Compatibility of H9N2 avian influenza surface genes
and 2009 pandemic H1N1 internal genes for
transmission in the ferret model
J. Brian Kimblea, Erin Sorrella, Hongxia Shaoa, Philip L. Martinb, and Daniel Roberto Pereza,1
aDepartment of Veterinary Medicine, University of Maryland, College Park and Virginia-Maryland Regional College of Veterinary Medicine, College Park, MD
20742; andbCenter for Advanced Preclinical Research, Science Applications International Corporation/National Cancer Institute, Frederick, MD 21702
Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved June 10, 2011 (received for review May 19, 2011)
In 2009, a novel H1N1 influenza (pH1N1) virus caused the first
influenza pandemic in 40 y. The virus was identified as a triple
reassortant between avian, swine, and human influenza viruses,
highlighting the importance of reassortment in the generation of
viruses with pandemic potential. Previously, we showed that
a reassortant virus composed of wild-type avian H9N2 surface
genes in a seasonal human H3N2 backbone could gain efficient
respiratory droplet transmission in the ferret model. Here we de-
termine the ability of the H9N2 surface genes in the context of the
internal genes of a pH1N1 virus to efficiently transmit via respira-
tory droplets in ferrets. We generated reassorted viruses carrying
the HA gene alone or in combination with the NA gene of a pro-
totypical H9N2 virus in the background of a pH1N1 virus. Four
reassortant viruses were generated, with three of them showing
efficient respiratory droplet transmission. Differences in replica-
tion efficiency were observed for these viruses; however, the
results clearly indicate that H9N2 avian influenza viruses and
pH1N1 viruses, both of which have occasionally infected pigs, have
the potential to reassort and generate novel viruses with respira-
tory transmission potential in mammals.
populations across Eurasia since the mid 1990s (1–4). Avian H9
viruses display a typical avian host range; however, many recent
isolates contain a leucine (L)—instead of glutamine (Q)—at po-
sition 226 in the receptor binding site of the HA protein, which
facilitates preferential binding to sialic acid receptors in an α2,6
conformation (SAα2,6), typical of human influenza viruses. L226-
containing H9N2 viruses show efficient replication in human air-
way epithelial cells and in the ferret model (5–7). Since the late
1990s, 12 cases of human H9N2 infection have been identified. In
addition, H9N2 viruses have been isolated sporadically but con-
sistently from pigs in Hong Kong special administrative region,
China, and South Korea (8–10). Clinically, human H9N2 infec-
tions present as typical seasonal influenza infections, potentially
allowing cases to go unreported and increasing the opportunities
forthe virus to transmit andreassort. Seroepidemiological studies
indicate that human infections are more prevalent than reported
(11–14). Experimentally, H9N2 surface genes reassorted with
a ferret model only after adaptation by serial passage and in-
corporation of amino acid changes on the surface and internal
In Mexico in 2009, an H1N1 virus emerged from swine into
the human population, quickly spread throughout the globe, and
became the first pandemic virus in more than 40 y. This novel
pandemic H1N1 (pH1N1) originated from a reassortment event
involving human, swine, and avian influenza viruses. Recent
studies indicate that this pH1N1 is present in swine populations
and continues to reassort with other swine influenza viruses (16).
ccording to the World Health Organization, H9, together
with H5 and H7 subtypes, are among the leading candidates
The original swine triple reassortant virus, as well as the ensuing
reassortant swine and the pH1N1, all contain the same internal
gene cassette (17, 18). This triple reassortant internal gene
(TRIG) cassette consists of the PB2 and PA from a North
American avian virus, the PB1 from a human H3N2 seasonal
virus, and the NP and NS from the classical swine H1N1. Many
of the currently circulating swine viruses contain the TRIG cas-
sette with a myriad of different surface genes. This finding has
lead to the hypothesis that the TRIG cassette is a stable collec-
tion of internal genes that allows for easy surface switching (17).
The continued presence of pH1N1 in swine, the propensity of
pH1N1 to reassort with other influenza viruses, the occasional
isolation of H9N2 viruses in swine and human populations, and
the “humanization” of the receptor binding preference of H9 HA
protein all underscore the real threat of a novel H9N2:pH1N1
reassortant with significant threat to the human population.
A novel pandemic virus must be antigenically distinct from
currently circulating influenza viruses to have a naive population
through which to spread (19). An H9N2:pH1N1 reassortant virus
with a H9 surface protein on a pH1N1 backbone would be an-
tigenically novel. The isolation of both subtypes of influenza
from swine and humans makes it feasible that a natural reas-
sortant between the two could occur. Here we describe the
ability of four H9N2:pH1N1 reassortant viruses to infect and
transmit in ferrets, an animal model that resembles human in-
fluenza infection and transmission. We found that these reas-
sortant viruses can transmit by respiratory droplet transmission
in ferrets, highlighting their pandemic potential.
