Identification, Characterization, and
Natural Selection of Mutations Driving
Airborne Transmission of A/H5N1 Virus
Martin Linster,1,3Sander van Boheemen,1,3Miranda de Graaf,1Eefje J.A. Schrauwen,1Pascal Lexmond,1
Benjamin Ma ¨nz,1Theo M. Bestebroer,1Jan Baumann,2Debby van Riel,1Guus F. Rimmelzwaan,1
Albert D.M.E. Osterhaus,1Mikhail Matrosovich,2Ron A.M. Fouchier,1,* and Sander Herfst1
1Department of Viroscience, Postgraduate School of Molecular Medicine, Erasmus Medical Center, 3015GE Rotterdam, the Netherlands
2Institute of Virology, Philipps-University, 35043 Marburg, Germany
Recently, A/H5N1 influenza viruses were shown to
acquire airborne transmissibility between ferrets
upon targeted mutagenesis and virus passage. The
critical genetic changes in airborne A/Indonesia/5/
05 were not yet identified. Here, five substitutions
proved to be sufficient to determine this airborne
transmission phenotype. Substitutions in PB1 and
PB2 collectively caused enhanced transcription and
virus replication. One substitution increasedHA ther-
mostability and lowered the pH of membrane fusion.
Two substitutions independently changed HA bind-
ing preference from a2,3-linked to a2,6-linked sialic
acid receptors. The loss of a glycosylation site in
HA enhanced overall binding to receptors. The
acquired substitutions emerged early during ferret
passage as minor variants and became dominant
rapidly. Identification of substitutions that are essen-
tial for airborne transmission of avian influenza
viruses between ferrets and their associated pheno-
types advances our fundamental understanding of
virus transmission and will increase the value of
future surveillance programs and public health risk
Since the first detection in the late 1990s (Claas et al., 1998),
highly pathogenic avian influenza (HPAI) A/H5N1 viruses
continue to circulate in poultry in Asia and the Middle East. Hun-
dreds of millions of domestic birds have died as a result of infec-
tion and during culling activities to control the spread of the
A/H5N1 virus. Occasional cross-species transmission events
have been reported for several species of wild birds, pigs, felids,
A/H5N1 virus infection in humans have been reported to the
World Health Organization from 16 countries, of which ?60%
had fatal outcome. Sustained human-to-human transmission
has not yet been described. However, the enzootic nature of
A/H5N1 virus, its broad host range, the large number of infected
hosts, and the observed accumulation of mammalian adaptive
substitutions in the virus could potentially increase the risk of a
future A/H5N1 virus pandemic.
For 15 years, one of the key questions for pandemic pre-
paredness has been whether the A/H5N1 virus might acquire
the ability to transmit via aerosols or respiratory droplets
(‘‘airborne transmission’’) among humans, a trait necessary for
the virus to become pandemic. It was recently shown that a fully
avian A/H5N1 virus can become airborne transmissible be-
tween ferrets (Herfst et al., 2012). Three other groups demon-
strated that reassortant viruses between A/H5N1 and 2009
pandemic A/H1N1 viruses that contain the H5 hemagglutinin
(HA) were also transmitted between ferrets or guinea pigs via
the airborne route (Chen et al., 2012; Imai et al., 2012; Zhang
et al., 2013b).
Herfst et al. (2012) introduced the well-known glutamic acid to
lysine substitution at position 627 (E627K) of the basic polymer-
ase 2 protein (PB2) that is associated with increased replication
in mammalian cells at relatively low temperatures (Aggarwal
et al., 2011; Subbarao et al., 1993; Taubenberger et al., 2005)
and was shown to be important for airborne transmission of
1918 A/H1N1 and 1999 A/H3N2 viruses between ferrets and
guinea pigs (Steel et al., 2009; Van Hoeven et al., 2009). In addi-
tion, two substitutions were introduced in the receptor binding
site (RBS) of HA that are known to switch receptor specificity
from ‘‘avian’’ a2,3-linked sialic acids (a2,3-SA) to ‘‘human’’
a2,6-SA (Matrosovich et al., 2000), glutamine to leucine at posi-
tion 222, and glycine to serine at position 224 (Q222L, G224S in
H5 HA numbering). These three substitutions or other polymer-
ase and RBS mutations with similar phenotypes were found in
all pandemic influenza viruses of the last century and were there-
of animal influenza viruses to humans to yield pandemic strains
(Chutinimitkul et al., 2010a; Sorrell et al., 2011). This ‘‘triple
mutant’’ virus was passaged ten times in the upper respiratory
tract of ferrets to yield mutant A/H5N1 viruses that were able
Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc. 329
to transmit via the airborne route between ferrets. In addition to
the three substitutions introduced by reverse genetics, two sub-
stitutions in HA (H103Y and T156A) were consistently found in all
transmitted A/H5N1 viruses. However, all airborne-transmitted
viruses had accumulated additional substitutions. The transmis-
sible virus with the lowest number of amino acid substitutions
compared to the A/H5N1 wild-type virus had a total of nine
Here, we describe the identification of a minimal set of substi-
tutions required for airborne transmission of influenza virus
terization of the phenotypic changes caused by each of these
substitutions. We show that the substitutions acquired upon
ferret passage of the triple mutant A/Indonesia/5/05 virus
emerged rapidly, which is suggestive for strong natural selec-
tion. The identification of previously unrecognized substitutions
is key to increasing our fundamental understanding of airborne
spread of influenza virus and may ultimately increase prognostic
capabilities and diagnostic value of surveillance studies neces-
sary for pandemic preparedness.
Airborne Transmission ofA/H5N1VirusbetweenFerrets
Is Determined by a Minimum of Five Amino Acid
To define a minimal number of substitutions in A/Indonesia/5/05
that confer airborne transmission between ferrets, transmission
experiments were performed as described previously (Munster
et al., 2009). First, a recombinant virus was produced based on
the consensus sequence of a previously identified virus that
was airborne transmissible and that contained the lowest num-
ber of substitutions (n = 9) compared to the wild-type virus
(PB2-E627K, PB1-H99Y, and PB1-I368V; HA-H103Y, HA-
T156A, HA-Q222L, and HA-G224S; and NP-R99K and NP-
S345N; Herfst et al., 2012). This virus was transmitted to two
out of two recipient ferrets, thus reproducing with a recombinant
clonal virus our earlier results with a virus isolate (Figure 1A, V1).
Next, we omitted either the two substitutions in nucleoprotein
(NP) (V2) or the two substitutions in PB1 (V3). Whereas the re-
combinant virus missing two substitutions in NP was transmitted
to two out of two animals, the virus missing two substitutions in
PB1 was not transmitted (Figure 1A, V2 and V3). To investigate
this further, the two substitutions in PB1 (H99Y and I368V)
were tested individually. The virus harboring I368V in addition
to the set of five substitutions consistently found in the airborne
transmitted viruses (PB2-E627K, HA-H103Y, HA-T156A, HA-
Q222L, HA-G224S) was not transmitted to recipient ferrets
(Figure 1A, V4), whereas the addition of PB1-H99Y yielded a re-
combinant virus that was detected in three out of four exposed
ferrets (Figure 1A, V5).
Starting with virus V5 that had six substitutions compared
to wild-type A/Indonesia/5/05 (HA-Q222L, HA-G224S, HA-
H103Y, HA-T156A, PB2-E627K, and PB1-H99Y), all individual
substitutions were omitted one by one (V7–V12). Viruses lack-
ing either the receptor-binding substitution HA-G224S or
HA-Q222L were detected in one and two out of four naive fer-
rets, respectively, upon exposure to inoculated ferrets (Fig-
ure 1B, V7 and V8). In contrast, when both HA-Q222L and
HA-G224S were omitted, the virus was not transmitted be-
tween ferrets (Figure 1B, V6). Viruses lacking HA-T156A, HA-
H103Y, PB1-H99Y, or PB2-E627K were not detected in
recipient ferrets upon exposure either (Figure 1B, V9–V12).
From this set of experiments, we conclude that PB2-E627K,
PB1-H99Y, HA-H103Y, HA-T156A, and either HA-Q222L or
HA-G224S in HA represent minimal sets of substitutions
required for airborne transmission of A/Indonesia/5/05 between
ferrets. Individual virus titers of nose and throat samples ob-
tained from all donor-recipient pairs are shown in Figure S1
Rapid Emergence of Substitutions Required for
Airborne Transmission of A/H5N1 in Ferrets
and H103Y and T156A in HA emerged during the ten repeated
Figure 1. Summary of Results to Determine
a Minimal Set of Substitutions Required for
Airborne A/H5N1 Virus Transmission be-
(A) Starting with virus V1 that represents the
airborne-transmitted virus with the lowest number
of amino acid substitutions (nine) as compared to
wild-type A/H5N1 (Herfst et al., 2012), the
requirement of substitutions in the PB1 and NP
segments was investigated. Recombinant viruses
V1-V5 are shown with eight gene segments, in
which colored squares represent the presence or
absence ofindicated substitutions.The proportion
of animals positive by virus isolation is indicated
(B) All substitutions of virus V5 were omitted indi-
vidually, and transmission was again tested in
ferrets for viruses V6–V12. Virus-shedding pat-
terns in donor and recipient ferrets for V1–V12 are
provided in Figure S1.
