Okayama/23/2004 (R292K mutant) (OE) viruses in single-step growth experiments (MOI of 5 TCID50/cell) (A) and
multiple-step growth experiments (MOI of 0.01 TCID50/cell) (B) and the A/Brisbane/59/2007 (H274 WT) () and A/Sydney/142/2007 (H274Y
mutant) (}) viruses in single-step growth experiments (C) and multiple-step growth experiments (D). Virus in the supernatant was titrated at the
indicated time points postinfection (log10 TCID50/ml). Each data point represents the mean log10 TCID50 from triplicate experiments. *,
statistically significant difference between WT and mutant virus (P  0.05).
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tectable virus shedding for 6 days postinfection (Fig. 4). R1
became infected within 24 h of being exposed to the infected
donors and, after being rehoused on day 3, subsequently in-
fected R2 by day 6. The two donor ferrets infected with a pure
population of the H274Y MUT virus shed virus for 4 to 6 days
postinfection. Although the proportion of H27Y MUT virus
remained very high (93% to 100%) in both donor ferrets over
the course of infection, the H274 WT virus was detected at low
levels in nasal washes from various time points. Repeat real-
time RT-PCR analysis of these samples confirmed these re-
sults. A low proportion of H274 WT virus was detected on
occasions in nasal washes from R1 (day 4 nasal wash contained
1% H274 WT virus) and R2 (day 6 nasal wash contained 6%
H274 WT virus).
FIG. 3. Infectivity and transmissibility of pure populations and mixtures of R292 WT and R292K MUT viruses in ferrets. The relative
proportion of viral mixtures is indicated in the panels where the percentage of R292 WT virus is displayed using white bars and the R292K MUT
proportion is displayed by black bars. No bars are present for samples when no virus was detected. R1 ferrets were introduced into the same cage
as donor ferrets on day 1. Asterisks indicate the day when R1 ferrets were influenza positive according to the point-of-care rapid test and moved
away from donor ferrets and housed with the R2 ferret. Retrospective RT-PCR analysis of nasal washes from the R1 ferret from the 20% WT­80%
MUT group and the 0% WT­100% MUT group detected influenza virus 1 to 2 days prior to the date when ferrets were rapid test positive and
housed with the R2 ferret.
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The within-host replication fitness cost for R292K and
H274Y. Figure 5 plots the mutant proportion over time and the
best-fit model curve for each of the donor ferrets in the R292K
experiment (Fig. 5A) and the H274Y experiment (Fig. 5B). A
positive value for , the difference in viral growth rates, indi-
cates that the mutant has a reduced replication fitness. Figure
6 plots the best-fit value for  for each ferret and the model-
fit-derived confidence interval for the estimate. Both the
R292K (Fig. 6A) and H274Y (Fig. 6B) mutants demonstrate
compromised replication fitness in the donor ferrets. The me-
dian replication fitness cost for R292K mutation was 0.36, and
for the H274Y mutation, it was 0.22. The distributions of
replication fitness costs for the two different mutant viruses
were not significantly different (Wilcoxon's rank-sum test, P 
0.24).
Table 1 presents results from a mixed-effects linear regres-
FIG. 4. Infectivity and transmissibility of pure populations and mixtures of H274 WT and H274Y MUT viruses in ferrets. The relative
proportion of viral mixtures is indicated in the panels where the percentage of H274 WT virus is displayed using white bars and that of H274Y
MUT is displayed by black bars. No bars are present for samples when no virus was detected. R1 recipient ferrets were introduced into the same
cage as donor ferrets on day 1. Asterisks indicate the day when R1 ferrets were influenza positive according to the point-of-care rapid test and
moved away from donor ferrets and housed with the R2 ferret. Retrospective RT-PCR analysis of nasal washes from the R1 ferret from the 100%
H274 WT­0% H274Y MUT group detected influenza 1 day prior to the date when ferrets were rapid test positive and housed with the R2 ferret.
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sion of the transformed data: ln {[1  p(t)}/gp(t)}  t. We
allow for a ferret- and strain-independent replication fitness
cost, an additional replication fitness cost if the mutant is
R292K (292), and a random effect for each ferret in the
estimation. That is, we concurrently fit, over all ferrets, i, the
model i  (  i292  i)t  i(t), where i is 0 for H274Y
ferrets and 1 for R292K ferrets, i has a mean of 0, and i(t) is
the ferret-specific time-dependent error. We see evidence for a
reduced replication fitness for all mutant viruses (  0.25
[0.01, 0.50]; P  0.056). Although the point estimate for the
replication fitness cost is approximately double for the R292K
mutant compared to the H274Y mutant (  292  0.25 
0.26 compared to   0.25), we do not have sufficient statistical
power to definitively claim an increased replication fitness cost
FIG. 5. The mutant proportion over time and the nonlinear model fit for each donor ferret. The replication fitness cost, , for the R292K
mutant (A) and H274Y mutant (B) is estimated for each ferret separately. The initial proportion is used to fix g in the model for each ferret.