Generation of H9N2 and H9N1 Influenza Viruses with Internal Genes
from pH1N1. All four viruses were made with the pH1N1 internal
genes (PB2, PB1, PA, NP, M, and NS) from A/Netherlands/602/
2009 (H1N1) (20). The surface genes came from either A/guinea
fowl/Hong Kong/WF10/1999 (H9N2) or from A/ferret/Mary-
land/P10_UMD/2008 (H9N2), herein referred to as WF10 and
P10, respectively (15, 21). WF10 has a typical avian host range
but has been shown to replicate efficiently in ferrets although
transmission among ferrets occurs only with animals in direct
contact (7, 15). The P10 virus is the result of 10 serial passages in
ferrets of an avian-human H9N2:H3N2 reassortant containing
the WF10 surface on a seasonal H3N2 (A/Memphis/14/1998)
backbone (15). The P10 virus has two mutations in the HA
Author contributions: J.B.K., E.S., and D.R.P. designed research; J.B.K., E.S., H.S., and P.L.M.
performed research; H.S. contributed new reagents/analytic tools; J.B.K., E.S., H.S., P.L.M.,
and D.R.P. analyzed data; and J.B.K., E.S., and D.R.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 19, 2011
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(T189A in HA1 and G192R in HA2) and one in the NA (I28V)
compared with the WF10. These amino acid changes were shown
to be crucial for efficient and reproducible respiratory droplet
transmission in ferrets (15). The four viruses generated in this
report had P10 HA and NA (2P10), P10 HA and pH1N1 NA
(1P10), WF10 HA and NA, (2WF10) or WF10 HA and pH1N1
NA (1WF10) (Fig. 1A).
Viruses grew to similar titers in Madin-Darby canine-kidney
(MDCK) cells, from 106.5to 107.2. However, 1WF10 showed
slower growth indicated by lower titers at 24 h postinfection (Fig.
1B). Wan et al. (7) had previously compared the plaque mor-
phology of wild-type WF10 to the seasonal H3N2 and the pre-
decessor of the P10 virus, 2WF10:6M98. Seasonal H3N2 showed
large plaques but WF10 formed pinpoint plaques (7). The
2WF10:6M98 and P10 plaques were of an intermediate size (15).
The 1P10 showed pinpoint plaques similar to wild-type WF10
(Fig. 1C) (7, 15); 2P10, 1WF10, and 2WF10 formed plaques of
intermediate size that were similar to the original 2WF10:6M98
or P10 viruses (Fig. 1C) (7, 15).
Infection, Pathology, Signs of Disease, and Transmission. The four
viruses established an infection in ferrets inoculated with 106
tissue culture infectious dose (TCID50) per ferret. Histopatho-
logical examination was performed on three H&E-stained sec-
tions of trachea and lung collected from directly inoculated
ferrets at 3 and 5 days postinoculation (dpi) (Fig. 2). Virus in-
fection in ferrets produced acute to subacute (5 dpi) tracheitis
and bronchointerstitial pneumonia that was more severe at 5 dpi.
The airways (trachea, bronchi, bronchioles) were minimally to
mildly affected. It was in the alveolar spaces/interstitium where
the lesions were most prominent. Acute to subacute bron-
chointerstitial pneumonia was characterized by expansion of the
alveolar septae with fibrin, edema, and a mixed inflammatory
infiltrate comprised of lymphocytes, plasma cells, and macro-
phages with fewer neutrophils. The lesions were moderate-to-
severe in 1P10 and 2P10 (Fig. 2), with occasional foci of alveolar
septal necrosis, multifocal hemorrhage, and alveolar hyaline
membranes. In the 1P10 ferrets the inflammation was more se-
vere, although there was less hemorrhage. The bronchointer-
stitial pneumonia was mild-to-moderate in 1WF10 and 2WF10
ferrets. Tracheal lesions consisted of minimal-to-mild acute in-
flammation characterized by submucosal edema with sparse in-
flammatory infiltrates. In 2WF10 ferrets the lesions were slightly
more advanced as there was mild multifocal subacute tracheitis
characterized by mild-to-moderate submucosal edema, with
multifocal intraepithelial lymphoplasmacytic infliltrates with
fewer neutrophils and macrophages. There was occasional in-
dividual epithelial cell necrosis, occasional cilia loss, and endo-
thelial cell hypertrophy in the submucosal blood vessels.
NP viral antigen was localized by immunohistochemistry in
lung and tracheal samples from 5 dpi. NP was clearly seen in the
alveolar septae in regions characterized by interstitial pneumonia
(Fig. S1). Areas of the lungs that did not show alveolar expan-
sion, particularly in regions of the 2WF10-infected lung, did not
show positive NP reaction. The airways, most notably the trachea
(Fig. S1), showed far less NP production. Typically, virus in the
trachea was mostly localized to the subepithelial mucosal layer,
with infrequent localization to the epithelial layer. Virus was not
located in the trachea of the WF10-infected ferrets and is con-
sistent with the minimal pathology seen.