330 Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc.
passages of the triple mutant A/Indonesia/5/05. To this end,
primers were designed to amplify virus genome fragments
covering these amino acid positions, and RT-PCR was per-
formed on RNA isolated from nasal turbinates, lungs, and nasal
swabs or washes collected from the ferrets after each passage.
Amplicons were sequenced using the Roche 454 GS Junior
PB1-99Y was already detected as a minor variant in the
nasal turbinates in passage 1 and increased during subse-
quent passages to become the dominant variant in all tis-
sues from passage 7 onward (Figure 2). HA-103Y was first
detected in the nasal swabs of passage 2, was detected again
in passages 5 and 6, and became the major variant from
passage 7 onward. Of note, nasal washes instead of nasal
turbinates were used to inoculate the ferrets from passage
6 onward, which may have contributed to rapid increase in
the proportion of mutants containing HA-103Y after passage
7 (and perhaps also PB1-99Y). HA-156A was detected as a
minor variant in passage 1 to rapidly become the dominant
variant from passage 3 onward. Thus, the three substitu-
tions that emerged during ferret passage and that con-
tributed to aerosol transmissibility of A/H5N1 virus arose as
early as after one or two passages and became dominant by
Nucleotide Variations in the Full Genomes of
A/H5N1wild-typeand A/H5N1HA Q222L, G224S PB2 E627K
during Ferret Passaging and Transmission
We next compared the full genome sequences of viruses
described in Herfst et al. (2012) present in passage 4, 8, and
10 nasal turbinates of ferrets inoculated with A/H5N1wild-typeor
A/H5N1HA Q222L, G224S PB2 E627K (Table S1). These samples
were selected because they contained high copy numbers of
viral RNA as determined by real-time PCR. Substitutions PB2-
T23P, PA-T363P, PA-P211T, PA-L498F, NP-G462E, and NA-
E239G emerged upon passage with both A/H5N1wild-typeand
A/H5N1HA Q222L, G224S PB2 E627Kbut did not become dominant
variants. In contrast, T156A became a major variant upon
passage of both viruses. Thus, whereas substitutions H99Y
in PB1 and H103Y in HA emerged only upon passage of
A/H5N1HA Q222L, G224S PB2 E627K, HA-T156A emergence was
independent of PB2-E627K,
which were introduced by reverse genetics. The latter three
substitutions remained dominant (>96.2%) throughout all
passages tested and were not detected throughout passage of
To study the effect of airborne transmission on the viral intra-
host nucleotide sequence variation, we compared the full virus
genome sequences present in nasal wash samples of ferrets
Figure 2. Single-Nucleotide Variations that Emerged upon Repeated Passaging of Influenza Virus A/H5N1HA Q222L, G224S PB2 E627Kin Ferrets,
as Detected by Deep Sequencing
Passage number is indicated on the x axis. Left and right y axes indicated proportion of the mutant among all reads (white bars) and the sequencing depth in
number of reads (black lines), respectively. The gray areas indicate that no virus sequences were amplified from these samples. S, virus stock used to inoculate
the first ferret (P1); NT, nasal turbinate; NS, nasal swab; NW, nasal wash.
Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc. 331
inoculated with A/H5N1HA Q222L, G224S PB2 E627Kafter ten pas-
sages in ferrets to nose swab samples of ferrets after two
consecutive airborne transmission events (ferrets F5 and F6,
Table S2). The number of nucleotide variations present in spec-
imens from F5 (n = 12) and F6 (n = 16) was substantially less as
Moreover, substitution PB1-99Y increased in frequency from
44.9% in passage 10 nasal wash to 100% and 73.3% in F5
and F6 after the two consecutive airborne transmission events.
Similarly, HA-103Y increased from 87.6% to 100% and 100%,
and HA-156A increased from 89.9% to 99% and 98.8%. These
collective data are indicative of a strong selection bottleneck
occurring on intrahost nucleotide sequence variation.
HA Substitutions Q222L, G224S, and T156A Affect
To study the impact of the HA substitutions associated with
airborne transmission on receptor preference, the attachment
patterns of A/Puerto Rico/8/1934 (PR8) viruses harboring wild-
type or mutant A/H5N1 HA proteins were first characterized
using formalin-fixed paraffin-embedded tissue sections of ferret
and human nasal turbinates, known to express ‘‘human-like’’
a2,6-SA receptors abundantly. PR8 virus with the wild-type H5
HA did not attach to nasal turbinate sections, whereas the
same virus with a control human H3 HA showed abundant
attachment, as expected (Figure 3A). Introduction of Q222L
and G224S in wild-type H5 HA resulted in abundant virus attach-
ment to the nasal turbinate sections, comparable to the H3 HA
control, as shown previously (Chutinimitkul et al., 2010a). Intro-
duction of H103Y and T156A in either the wild-type H5 HA or
HAQ222L, G224Sdid not result in obvious changes in these pat-
terns of virus attachment. The attachment patterns of HAs to
the ferret and human nasal turbinate tissue sections were
We used two solid-phase enzyme-linked receptor-binding as-
says to determine HA binding of virus immobilized on a 96-well
Figure 3. Receptor Binding Properties of Wild-Type and Mutant A/H5N1 HA Proteins
(A) Attachment patterns of viruses expressing wild-type or mutant H5 HA to tissue sections of ferret and human nasal turbinates. Red color represents binding of
influenza viruses. Images were chosen to reflect representative attachment patterns.
(B) Direct binding of viruses expressing wild-type or mutant H5 HA to fetuin containing either a2,3-SA (red bars) or a2,6-SA (blue bars). A/dk/Bav/1/77 and A/HK/
1/68represent avianandhumanprototypestrainsA/duck/Bavaria/1/1977(A/H1N1)andA/HongKong/1/1968(A/H3N2),respectively.Errorbarsrepresent theSD
of the mean values (n = 2). See also Figure S2.
(C) Agglutination of TRBCs by viruses expressing wild-type or mutant H5 HA. TRBCs were left untreated, stripped from SA using Vibrio cholerae neuraminidase
(VCNA), or modified to contain either a2,3-SA or a2,6-SA. Numbers show the HA titers determined with the indicated TRBCs using various mutant viruses.
A/Netherlands/213/03 served as a typical human virus with a2,6 SA preference.
332 Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc.
binding inhibition assay (Matrosovich and Gambaryan, 2012).
PR8 viruses that harbor wild-type or mutant H5 HAs were
tested in direct binding assays with receptor analogs 30-fetuin
and 60-fetuin (Figure 3B). Introduction of the Q222L and G224S
in HAwild-typeresulted in a switch in receptor binding specificity
from a2,3-SA to a2,6-SA as expected, with no residual a2,3-
SA binding. Introduction of H103Y and T156A in HAQ222L, G224S
resulted in increased binding to a2,6-SA receptors but also
low binding to a2,3-SA. Introduction of H103Y and T156A in
ing to a2,6-SA. Thus, in both the context of HAwild-type and
HAQ222L, G224S, substitutions H103Y and T156A resulted in
increased binding avidity and limited dual receptor specificity.
A direct binding assay performed with 30-sialyl-N-acetyllactos-
amine- and 60-sialyl-N-acetyllactosamine-containing synthetic
sialylglycopolymers rather than 30-fetuin and 60-fetuin yielded
similar results (Figure S2A). In a fetuin-binding inhibition assay,
we compared binding of the viruses to a panel of sialylglycopol-
ymerscontaining several different sialyloligosaccharide moieties
(Figure S2B). In this assay, the wild-type and mutant H5 viruses
in general bound less avidly as compared to the control avian
H1N1 virus A/Duck/Bavaria/1/1977 and human H3N2 virus
ger to various a2,3-SA analogs than to a2,6-SA, whereas
HAQ222L, G224Sand HAQ222L, G224S, H103Y, T156Ashowed the oppo-
site binding preference (Figure S2B), thus yielding similar results
as the direct binding assays.