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for the R292K mutant compared to the H274Y mutant. The
effect of individual ferrets is substantial, as can be seen by the
estimates for the variance of the random effect, i. If we re-
strict the analysis to only the 100% MUT­0% WT infections,
then we do find a statistically significant effect for the mutant
type, in which the R292K mutation clearly has a greater rep-
lication fitness cost than the H274Y mutation (results not
shown, but compare the lower right panels in Fig. 5A and B).
The transmission fitness for R292K and H274Y. Figures 3
and 4 show a clear difference in the abilities of the R292K
MUT and H272Y MUT viruses to transmit between ferrets.
On no occasion was an R292K mutant transmitted (Fig. 3),
even when the mutant dominated infection at the time of
transmission (e.g., see Fig. 3, 20% WT­80% MUT donors,
infection of R1 on day 2). In contrast, we see transmission of
the H274Y mutant in all but one of the eight possible trans-
mission events. Figure 7 summarizes the transmission obser-
vations presented in Fig. 3 and 4, plotting the infectee's mutant
proportion as a function of the infector's mutant proportion.
The H274Y data are consistent with a linear relationship with
slope 1; that is, there is no evidence for a transmission fitness
cost or advantage for the H274Y mutant, unlike the R292K
mutant which did not transmit, as illustrated by the data points
for that experiment lying on the y  0 axis in Fig. 7.
The scatter about the line y  x for the H274Y strains in Fig.
7 indicates that the transmitted proportion, while qualitatively
predicted by the infector's proportion, is subject to significant
stochastic (random) variation. In particular, no mutant virus
FIG. 6. Summary of the within-host replication fitness of the mu-
tant viruses in donor ferrets. Shown is a summary of the fitness cost, ,
for each donor ferret, for the R292K mutant (A) and H274Y mutant
(B). The error bars indicate the 95% confidence interval for  for each
ferret, based on the asymptotic normal distribution for the parameter
estimate.
TABLE 1. Results from the mixed-effects linear regression on the
transformeda
proportions data
Predictor Coefficient 95% CIb
P value
 0.248 (0.007, 0.503) 0.056
292 0.260 (0.095, 0.616) 0.152
Random-effects
parameter
SD estimate 95% CI on SD
i
c
0.335 (0.212, 0.528)
Residual 0.900 (0.770, 1.054)
a
See Materials and Methods for details.
b
95% CI, 95% confidence interval.
c
The random effect, i, with a mean of zero and estimated standard deviation,
allows for per-ferret random variation.
FIG. 7. Summary of the transmission fitness of mutant viruses. The
abscissa shows the mutant proportion of the infecting ferret's viral load
on the day preceding confirmed transmission. The ordinate shows the
mutant proportion of the infected ferret's viral load within the first
24 h postinfection. For donor (D1 or D2)-to-R1 ferret transmission
events, we take the average proportion in the two donors. Open circles
(E) are the donor-to-R1 transmission events from the R292K experi-
ment. Open squares () are the donor-to-R1 transmission events, and
open triangles () are the R1-to-R2 transmission events in the H274Y
experiment. Note that there are three points (1 circle, 1 square, and 1
triangle) at coordinates 0, 0. The dotted line shows the relationship y 
x, a visual guide to help demonstrate the qualitative proportional-
transmission relationship for the H274Y data.
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was detected in the R2 recipient ferret following infection of
donor ferrets with a mixture containing 20% H274Y MUT
virus [Fig. 4 and 7, triangle at (x, y)  (0.1, 0)]. A mixed-
infection transmission chain has collapsed to a pure infection
chain by chance, suggesting that the mutant may be competi-
tively excluded from the ferret population over an extended
number of generations. In contrast when donor ferrets were
infected with a mixture containing either 50% or 80% H274Y
MUT virus, the resistant virus remained present in the mix-
tures through to the R2 recipient ferret. For these two mixtures
(Fig. 4 and Fig. 7), the two donor-to-R1 recipient transmission
events show fluctuations in the proportion in opposite direc-
tions (60% down to 33% and 35% up to 68%). Potential
sources for this variation in transmitted proportion include
inherent stochastic variation in the transmitted inoculum
and/or animal host variability.