In a third approach, we assessed the binding of PR8 viruses
expressing wild-type or mutant H5 HA in a HA assay using
normal turkey red blood cells (TRBC) that contain both a2,3-
SA and a2,6-SA or modified TRBC that contain either a2,3-SA
or a2,6-SA alone or no SA (Figure 3C). As shown previously,
introduction of Q222L and G224S in H5 HA resulted in a switch
in receptor binding preference from a2,3-SA to a2,6-SA (Chutini-
mitkul et al., 2010a). Additional introduction of the H103Y
and T156A substitutions required for airborne transmission
increased the HA titers to both a2,3-SA and a2,6-SA containing
TRBC. Each of the individual receptor-binding site substitutions
Q222L and G224S in the context of changes H103Y and T156A
displayed binding to a2,6-SA containing TRBC, which is in
agreement with the fact that single RBS substitutions were suf-
ficient for airborne transmission (Figure 1B).
Introduction of H103Y in HAwild-type, HAT156A, HAQ222L, G224S,
and HAT156A, Q222L, G224Sdid not result in consistent changes in
HA titers. In contrast, introduction of T156A in HAwild-type,
HAH103Y, HAQ222L, G224S, and HAH103Y, Q222L, G224Sresulted in
dual receptor specificity as indicated by a consistent R2-fold in-
crease in HA titers, irrespective of the HA used.
Collectively, we conclude from these studies that H103Y had
no discernable effect on receptor binding preference, whereas
T156A increased overall virus binding to both a2,3-SA and
a2,6-SA, thus resulting in dual receptor specificity.
HA Substitution H103Y Affects Acid and Temperature
Upon virus attachment to SA receptors on the cell surface and
internalization into endosomes, a low-pH-triggered conforma-
tional change of HA mediates fusion of the viral and endosomal
membranes to release the virus genome in the cytoplasm (Shaw
and Palese, 2013). We measured the pH threshold required for
fusion of wild-type and mutant HAs. Vero cells were transfected
with HA-expression plasmids and exposed to trypsin to
cleave and activate the HA, followed by acidification of the cell
culture at a pH range of 5.2 to 6.0. Visual inspection of the cell
cultures for the presence of syncytia (multinucleated cells) was
used to determine the pH threshold required for fusion (Fig-
ure 4A). Fusion of HAwild-typewas triggered at pH % 5.6, similar
to the threshold pH of the control H5 HA of A/Hongkong/
156/97 (pH % 5.8). HA of the control human H3N2 virus
A/Netherlands/213/03 required a lower pH (%5.2) for fusion to
occur. These data are in agreement with the observation that
avian influenza virus HAs generally trigger fusion at a higher pH
than human virus HAs (Galloway et al., 2013). The three substitu-
tions that affect receptor binding (T156A, Q222L, G224S; see
above) did not result in a reduction of the threshold pH for fusion
as compared to HAwild-type. In contrast, upon introduction of
H103Y in HAwild-typeand HAT156A, Q222L, G224S, fusion was trig-
gered only at pH 5.2 and lower. Moreover, HAH103Y, T156A, Q222L
and HAH103Y, T156A, G224Salso triggered fusion at relatively low
pH (pH % 5.2 and pH % 5.4, respectively).
As a second readout for fusion, we used a ‘‘cell content mixing
assay,’’ in which two populations of Vero cells were transfected
with HA and either a chloramphenicol-acetyl-transferase
construct under control of the HIV-1 promoter (LTR-CAT) or an
HIV-1 transactivator construct (pTat). Upon mixing of the two
cell populations and after acidification of the cell culture at a
pH range of 5.2 to 6.0, fusion was quantified by measuring
CAT expression that is dependent on the expression of Tat.
Also in this assay, introduction of H103Y in HAwild-type and
HAT156A, Q222L, G224Sresulted in a lower threshold pH for fusion,
as indicated by the dotted lines in Figures 4B and 4C, respec-
tively. The difference in pH required for fusion between HA with
andwithout H103Ywassimilar tothatobservedforthereference
A/H3N2 and A/H5N1 HAs (Figure 4D).
The switch of influenza virus HA from a metastable nonfuso-
genic to a stable fusogenic conformation can also be triggered
at neutral pH when the HA is exposed to increasing temperature.
Thisconformational change of HAis biochemically indistinguish-
able from the change triggered by low pH (Carr et al., 1997) and
results in a loss in the ability to bind receptor. To further investi-
gate HA stability, PR8 viruses harboring wild-type or mutant HAs
were incubated at increasing temperatures, after which the abil-
ity of the viruses to agglutinate TRBCs was quantified. The PR8
virus with the H3 HA of A/Netherlands/213/03 retained HA activ-
ity even upon treatment for 30 min at 60?C. In contrast, PR8 with
H5 HA of A/Indonesia/5/05 lost HA activity upon treatment at
56?C for 30 min. Irrespective of the presence or absence of
substitutions affecting receptor binding (HAwild-type, HAT156A,
HAQ222L, G224S, and HAT156A, Q222L, G224Swere tested), H103Y re-
sulted in increased temperature stability as measured in the HA
assay (Figure 4E).
Collectively, these data indicate that H103Y has a stabilizing
effect on the HA of A/Indonesia/5/05—with respect to both low
pH and high temperature treatment—irrespective of the pres-
ence or absence of substitutions that affect receptor binding.
Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc. 333
PB2-E627K and PB1-H99Y Affect Polymerase Activity
The influenzaviruspolymerase complex—consisting ofthe poly-
merase proteins PA, PB1, and PB2—transcribes the negative
sense viral RNA ((?)vRNA) in mRNA and positive sense copy
newly synthesized (?)vRNA. To read out polymerase complex
function, we used a (?)vRNA reporter construct consisting of
the firefly luciferase open reading frame flanked by the noncod-
ing regions of segment 8 of influenza A virus (de Wit et al., 2010).
Upon cotransfection of the reporter with expression plasmids
encoding PB1, PB2, PA, and NP, the (?)vRNA reporter is tran-
scribed and the firefly luciferase protein is expressed. A plasmid
that constitutively expresses Renilla luciferase was cotrans-
fected as an internal control to standardize transfection effi-
ciency and sample processing. Substitution E627K in PB2
resulted in a 12-fold increase in firefly luciferase expression as
compared to the wild-type PB2. Substitution H99Y in PB1 re-
sulted in a 3-fold increase. The polymerase complex with both
PB2-E627K and PB1-H99Y yielded a 25-fold increase in firefly
luciferase expression as compared to the polymerase complex
of the wild-type virus (Figure 5A).
To study the levels of transcription of (?)vRNA, (+)cRNA, and
extension assays using total cellular RNA isolated upon virus
inoculation of MDCK cells. Three hours after inoculation with
A/H5N1wild-type, all threeRNA species were detected (Figure 5B).
Introduction of E627K in PB2 resulted in elevated levels of the
viral RNAs, predominantly increasing the amount of (?)vRNA
and (+)cRNA, while marginally changing mRNA. Substitution
H99Y in PB1 resulted in slightly reduced levels of all detected
RNA species as compared with A/H5N1wild-type. Upon inocula-
tion with a virus containing both PB2-E627K and PB1-H99Y,
Figure 4. Analysis of pH Threshold for Fusion and Thermostability of Wild-Type and Mutant A/H5N1 HA Proteins
(A) Syncytium formation in MDCK cells upon expression of wild-type or mutant HA proteins after exposure to different pH. The red line marks the range of pH
values at which fusion was detected microscopically. HA of A/HongKong/156/97 (H5N1) and A/Netherlands/213/03 (H3N2) were included as typical avian and
human control viruses.
(B–D) Quantification of fusion as measured by the expression of a CAT reporter gene in a cell content mixing assay for (B) influenza virus A/Indonesia/5/05
HAwild-type(solid line) and HAH103Y(dotted line); (C) HAT156A, Q222L, G224S(solid line) and HAH103Y, T156A, Q222L, G224S(dotted line); and (D) A/HongKong/156/97
(A/H5N1, solid line) and A/Netherlands/213/03 (A/H3N2, dotted line). Arrows indicate the pH threshold value at which syncytia were detected visually in (A).
(E) HA protein stability as measured by the ability of viruses to agglutinate TRBCs after incubation at indicated temperatures for 30 min. Colors indicate the HA
titers upon treatment at various temperatures for 30 min as shown in the legend.
334 Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc.
the ratio between (?)vRNA, (+)cRNA, and mRNA was similar as
observed for A/H5N1wild-typebut with higher overall levels of all
three RNA species.
As a third test of polymerase function, we studied in vitro repli-
cation of the recombinant A/H5N1 viruses with and without
polymerase substitutions by measuring plaque sizes upon
inoculation of MDCK cells. At 48 hr after inoculation, cells were
fixed and stained with an anti-NP antibody, and the number of
pixels representing ?100 to 500 plaques on digital images was
quantified. As compared to A/H5N1wild-type, A/H5N1PB2 E627K
and A/H5N1PB1 H99Y displayed a slight reduction in plaque
(A/H5N1PB2 E627K, PB1 H99Y) yielded plaques that were signifi-
cantly larger than those observed for A/H5N1wild-type(Figure 5C).