DISCUSSION
The NAIs are an important class of antiviral drugs for the
treatment and prevention of influenza virus infection. How-
ever, as is the case for many antiviral drugs, the emergence of
resistant variants is a major concern. To accurately assess the
risk that a resistant virus will emerge and spread throughout
the community, it is important to determine the relative viral
fitness of a strain. Previous studies have assessed viral fitness of
NAI-resistant viruses using both in vitro and in vivo models
(12) and have found that many NAI-resistant variants have
compromised fitness, with impaired growth in vitro and re-
duced infectivity and transmissibility in animal experiments
(22).
In vitro studies that determine the rate of viral replication
and viral titers over time are relatively easy to perform, but
previous studies suggest that these data do not accurately re-
flect the replication fitness of a strain in an animal model (7,
13, 29, 30, 36). This could be because in vitro replication ki-
netics experiments may not be impacted by the defective NA
enzyme activity of mutant viruses as there are fewer host an-
tiviral mechanisms present in cell lines (19). In the present
study, the MDCK and MDCK-SIAT1 single-step experiments
detected a significant difference between the growth of the
R292 WT and R292K MUT viruses, but no difference was seen
in the multiple-replication-cycle experiments. In contrast, the
in vitro growth characteristics of the H274 WT and the H274Y
MUT viruses were identical in both cell lines in single-step and
multiple-replication assays.
Ferrets, in contrast to mice, have a type and distribution of
human influenza receptors (-2,6-glycosidic linkages) in their
respiratory tracts similar to those in humans, and as such the
ferret model is considered to be one of the best animal models
to assess human influenza virus growth and transmissibility
(28). Previous studies have assessed viral fitness of NAI-resis-
tant viruses in a ferret model by infecting animals with a pure
population of either a wild-type or mutant virus and comparing
the minimum dose required to obtain an infection, the dura-
tion of infection, and how readily the viruses will transmit to
other naïve ferrets. In this study, a novel method was used to
assess viral replication and transmission fitness, in which fer-
rets were infected with either pure populations or various
mixed populations of wild-type and mutant viruses. The result-
ant viral proportion of wild-type and mutant virus present in
nasal washes in each animal was determined every 1 or 2 days
during infection of both donor and recipient ferrets. A com-
petitive-mixtures model such as this should mean that if one
strain replicates at a faster rate than the other (i.e., it has an
increased replication fitness), a larger number of progeny vi-
ruses will be produced and therefore infect a higher proportion
of the finite number of cells available for infection prior to
immune clearance, resulting in one strain outgrowing the other
over time. Furthermore, we have used a simple mathematical
model of the replication kinetics to capture this outgrowth
behavior and then calculated (model-based) estimates of the
replication fitness cost using a mixed-effects statistical model.
The competitive-mixtures model was able to clearly identify
the compromised within-host replication fitness of both the
R292K and H274Y mutant viruses. Within the animals in-
fected with 100% R292K MUT virus, mutation during viral
replication resulted in R292 WT viruses rather than R292K
MUT strains being selected (due to the greater replication
fitness of the wild-type virus compared to the mutant strain),
which led to the dominance of the wild-type virus in the pop-
ulation. Because the transmission fitness of the R292K mutant
was so low (our point estimate is that it is zero), only after the
wild-type had begun to outgrow the mutant did we see trans-
mission to recipient ferrets (e.g., in Fig. 3, 0% for R292 WT
versus 100% for R292K MUT). Previous studies have also
demonstrated the compromised transmission fitness of the
R292K mutant (9), and as such for this type of mutant, there
may be less need to conduct the competitive-mixtures experi-
ment compared to using the standard ferret experiments that
involve infection with only the pure viral populations. How-
ever, the competitive-mixtures model does appear to be a more
powerful model when comparing two viruses that have similar
within-host replication or transmission fitness. When ferrets
were infected with pure populations of either H274 WT or
H274Y MUT virus, both viruses were infectious in the donors,
and the mutant demonstrated no detectable replication fitness
cost (Fig. 5B, lower right panel). However, replication fitness
differences did appear to be present when analyzing the pro-
portions of the mutant and wild-type viruses in the ferrets
infected with the mixtures (Fig. 6 and Table 1), suggesting that
the H274Y MUT, like the R292K MUT virus, had a lower
replication fitness cost than the wild type within the host. Our
inability to identify a difference in replication fitness cost ()
between R292K and H274Y may be attributable to a number
of factors but is primarily due to a lack of statistical power.
The oseltamivir-resistant H274Y MUT virus used in this
study is representative of the viruses that became the predom-
inant circulating seasonal A(H1N1) strain during 2007 and
2008. The increased frequency of the H274Y mutant A(H1N1)
viruses in the human population, compared to the H274 WT
A(H1N1) viruses (11), suggests that these strains have a fitness
level equal to or greater than that of the circulating wild type.