From these assays, we conclude that PB2-E627K and PB1-
H99Y collectively resulted in increased levels of (?)vRNA,
(+)cRNA, and mRNA transcription and increased virus replica-
tion in MDCK cells.
Here, we show that, of the nine substitutions observed in an
airborne transmissible A/H5N1 virus (Herfst et al., 2012), two
alternative sets of five mutations are identified (E627K in PB2;
H99Y in PB1; H103Y, T156A, and either Q222L or G224S in
HA), either of which is sufficient to confer ferret transmissibility
on A/Indonesia/5/05. Keeping in mind that the design of this
ferret transmission model is qualitative rather than quantitative
and that such studies need to take the principles of replace-
ment, reduction, and refinement in animal experiments into
account (Belser et al., 2013a; Russell and Burch, 1959), it
should be noted that the present study is limited by the number
of animals that were used and was purposely designed to define
a minimal set of substitutions rather than the definitive minimal
set of substitutions required for airborne transmission in ferrets.
Starting with the recombinant A/H5N1 virus harboring nine sub-
stitutions, omission of two substitutions in NP still yielded virus
transmission to five out of six airborne-exposed ferrets (V2 and
V5). Subsequently, a virus lacking PB1-H99Y (V4) was not de-
tected by virus isolation and serology in two out of two
airborne-exposed ferrets, whereas a virus lacking PB1-I386V
was detected by virus isolation in three out of four airborne-
exposed ferrets, thus providing evidence that H99Y and not
I386V was required for airborne transmission. In the subsequent
experiment, all individual substitutions from the remaining set of
six (PB2-E627K, PB1-H99Y, HA-H103Y, HA-T156A, HA-Q222L,
and HA-G224S; Figure 1B) were eliminated one by one. Viruses
lacking either HA-Q222L or HA-G224S still resulted in airborne
transmission in two and one of four ferrets tested, indicating
that a single receptor-binding site substitution was sufficient
for transmission. In contrast, in transmission experiments using
viruses from which each of the other single substitutions were
eliminated, virus was not detected in exposed ferrets. However,
some of these exposed animals seroconverted despite a lack of
virus detection. Apparently, although transmission may occur
for some viruses with five substitutions as measured by
serology or single time points of virus detection (Figure S1),
the viruses were insufficiently replication competent to cause
robust infection and seroconversion consistently in the exposed
ferrets. As a consequence, refining the minimal set of substitu-
tions required for airborne transmission would require substan-
tial numbers of ferret pairs, given the statistical considerations
for this type of experiment (Belser et al., 2013a; Nishiura et al.,
Figure 5. Effect of H99Y in PB1 and E627K in PB2 on Polymerase Activity and Virus Replication
(A) Minigenome reporter assay. Plasmids encoding PB2, PB1, PA, and NP were cotransfected with a vRNA reporter encoding firefly luciferase. Luminescence of
firefly luciferase was standardized using a plasmid constitutively expressing Renilla luciferase. Results are calculated as relative light units (firefly luciferase/
Renilla luciferase) and plotted as fold increase over wild-type. Error bars indicate the SD from the average of two independent experiments.
(B) Primer extension assay. MDCK cells were inoculated at an MOI of 1 with wild-type or mutant A/Indonesia/5/05 viruses. At 3 hr postinoculation, cells were
lysed, and viral RNA levels were determined by primer extension analysis using primers specific for mRNA, cRNA, or vRNA of PB1. Determination of the 5S RNA
levels served as an internal loading control.
(C) Plaque assay. MDCK cells were inoculated with A/Indonesia/5/05 viruses containing the indicated substitutions. After 48 hr, plaque formation was visualized
by influenza NP-specific staining. Digital images were analyzed using ImageQuant TL software to determine plaque size. The surface of individual plaques in
panels, the mutations in PB1 and PB2 are indicated, with dashes representing the wild-type sequences.
Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc. 335
The substitutions required for airborne transmission in ferrets
were determined in in vitro assays to either affect HA binding
to receptors, HA stability, or activity of the polymerase complex.
Two substitutions introduced by reverse genetics (Q222L and
G224S) are known to change the receptor binding preference
of the H5 HA from avian-like a2,3-SA to human-like a2,6-SA
(Chutinimitkul et al., 2010a). This change in receptor binding
preference, either through single or double substitutions, was
required for airborne transmissibility, which is in agreement
with loss-of-function transmission studies using 1918 A/H1N1
and 1957 A/H2N2 viruses (Pappas et al., 2010; Tumpey et al.,
2007). Although HAs with single Q222L or G224S substitutions
were less efficient in binding a2,6-SA-containing TRBC as
compared to the double mutant, viruses with the single RBS
substitutions were still transmissible. HA substitution T156A
increased virus binding to both a2,6-SA and a2,3-SA in quantita-
tive binding assays. Imai et al. (2012) showed that substitution
N154D in HA also affected transmission of a reassortant H5
virus. T156A and N154D result in the loss of the same glycosyl-
ation site in HA, suggesting that loss of this glycosylation site
rather than the specific amino acid substitutions were important
for the change in phenotype. Loss of glycosylation in H5 HA in
combination with substitutions Q222L and G224S was previ-
ously shown to enhance virus replication in ferrets (Wang et al.,
2010). The airborne-transmissible H5 virus of Imai et al. (2012)
also contained Q222L in HA but had N220K as a second substi-
tution in the RBS. In a third study, Q222L/G224S, along with
Q192R in the context of A/Vietnam/1203/04 HA, also resulted
in slightly increased transmission in ferrets (Chen et al., 2012).
These studies thus indicate that changes in receptor specificity
critically contribute to airborne transmission of H5 viruses in fer-
rets. Affinity measurements using A/Indonesia/5/05, A/Vietnam/
1203/04 and A/Vietnam/1194/04 HAs with substitutions associ-
ated with airborne transmission revealed a binding preference
for human receptors (Figure 3) (de Vries et al., 2014; Lu et al.,
2013; Xiong et al., 2013; Zhang et al., 2013a). Structural studies
of HA further showed that human and avian receptor analogs
were bound in the RBS in the same ‘‘folded-back’’ conformation
as seen for HA from H1, H2, and H3 pandemic viruses, which is
Xiong et al., 2013; Zhang et al., 2013a). In these studies, the
affinity of the mutant H5 HA was relatively low as compared to
HA of human H2 and H3 viruses (Liu et al., 2009; Skehel and
Wiley, 2000; Xiong et al., 2013; Zhang et al., 2013a). Although
it was speculated that an N-linked glycan at the tip of HA might
sterically hinder SA binding (Xiong et al., 2013), direct evidence
from structural studies on HA of the airborne transmissible
viruses is still lacking.
Arguably, one of the more intriguing findings of the Imai et al.
(2012) and Herfst et al. (2012) studies is the requirement of
HA mutations that affect stability in terms of temperature and
pH. H103Y in A/Indonesia/5/05 HA resulted in increased
temperature stability and requirement of lower pH treatment to
trigger membrane fusion, similar as described for T315I by
Imai et al. (2012). H103Y was recently shown to increase the
thermostability of HA (de Vries et al., 2014), and temperature-
dependent circular dichroism spectroscopic experiments re-
vealed hydrogen bond formation between 103Y and 413N in
adjacent monomers that stabilized the trimeric protein (Zhang
et al., 2013a). In contrast, T315I stabilized the position of the
fusion peptide within the HA monomer (Xiong et al., 2013), indi-
ity. Galloway et al. (2013) suggested that an altered pH of fusion
may be associated with virus adaptation to new hosts. Further-
more, substitutions that decrease the pH of fusion increased
virus replication in the upper respiratory tract of ferrets and in
mice (Krenn et al., 2011; Shelton et al., 2013; Zaraket et al.,
2013). Although the contribution of H103Y and T315I to increase
airborne transmission between ferrets may be related to the pH
of fusion or thermostability, these properties may merely be a
surrogate for another—as yet unknown—phenotype, such as
stability of HA in aerosols, resistance to drought, stability in
mucus, or altered pH in the host environment.
The requirement of increased polymerase activity to yield an
airborne transmissible H5 virus was also expected (Sorrell
et al., 2011). PB2 E627K has been identified as a major determi-
nant of host adaptation of pandemic influenza viruses (Aggarwal
et al., 2011; Steel et al., 2009; Subbarao et al., 1993; Tauben-
berger et al., 2005; Van Hoeven et al., 2009). Here, E627K also
ever, we identified a substitution acting in concert with E627K to
increase polymerase activity. Like PB2 E627K, PB1 H99Y alone
resulted in increased polymerase activity in minigenome assays
butdecreased virusreplication. Whencombined, thesetwosub-
stitutions had a synergistic effect in minigenome assays and
increased virus replication. In primer extension assays, PB2
E627K predominantly caused an increase in vRNA and cRNA,
changing the ratio between RNA replication and mRNA tran-
scription. Addition of PB1 H99Y lowered vRNA and cRNA while
further increasing mRNA levels, yielding a similar ratio as
observed for A/H5N1wild-type but at overall increased levels.
Here, we thus describe an ‘‘adaptive’’ substitution in PB1 that
potentially improved the levels of, and balance among, vRNA,
mRNA, and cRNA in concert with PB2-E627K in A/Indonesia/
5/05. Beyond HA, only these two amino acid substitutions
were required to generate airborne-transmissible A/H5N1 virus.
Such conclusion cannot be obtained from the A/H5 virus trans-
mission experiments that use reassortant viruses. Zhang et al.
(2013b) showed that the PA or NS1 genes of a pH1N1 virus
contributed to airborne transmission of reassortant A/H5N1
viruses in guinea pigs. At present, it is unclear how the guinea
pig and ferret models compare with respect to A/H5 virus trans-
Although some substitutions leading to an airborne phenotype
have been observed in nature, the required combination of sub-
stitutions has not yet been detected (Herfst et al., 2012; Neu-
mann et al., 2012; Russell et al., 2012). Keeping in mind that
the ferret model may not be predictive for airborne A/H5N1 virus
emergence in humans, it is of interest to note that, upon acquisi-
tion of PB2 E627K and the receptor binding changes Q222L/
G224S, the additional substitutions associated with the airborne
phenotype of A/H5N1 emerged within only one or two passages
in ferrets, became dominant after six passages, and appeared to
be selected for during transmission events. Other sets of muta-
tions than those identified here that similarly result in altered re-
ceptor specificity and stability of HA and polymerase function in
336 Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc.
mammalian cells may also be sufficient to increase airborne
transmission. The specific mutations to increase transmission
may be dependent on virus strain or subtype (Tharakaraman
et al., 2013). Given that some of the required substitutions
(PB2 E627K and HA T156A) are commonly found in field isolates
identified substitutions, emergence of transmissible A/H5N1
influenza A viruses in nature cannot be excluded (Russell et al.,
2012). In this light, the A/H7N9 virus outbreak in China is a cause
of concern, as some A/H7N9 field strains contain E627K in PB2
the tip of HA (Liu et al., 2013). These viruses were shown to have
increased preference for a2,6-SA and decreased binding to
a2,3-SA (Kageyama et al., 2013; Richard et al., 2013; van Riel
et al., 2013). Although airborne A/H7N9 virus transmission in fer-
rets was shown to be limited (Lam et al., 2013; Richard et al.,
2013; Shi et al., 2013; Zhu et al., 2013), it cannot be excluded
that fully avian viruses adapt upon repeated passage in mam-
mals to gain transmissibility. In analogy to H5, it can be specu-
lated that the A/H7N9 virus needs to acquire increased binding
preference for a2,6-SA over a2,3-SA, increased HA stability,
and increased polymerase activity. For both A/H5N1 and
A/H7N9, appropriate surveillance for emergence of mutations
thataffect HA receptor binding, HAstability, polymerase activity,
and transmission is thus warranted. It has been argued that
different sets of substitutions may lead to similar virus pheno-
types, and hence, sequence-based virus surveillance may be
misleading. However, such surveillance may be improved by
including phenotyping assays—using relatively simple methods
as described here—in the future. Such surveillance may be
further improved by deep sequencing, in addition to sequencing
consensus virus genomes.
Although it is clear that studies in ferrets may not be predictive
for influenza virus outbreaks in humans, the ferret transmission
model is one of the best models for influenza available today
(Maher and DeStefano, 2004). Experiments like the ones pre-
sented here are crucial to increase our basic understanding of
airborne virus transmission, as such knowledge is currently
very limited. The results of this study do not imply that an H5
influenza pandemic is imminent but warrant an intensified and
broadened approach to detect emerging influenza viruses early
and take immediate action once viruses naturally gain functions
that might enable them to become a pandemic threat.
See Supplemental Information for additional details and references.
Viruses and Cells
Madin-Darby caninekidney(MDCK)cells, 293T(human kidneyepithelial cells),
and subclone 118 of Vero-WHO cells (African green monkey kidney epithelial
cells) were used for virus propagation, plasmid transfections, and fusion
Influenza virus A/Indonesia/5/05 (A/H5N1) was isolated from a human case
of HPAI virus infection. All viruses used in this study were generated upon
transfection of 293T cells with reverse genetics plasmids. Recombinant
viruses were propagated in MDCK cells, and viral titers were determined by
end-point titration. For binding and stability assays, recombinant viruses
with seven gene segments of A/PR/8/34 and the wild-type or mutant HA
segment of A/Indonesia/5/05 were used.
Ferret Model for Airborne Transmission
Ethical, biosafety, and biosecurity considerations related to the experiments
are described in detail in the Supplemental Information.
One- to 2-year-old ferrets (mustela putorius furo), free of antibodies against
H5HA,wereinoculatedwithvirus.Thenextday,asecondferret washoused in
grids, allowing the transfer of air between cage mates but preventing direct
contact. Nose and throat swabs of inoculated and naive animals were taken
every other day to test for virus presence. Blood from naive contact animals
was collected 14 days after first exposure to inoculated animals and tested
for the presence of antibodies against H5 HA.
Given that airborne transmission in ferrets has never been observed for
A/Indonesia/5/05 or any other avian A/H5N1 virus (Table S3), we define every
single event of virus detection in naı ¨ve ferrets as ‘‘airborne transmission.’’ The
use of small group sizes could result in an underestimation of airborne
transmission. Thus, although ‘‘not transmissible’’ does not mean ‘‘will never
transmit,’’ ‘‘transmissible’’ is a clearly defined virus phenotype.
Viral RNA was extracted from ferret samples upon virus passaging, converted
to cDNA, and amplified by PCR using primers covering the full viral genome.
PCR fragments for each virus were pooled in equal concentrations, and
libraries were created for each virus. Emulsion PCR and GS Junior sequencing
runs were performed according to instructions of the manufacturer (Roche).
Sequence reads were sorted by bar code, trimmed at 30 nucleotides from
the 30and 50ends to remove primer sequences, and the 30ends were further
trimmed to improve quality using a Phred score of 20. Reads were aligned to
reference sequence A/Indonesia/5/05 using CLC Genomics software 4.6.1.
The threshold for mutation detection was manually set at 1%.
Virus Binding Assays
Formalin-fixed, paraffin-embedded tissues from ferrets and humans were
used for virus histochemistry. Sucrose-purified viruses were labeled with fluo-
The FITC label was detected with a peroxidase-labeled anti-FITC antibody,
and the signal was amplified using a Tyramide Signal Amplification System.
Peroxidase was revealed with 3-amino-9-ethyl-cabazole, and tissues were
counterstained with hematoxylin and embedded in glycerol-gelatin.
For direct fetuin binding assays, 96 well plates were first coated with bovine
fetuin and subsequently incubated with BPL-inactivated viruses, washed, and
blocked with desialylated bovine serum albumin. The virus-coated wells were
incubated with2-fold dilutions of receptor analogs (resialylated fetuin prepara-
tions containing either a2,3-linked SAs [30Fetuin] or a2,6-linked SAs [60Fetuin]
labeled with horseradish peroxidase). Plates were washed, and tetramethyl-
benzidine substrate was added, after which the absorbance at 450 nm was
determined. The association constants (Kass) of virus complexes with analogs
were determined from the slopes of Scatchard plots.
TRBC by incubation with Vibrio cholerae neuraminidase. Complete removal of
SAs was confirmedby loss of HA using control viruses.Resialylation was done
using a2,3-(N)-sialyltransferase or a2,6-(N)-sialyltransferase to produce a2,3-
TRBC and a2,6-TRBC, respectively. Resialylation of either a2,3 or a2,6 was
confirmed using viruses with known receptor specificity. Viruses were tested
in standard HA assay using native and resialylated TRBCs.
HA-mediated membrane fusion was tested by transfecting populations of
Vero-118 cells with plasmids expressing HA and b-galactosidase (b-gal) along
with either pLTR-CAT (a chloramphenicol acetyltransferase [CAT] gene under
the control of the human immunodeficiency virus type 1 long terminal repeat)
or pTat (expressing the HIV-1 transactivator of transcription Tat). One day after
transfection, both cell populations were harvested, pooled, and replated. The
were harvested 24 hr after the pH pulse, and CAT and b-gal expression were
quantified by enzyme-linked immunosorbent assays. As an alternative to
CAT quantification, cells were fixed, washed, and stained with Giemsa for
Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc. 337
Viruses were incubated for 30 min at different temperatures before performing
anHAassay usingTRBCs. Two-fold dilutions of virusinPBS containing 0.25%
red blood cells were prepared in a U-shaped 96 well plate and were incubated
for 1 hr at 4?C, and agglutination was recorded.