In this study, we identified the mutant strain to have similar
transmission fitness compared to the wild type but slightly
reduced within-host replication fitness. These differences may
be due to the limitations in the ferret model such as the
relatively small numbers of ferrets that are able to be used in
each experiment and animal-to-animal variability as, unlike
mice, the ferrets used in these experiments are derived from an
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outbred population. However, a repeat of the H274Y compet-
itive-mixtures experiment using the same strains generated
results similar to those presented here (data not shown). An-
other limitation of this study and other similar studies is that
particular wild-type and mutant strains are selected as being
representative of their respective phenotypes/genotypes, and
the potential exists that the choice of other representative
strains may have produced slightly different results from those
observed. As described previously, fitness differences between
virus strains with the same resistance mutations have made in
vivo comparisons of NAI-resistant mutant viruses difficult (36).
To address this, future studies could use reverse genetics and
site-directed mutagenesis to generate mutant and wild-type
viruses that have an identical genome other than a single mu-
tation in the NA gene that confers resistance. However, this
approach also has its limitations, as it precludes the effects of
compensatory mutations in other genes that may have oc-
curred through natural evolution and may be necessary for
maintaining viral fitness in humans.
The coinfection of ferrets with two different viruses as used
in this study raises the possibility of gene segment reassortment
leading to the generation of progeny viruses with genes derived
from both of the parent strains. Previous studies, however,
suggest this outcome is relatively rare. In ferrets coinfected
with pandemic A(H1N1) 2009 and seasonal influenza A vi-
ruses, no reassortant viruses were detected (25), while a dif-
ferent study investigating coinfection of ferrets with A(H5N1)
and A(H3N2) strains found that only 9% of recovered viruses
contained genes from both parent viruses (15). Due to the high
genetic similarity between the wild-type and mutant viruses
used in the present study (the H274 WT and H274Y MUT
strains differed by only nine amino acid differences across the
entire genome), it is likely that any reassortants would not
display a significant fitness difference from the parent strains,
particularly given that none of the residue differences between
the wild-type and mutant strains were located in sites known to
affect growth, transmissibility, or virulence. The high genetic
similarity between the progenitor strains also means that de-
tection of reassortants is considerably more difficult than when
analyzing progeny viruses from ferrets coinfected with two
completely different strains [e.g., A(H5N1) and A(H3N2)]. It is
also important to note that the degree of reassortment has no
influence on the modeling component of this study. Mathe-
matical models used to make assessments of antiviral deploy-
ment policy require information only on whether a strain is
resistant or sensitive and the relative transmission fitness of
that resistant mutant (compared to the wild-type sensitive
strain) and do not require detailed genetic characterization
(18, 21, 35).
This study has produced estimates for the replication fitness
cost () that will be used to determine the rate at which
resistant-strain infections revert to sensitive-strain infections in
mathematical models of influenza spread in human popula-
tions. Modeling studies to date have not allowed for this re-
version process due to a lack of data. Furthermore, these
models, while including analyses with a relative transmission
fitness equal to 1, have also made inferences based on an
assumed reduced transmission ability for the resistant strain.
Figure 7 demonstrates that the H274Y MUT strain likely has
a transmissibility equal to that of the wild type. Outcomes from
existing models should be reevaluated in light of the findings
presented here.
The replication and transmission fitness of NAI-resistant
influenza A viruses was investigated in this study using a novel
competitive-virus-mixtures ferret model, which enables the be-
havior of a mixed-virus population, comprising mutant and
wild-type viruses, to be monitored during the course of infec-
tion and over several transmission events. This method appears
to provide greater insight into the relative fitness of two dif-
ferent strains, compared to more traditional methods that in-
fect ferrets with only pure populations of sensitive or resistant
viruses. Given the importance of oseltamivir as the currently
most prescribed NAI for the control of influenza infections, it
is critical that the fitness of the H274Y variant and other
NAI-resistant viruses be further investigated to assess their
potential to spread widely throughout the community. For
example, since the emergence of the A(H1N1) pandemic in
2009, over 200 A(H1N1) pandemic strains have been shown to
contain the H274Y mutation conferring oseltamivir resistance
(34). Although there has been some evidence of transmission
of these resistant strains, at this stage it appears to occur only
between close contacts (8, 17). It would be of significant value
to test these viruses for transmission efficiency in models such
as the one described here. The results from this preliminary
study suggest that the competitive-mixtures ferret model is a
valuable tool to compare the relative fitness of two influenza
virus strains and can provide real insights into the infectivity
and transmissibility of NAI-resistant strains.
ACKNOWLEDGMENT
The Melbourne WHO Collaborating Centre for Reference and Re-
search on Influenza is supported by the Australian Government De-
partment of Health and Ageing.
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