A model vRNA, consisting of the firefly luciferase flanked by the noncoding
regions of segment 8 of influenza A virus, under the control of a T7 RNA poly-
merase promoter, was transfected into 293T cells, along with plasmids ex-
luciferase expression plasmid. 24 hr after transfection, firefly and Renilla
luminescence was measured. Relative light units were calculated as the ratio
of firefly and Renilla luciferase.
MDCK cells were inoculated with virus and grown with an Avicel (FMC
biopolymers, Brussels) overlay. Two days later, cells were washed, fixed, per-
meabilized, and stained with anti-NP monoclonal antibody and goat-anti-
mouse-HRP. True blue reagent (KPL) was added to the cells, and digital
images were taken to quantify plaque size using ImageQuant TL software
(GE Healthcare Life Sciences).
Primer Extension Assay
MDCK cells were inoculated with virus, and RNA was isolated 3 hr later using
Trizol reagent (Invitrogen). Radioactive-labeled primers specific for mRNA/
cRNA, vRNA of PB1, and 5S rRNA were annealed to prime a reverse transcrip-
tion reaction. The reaction was stopped, and transcription products were
separated on 6% polyacrylamide gels containing 7 M urea in trisborate-
EDTA buffer and detected using autoradiography films.
Supplemental Information includes Extended Experimental Procedures, two
figures, and four tables and can be found with this article online at http://dx.
M.L. and S.v.B. are PhD students who were jointly responsible for the critical
first series of experiments, the analysis of data, and drafting the manuscript
under the supervision of S.H. In particular, M.L. identified a minimal set of mu-
tations required for airborne transmission, whereas S.v.B. was responsible for
sequence analyses and bioinformatics. Together, with S.H., they initiated the
follow-up phenotypic analyses in collaboration with the other authors.
We thank Dennis de Meulder for excellent technical assistance, Nicolai
Bovin (Institute of Bio-organic Chemistry, Moscow, Russia) for providing
sialylglycopolymers, and Malik Peiris (University of Hong Kong) for providing
A/Indonesia/5/2005 with permission from I. Kandun of the Indonesian
government.This workwas supported
HHSN266200700010C, EU FP7 grants EMPERIE (223498), ANTIGONE
(278976), FLUPIG (258084), and PREDEMICS (278433). MdG was funded in
part by a Marie Curie fellowship (PIEF-GA-2009-237505). All experiments
involving A/H5N1 virus were performed before January 2012 in agreement
with a moratorium on A/H5N1 gain-of-function research and restarted after
February 2013 but without NIH/NIAID funding. Special arrangements are in
place with the NIH and the contractor at Icahn School of Medicine at Mount
Sinai, New York, for sharing the viruses (and plasmids) in the present paper.
A.D.M.E.O. and G.F.R. are CSO and part-time employees of ViroClinics Bio-
sciences B.V. A.D.M.E.O. has advisory affiliations on behalf of ViroClinics
Biosciences B.V. with GlaxoSmithKline, Novartis, and Roche. A.D.M.E.O.
and R.A.M.F. are holders of certificates of shares in ViroClinics Biosciences
B.V. To avoid any possible conflict of interests, Erasmus MC policy dictates
Personeelsparticipaties. The board of this foundation is appointed by the
Board of Governors of the Erasmus MC and exercises all voting rights with re-
gard to these shares.
Received: November 26, 2013
Revised: February 17, 2014
Accepted: February 24, 2014
Published: April 10, 2014
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Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc. 339
EXTENDED EXPERIMENTAL PROCEDURES
All experiments involving highly pathogenic A/H5N1 viruses were conducted at enhanced animal biosafety level 3 (ABSL3+). The
ABSL3+ facility of Erasmus MC consists of a negative pressurized (?30 Pa) laboratory in which all in vivo and in vitro experimental
work is carried out in class 3 isolators or class 3 biosafety cabinets, which are also negative pressurized (<-200 Pa). Although the
laboratory is considered ‘‘clean’’ because all experiments are conducted in closed class 3 cabinets and isolators, special personal
protective equipment, including laboratory suits, gloves and FFP3 facemasks is used. Air released from the class 3 units is filtered by
High Efficiency Particulate Air (HEPA) filters and then leaves the facility via a second set of HEPA filters. Only authorized personnel
that received the appropriate training can access the ABSL3+ facility. All personnel working in the facility is vaccinated against sea-
sonal and A/H5N1 influenza viruses. For animal handling in the facilities, personnel always work in pairs. The facility is secured by
procedures recognized as appropriate by the institutional biosafety officers and facility management at Erasmus MC and Dutch
and United States government inspectors. Antiviral drugs (oseltamivir and zanamivir) and personnel isolation facilities are directly
available to further mitigate risks upon incidents.
Prior to submission of the manuscript, all authors read and commented on all aspects of the study (design, realization, scientific
value, ethical considerations, integrity of data, presentation of experimental outcomes, biosafety and biosecurity aspects etc.).
Weconcludedthat researchinvolving airborne transmissible A/H5N1influenza virusesmustbeconsidered DUR(dual-use research),
but that the present study does not qualify as DURC (dual-use research of concern), since no increase in virulence or transmissibility
compared to previously published studies is described. Furthermore, we also cannot foresee any direct misapplication of the pre-
sented work resulting in a threat to public health. In contrast, the gain of knowledge and future applications of this work will be bene-
ficial for science and society.
The manuscript was sent for clearance to the Biosafety Office of Erasmus MC, representing the institutional Biosafety Advisory
Board. Subsequently, we requested (under formal protest) an export license issued by the Dutch government for communication
Committee and the Ethical Advisory Board of the EU FP7 program ‘‘ANTIGONE’’ endorsed publication of the manuscript and
delivered comments that were carefully incorporated in the manuscript. NIAID as another funding agency requested an appendix
5 procedure to account for possible biosafety and biosecurity risks and to develop a communication plan. The final version of the
manuscript was sent to the funders and all individuals involved in the evaluation process, after which the manuscript was submitted
supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin, 100 mg/ml streptomycin, 2 mM glutamine, 1.5 mg/ml sodiumbi-
carbonate (Lonza), 10 mM HEPES (Lonza), and non-essential amino acids (MP Biomedicals Europe, Illkirch, France). 293T cells were
cultured in Dulbecco modified Eagle’s medium (DMEM, Lonza) supplemented with 10% FCS, 100 IU/ml penicillin, 100 mg/ml strep-
tomycin, 2 mM glutamine, 1 mM sodium pyruvate, and non-essential amino acids. Subclone 118 of Vero-WHO cells (Vero-118) was
cultured in Iscove’s modified Dulbecco’s medium (IMDM; BioWhittaker) supplemented with 10% fetal calf serum (FCS), 100 IU/ml
penicillin, 100 mg streptomycin and 2 mM glutamine (Kuiken et al., 2004).
Influenza virus A/Indonesia/5/05 (A/H5N1) was isolated from a human case of HPAI virus infection and passaged once in embryo-
nated chicken eggs followed by one passage in MDCK cells. All eight gene segments were amplified by reverse transcription poly-
merase chain reaction and cloned in a modified version of the bidirectional reverse genetics plasmid pHW2000 (Chutinimitkul et al.,
2010b; de Wit et al., 2004). Substitutions of interest (H103Y, T156A, Q222L and G224S in HA, E627K in PB2, R99K and S345N in NP
and H99Y and I368V in PB1) were introduced by reverse genetics using the QuikChange multi-site-directed mutagenesis kit
(Stratagene, Leusden, Netherlands) according to the instructions of the manufacturer. Recombinant viruses were produced upon
transfection of 293T cells and virus stocks were propagated and titrated in MDCK cells. For binding assays, and stability assays,
reassortant viruses consisting of seven gene segments of influenza virus A/PR/8/34 and the HA segment of A/H5N1 were produced
using a previously described reverse genetics system for influenza virus A/PR/8/34 (de Wit et al., 2004). For binding studies, viruses
20% sucrose cushion and stored in 0.02% sodium azide (Sigma-Aldrich).
Virus Titration in MDCK Cells
Virus titers were determined by end-point titration in MDCK cells. MDCK cells were inoculated with tenfold serial dilutions of virus
Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc. S1
10 mM HEPES, non-essential amino acids, and 20 mg/ml trypsin (Lonza). Three days after inoculation, supernatants of infected cell
cultures were tested for agglutinating activity using TRBCs as an indicator of virus replication in the cells. Infectious virus titers were
calculated from 4 replicates (nose swabs, and throat swabs) or 10 replicates (virus stocks) by the method of Spearman-Karber.
Airborne Transmission of A/H5N1 Virus in the Ferret Model
An independent animal experimentation ethical review committee (Dutch Stichting Dier Experimenten CommissieConsult) approved
all animal studies. 64 female ferrets between 1 and 2 years of age were obtained from an accredited ferret breeder. All animals were
tested for the presence of antibodies against A/H5, A/H3 and A/H1 prototype influenza A viruses and Aleutian Disease Virus, were
microchipped and received hormonal treatment to prevent estrus.
Aerosol or respiratory droplet transmission experiments were performed as described previously (Munster et al., 2009). In short, 2
or 4 seronegative female adult ferrets (Mustela putorius furo) were inoculated intranasally with 106TCID50of virus by applying 250 ml
placed opposite to each inoculated ferret. Each transmission pair was housed in a separate transmission cage designed to prevent
direct contact between the inoculated and naive ferrets but allowing airflow from the inoculated to the naive ferret. Nose and throat
swabs were collected on 1, 3, 5, and 7 days postinoculation (dpi) for inoculated ferrets and on 1, 3, 5, 7, and 9 days post exposure
(dpe) for the naive ferrets. Virus titers in swabs were determined by end-point titration in MDCK cells.
Clinical manifestations observed in animals upon inoculation of a virus suspension or infection via aerosols or respiratory droplets
included ruffled fur, loss of appetite, and lethargy. Two animals that died in the course of the experiments were examined for the
presence of influenza virus in respiratory organs and were found to be free of detectable levels of viral protein. We concluded that
these ferrets died due to reasons unrelated to the effect of virus infection. No animals required withdrawal from the study on animal
welfare grounds. All animals were humanely killed at the end of the in-vivo phase of the study.
Statistical Considerations for Ferret Transmission Experiments
Given that airborne transmission in ferrets has never been observed for A/Indonesia/5/05 or any other avian A/H5N1 virus (Table S3),
we define every single event of airborne transmission of mutant A/Indonesia/5/05 virus as detected by the presence of virus in naive
studies in ferrets, extending beyond the analysis by Belser et al. (2013b) and discussions therein. A search for published literature via
http://www.ncbi.nlm.nih.gov/pubmed/ using search terms ’’influenza,’’ ‘‘transmission,’’ and ‘‘ferret’’ was performed on 27-12-2013.
Studies that did not report airborne transmission between ferrets based on virus replication data or that tested swine viruses exclu-
sively were discarded from the set. We added two articles manually that were not retrieved automatically in this search and that we
were aware of (Jones etal.,2014; Kimble etal.,2014). Fromthe total of 48full-text articles thatwere selected, wereport the wild-type
viruses that were tested, the group size of the experiment and the number of airborne transmission events that were reported (Table
S3). From this analysis, it was clear that wild-type A/H5N1 viruses have never been shown to transmit via the airborne route between
other avian influenza viruses (with the exception of 2013 A/H7N9 viruses) were shown to be not transmitted between 73 ferret pairs
tested in 10 studies, with the exception of 1 study reporting transmission of a single avian A/H1N1 virus. In sharp contrast, human
influenza viruses were transmitted in 132/149 ferret pairs in 32 published studies. Despite the small group sizes in most published
studies, human influenza viruses were transmitted in 32/32 studies, while avian A/H5N1 viruses were transmitted in 0/7 studies.
The 2013 A/H7N9 virus (which has several mammalian adaptation markers) was transmissible in 19 of 44 ferret pairs in 6 published
with A/H7N9 virus were comparable (transmission in 1/3, 4/8, 9/18, 3/9, 1/3, 1/3 ferret pairs in the individual studies). Thus, although
the statistical power oftransmission studiesasperformed here isinsufficient foraquantitative analysis oftransmission efficiency due
to small group size, sufficiently robust data are generated to draw qualitative conclusions. We can therefore interpret any event of
airborne transmission of mutant A/H5N1 virus as an indicator of increased transmission over wild-type A/H5N1 (never found to be
transmitted), while we cannot draw strong conclusions about mutant A/H5N1 viruses that did not transmit between small numbers
of ferret pairs.
The presence of antibodies elicited against the tested viruses was confirmed by HA inhibition (HI) assay using standard procedures
(WHO, 2002) and A/Indonesia/5/05HA H103Y, T156A, Q222L, G224Sas a test antigen. Briefly, ferret antisera were prepared upon intranasal
inoculation and collecting blood 14 days later. Antisera were pre-treated overnight with receptor destroying enzyme Vibrio cholerae
neuraminidase (VCNA) at 37?C, and incubated at 56?C for 1h the next day. Twofold serial dilutions of the antisera, starting at a 1:20
dilution, were mixed with 25 ml of a virus stock containing 4 hemagglutinating units and were incubated at 37?C for 30 min. Subse-
quently, 25 ml 1% TRBCs was added and the mixture was incubated at 4?C for 1h. HI was read and was expressed as the reciprocal
value of the highest dilution of the serum that completely inhibited agglutination of virus and erythrocytes.
S2 Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc.
Analysis of Virus Nucleotide Sequence Variation Using a 454 Sequencing Platform
Viral RNA was extracted from ferret tracheal swabs, nasal swabs or nasal washes upon passaging with influenza viruses
A/H5N1wild-typeand A/H5N1HA Q222L, G224S PB2 E627K, using the High Pure RNA Isolation Kit (Roche). A SuperScript III One-Step
reverse transcription kit (Invitrogen) was used to synthesize cDNA from extracted RNA. The RT mixture contained 46 ml of RNA
extract, 4 ml (2 pmol/mL) primer AGCRAAAGCAGG, 1 ml (40 U/ml) Ribonuclease Inhibitor (Promega), and 4 ml (10 mM each) deoxynu-
cleoside triphosphates (Roche) in a 55 ml volume. After a 5 min incubation at 65?C for optimal primer hybridization to template, 16 ml
(10x) First-Strand buffer, 14 ml (0.1 M) DTT, 1 ml (40 U/ml) Ribonuclease Inhibitor (Promega) and 4 ml (200 U/mL) SuperScript III Reverse
Transcriptase was added to the mixture in a 80 ml volume. The RT mixture was sequentially incubated at 25?C for 5 min and 50?C for
1 hr to obtain cDNA. cDNA was subjected to polymerase chain reaction (PCR), using 32 primer sets (Table S4) that cover the full viral
genome (Salzberg et al., 2007).
The PCR mixtures contained 10 pmol of each forward and reverse primer, 3 ml of cDNA, 1 ml (10 mM each) deoxynucleoside
triphosphate, 5 ml 10 3 PCR Gold buffer, 5 ml (25 mM) MgCl2, and 1 ml (2.5 U/mL) AmpliTaq Gold DNA Polymerase (Applied Bio-
systems, Bleiswijk, The Netherlands). Water was then added to achieve a final volume of 50 mL. The PCR mixture was incubated
at 95?C for 6 min, followed by 40 cycles at 95?C for 30 s, 45?C for 30 s, 72?C for 1 min, and a final extension at 72?C for 5 min.
PCR fragments were visualized by blue light after electrophoresis on a 1% agarose gel containing 1 3 SYBR? Safe DNA Gel Stain
(Life Technologies, Bleiswijk, The Netherlands) in 1 3 Tris-borate buffer (pH 8.0). SmartLadder (Eurogentec) was used to estimate
amplicon size. Fragments ranged from 426 to 627 nucleotides in length.
Fragments with the correct size, were extracted from the gel using the MinElute Gel Extraction Kit (QIAGEN, Venlo, The
Netherlands) according to the manufacturers protocol, and subsequently DNA concentrations were measured using a NanoDrop
1000 Spectrophotometer (Thermo Scientific).
Fragments for each virus were pooled in equal concentrations and libraries were created for each virus according to the manufac-
turer’s protocol without DNA fragmentation (GS FLX Titanium Rapid Library Preparation, Roche). The emPCR (Amplification Method
Lib-L) and GS Junior sequencing runs were performed according to instructions of the manufacturer (Roche). Sequence reads from
the GS Junior sequencing data were sorted by barcode and aligned to reference sequence A/Indonesia/5/05 using CLC Genomics
end of the reads were trimmed to improve quality, using a Phred score of 20. The threshold for detection was manually set at 1%.
Viruses were purified on a sucrose gradient and labeled with fluorescein isothiocyanate (FITC, Sigma-Aldrich) as described previ-
ously (van Riel et al., 2007). Briefly, MDCK cells were inoculated and virus-containing supernatant was concentrated using a sucrose
cushion and purified on sucrose gradients before inactivation by dialysis against 0.1% formalin and labeling with an equal volume of
0.1 mg/ml FITC.
All selected tissues were free of histological evidence for infection. Virus histochemistry was performed as described previously
(van Riel et al., 2007). Briefly, formalin-fixed paraffin-embedded tissues were deparaffinized with xylene and rehydrated with graded
alcohol. 50-100 hemagglutinating units of FITC-labeled influenza viruses were incubated with the respective tissue sections over-
night at 4?C. The FITC label was detected with a peroxidase-labeled rabbit anti-FITC antibody (Dako, Heverlee, Belgium) and the
signal was amplified using a Tyramide Signal Amplification System (Perkin Elmer, Groningen, The Netherlands) according to the in-
structions of the manufacturer. Peroxidase was revealed with 3-amino-9-ethyl-cabazole (Sigma-Aldrich) and tissues were counter-
stained with hematoxylin and embedded in glycerol-gelatin (Merck, Darmstadt, Germany). Attachment of influenza virus to tissues
was visible as granular to diffuse red staining of the epithelium.
Direct Receptor Binding Assay
For direct receptor binding assays and fetuin binding inhibition assay, virus working dilutions were prepared as described previously
(Matrosovich and Gambaryan, 2012). In addition, equal absorption of the wild-type and mutant A/H5N1 viruses in the wells of micro-
plates was confirmed using a direct enzyme-linked immunosorbent assay with PR/8 neuraminidase-specific antibodies (BEI re-
sources, Manassas, USA). Biotinylated synthetic poly-N-(2-hydroxyethyl)acrylamide-based sialylglycopolymers (SGPs) containing
20 mol% of Neu5Aca2,3Galb1-4GlcNAcb (30SLN) or Neu5Aca2,6Galb1-4GlcNAcb (60SLN) (Lectinity Holding, Inc., Moscow, Russia)
were used as receptor analogs. Also re-sialylated fetuin preparations, containing either a2,3-linked SAs (30Fetuin) or a2,6-linked SAs
(60Fetuin) labeled with horseradish peroxidase (HRP) were tested for their binding to the viruses. Ninety-six well plates were coated
overnight at 4?C with bovine fetuin (5 mg/ml) in PBS, washed with distilled water and dried at room temperature. Fetuin-coated plates
were incubated overnight at 4?C with BPL-inactivated viruses in PBS, washed 3 times with PBS and blocked with 0.1% solution of
desialylated bovine serum albumin (BSA-NA, Sigma-Aldrich) in PBS. After washing 3 times with ice-cold washing buffer (PBS with
0.05%Tween-80), thereplicate virus-coated wellswere incubated withtwo-fold dilutions of thereceptor analogs inincubation buffer
after which substrate (tetramethylbenzidine) was added to detect HRP activity. To detect binding of biotinylated SGPs, plates were
washed 3 times with ice-cold washing buffer, followed by incubation with streptavidin-HRP. After subsequent washing, tetramethyl-
benzidine was added to quantify binding. The absorbencies at 450 nm were determined, transferred to a PC and processed with
Microsoft Excel software. The data were converted to Scatchard plots (A450/C versus A450), where C is the concentration of the
Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc. S3
receptor analog in solution and A450 is the absorbency in the corresponding well. Concentrations were expressed in arbitrary units
(AU) for 30and 60fetuin and in mM of sialic acid for SGPs. The apparent association constants (Kass) of virus complexes with analogs
were determined from the slopes of the Scatchard plots.
Fetuin Inhibition Assay
the level of its competition with fetuin-HRP (Matrosovich and Gambaryan, 2012). As receptor analogs, several SGPs were used
(Lectinity Holding, Inc., Moscow, Russia), which contained the following sialyloligosaccharide ligands: Neu5Aca2,3Galb1-4GlcNAcb
4(Fuca1-3)GlcNAcb (SLex), Neu5Aca2,3Galb1-4(Fuca1-3)(6-O-HSO3)GlcNAcb (Su-SLex), Neu5Aca2,3Galb1-3GlcNAcb (SLec),
Neu5Aca2,3Galb1-3(6-O-HSO3)GlcNAcb (Su-SLec) and Neu5Aca2,3Galb1-3(Fuca1-4)GlcNAcb (SLea). Viruses were first absorbed
in fetuin-coated plates as described above. Subsequently, two-fold dilutions of SGPs in HRP-labeled fetuin-HRP-containing buffer
were added to the wells, followed by incubation for 1 hr at 4?C. After washing, tetramethylbenzidine was added and peroxidase ac-
tivity was measured. Association constants were calculated from the binding inhibition data as described previously (Matrosovich
and Gambaryan, 2012).
Modified Red Blood Cell Assay
Modified TRBC’s were prepared as described (Nobusawa et al., 2000) with slight modifications. Briefly, all SAs were removed from
the surface of TRBC by incubation of 62.5 ml of 20% TRBC in PBS with 50 mU of VCNA (Roche, Almere, The Netherlands) in 8 mM
calcium chloride at 37?C for one hour. Complete removal of SAs was confirmed by loss of HA of treated TRBC using control viruses.
Resialylation was done using 0.25 mU a2,3-(N)-sialyltraferase (Calbiochem, California, USA) or 12 mU a2,6-(N)-sialyltransferase
(Calbiochem, California, USA) and 1.5 mM CMP-SA (Sigma-Aldrich, Zwijndrecht, the Netherlands) at 37?C in 75 ml for 2 hr to produce
a2,3-TRBC and a2,6-TRBC respectively. After washing, the TRBCs were resuspended in PBS containing 1% BSA to a final concen-
tration of 0.5%. Resialylation of either a2,3 or a2,6 was confirmed by HA using viruses with known receptor specificity. Viruses were
tested in standard HA assay using native and resialylated TRBCs. In brief, twofold dilutions of virus were made in PBS. An equal vol-
ume of 0.5% TRBCs was added and incubated at 4?C for one hour before reading the HA titer.
Fusion was tested as previously described (Herfst et al., 2008) in a cell content mixing (CM) assay in which two 10 cm dishes con-
taining Vero-118 cells were each transfected with 5 mg of pCAGGS-HA and 1 mg of pTS27 plasmid, a constitutive b-galactosidase
(b-Gal) expression vector, using Xtremegene transfection reagent (Roche). The cells in one dish were cotransfected with 4 mg of
pLTR-CAT (containing the chloramphenicol acetyltransferase [CAT] gene under the control of the human immunodeficiency virus
type 1 [HIV-1] long terminal repeat [LTR]), and the cells in the other dish were cotransfected with 4 mg of pTat (expressing the
HIV-1 transactivator of transcription Tat). One day after transfection, both cell populations were harvested using trypsin-EDTA,
pooled, and plated in a six-well plate format. The next morning, cells were exposed to PBS at different pH for 10 min. Cell lysates
were harvested 24 hr after the pH pulse, and CAT and b-Gal expression were quantified by enzyme-linked immunosorbent assays
(Roche). HA-mediated fusion of cellular membranes resulted in polykaryon formation and Tat-mediated transactivation of the HIV-1
LTR resulted in induction of CAT expression. Alternatively to CAT quantification, cells were fixed using 70% ice-cold acetone,
washed and stained using a 20% Giemsa mixture for microscopy (Merck Millipore, Darmstadt, Germany).
HA Stability Assay
Viruses were incubated for 30 min at different temperatures before performing an HA assay using TRBCs. Two-fold dilutions of virus
in PBS containing 0.25% red blood cells were prepared in a U-shaped 96 well plate and were incubated for one hour at 4?C and
agglutination was recorded.
A model vRNA, consisting of the firefly luciferase open reading frame flanked by the noncoding regions (NCRs) of segment 8 of influ-
enza A virus, under the control of a T7 RNA polymerase promoter was used for minigenome assays (de Wit et al., 2010). The reporter
plasmid (0.5 mg) was transfected into 293T cells in 6-well plates, along with 0.5 mg of each of the pHW2000 plasmids encoding PB2,
plasmid pRL (Promega, Leiden, Netherlands) as an internal control. 24 hr after transfection, luminescence was measured using a
Dual-Glo Luciferase Assay System (Promega) according to the instructions of the manufacturer in a TECAN Infinite F200 machine
(Tecan Benelux bv, Giessen, Netherlands). Relative light units (RLU) were calculated as the ratio of firefly and Renilla luciferase
The assay was performed as described (Matrosovich et al., 2006). In brief, MDCK cells (106per well) were seeded in a 6 well plate
to reach 90% confluency the next day. One hour after inoculation with 103tcid50/ml, the inoculum was replaced with 1:1 mixture of
S4 Cell 157, 329–339, April 10, 2014 ª2014 Elsevier Inc.