The four viruses grew to similar titers in inoculated (DI) fer-
rets and were cleared in a similar time frame (Fig. 3, red lines).
Additionally, all four viruses were able to transmit to direct
contact (DC) ferrets (Fig. 3, blue lines), which shed virus and
cleared the infection in a similar fashion. Differences were seen,
however, in the respiratory contact (RC) ferrets (Fig. 3, green
lines). Only three viruses were able to transmit in RC ferrets.
Differences were apparent in the efficiency of respiratory droplet
transmission. The 1P10 transmitted the fastest (4–6 dpi) and
grew to the highest titers of any of the RC groups (Fig. 3A). The
2P10 transmitted slower (6–8 dpi) and grew to slightly lower
titers (Fig. 3B). The 1WF10 failed to transmit to ferrets via re-
spiratory droplets (Fig. 3C). Finally, the 2WF10 showed a more
diverse transmission profile (Fig. 3D). The first RC ferret be-
came positive for infection 6 d before the other RC, but the virus
grew to a lower titer. Ferrets lost weight and had at least 1 d of
observed fever (Table S1), with the exception being the two RC
ferrets for the 1WF10 virus that did not become infected. Ad-
ditional signs of disease observed included increase in sneezing,
mild depression, and mild diarrhea. Despite the small number of
animals used, our studies clearly show significant compatibility
between the HA of H9 influenza viruses and the pH1N1 back-
bone to produce pathology and transmission in the ferret model.
Molecular Changes Associated with Transmission. Nasal washes
from RC ferrets on the day of peak shedding were passed once in
MDCK cells and then sequenced to determine if genetic changes
had arisen that could account for differences seen in titer and
transmission between and within the infected groups. The virus
recovered from 1P10 was found to have accrued no mutations in
the ferrets. The 2P10 showed two nonsynonymous mutations that
were identical in the two RC ferrets. The alanine residue at
from WF10 (H9N2). (B) Replication of four reassortant viruses in MDCK cells. Six-well plates of confluent MDCK cells were inoculated with 0.1 multiplicity of
infection of either 1P10 (red), 2P10 (green), 1WF10 (blue), or 2WF10 (black). Supernatants were harvested twice a day for 3 d and titered. Mean titer and SD
were calculated. (C) Plaque morphology in MDCK cells for 1P10, 2P10, 1WF10, and 2WF10. Cells were infected with 1 × 10−6dilution of stock virus except for
the 1P10, in which a 1 × 10−5dilution was used because of smaller plaque size.
Characteristics of H9N2:pH1N1 viruses in vitro. (A) Genes colored black come from pdmH1N1. Red genes originate from P10 (H9N2). Green genes are
Kimble et al.PNAS
| July 19, 2011
| vol. 108
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position 30 of the NA protein was mutated to a threonine
(A30T) and the threonine residue at position 58 of PB2 was
altered to an isoleucine (T58I). The 2WF10 also showed two
nonsynonymous mutations. Serine at position 261 of the PB1 was
changed to an asparagine (S261N) and was seen in both RC
ferrets. Finally, valine at position 104 of the HA protein was
switched to alanine (V104A). The V104A mutation was only
seen in the 2WF10 RC ferret with significantly reduced speed of
transmission (Fig. 3D, green line with triangle).
mutations was performed on viral RNA samples obtained directly
from nasal washes from every positive sample (Fig. 4). The NA
A30T mutation in the RC2P10 group was found in both ferrets on
the first day virus was shed. There was a reversion to wild-type
(A30) on the final 2 d of shedding for one RC ferret, but the other
consistently shed virus with the mutation (T30) on all days. The
mutation was never detected in the DI or DC ferrets. The PB2
T58I mutation of the RC2P10 group arose posttransmission
for both RC ferrets. Likewise, the PB1 S261N mutation in the
RC2WF10 ferrets appeared days after transmission. The HA
V104A mutation seen in only one RC2WF10 ferret occurred in
the DC2WF10 ferret associated with it on the last day of shedding
(9 dpi). The RC2WF10 ferret began shedding virus with the A104
wild-type (V104) for the final 2 d of shedding.
In this study we tested the compatibility between the HA of an H9
subtype influenza virus and the rest of genes from a prototypical
H9N2 are compatible for reassortment and capable of creating
viruses with the ability to infect and transmit via respiratory con-
tact in ferrets. Previous results indicate that an H9N2:H3N2
(2WF10:6M98) reassortant could infect and transmit to DC fer-
rets. However, transmission to RC ferrets only occurs after serial
passage and adaption in ferrets (P10) (15). Interestingly, we show
that the wild-type WF10 surface genes in the background of the
pH1N1 viruscan infect andtransmitto RCferretswithno need of
efficient transmission of pH1N1 viruses in the ferret model. Nev-
ertheless, adapting the H9N2 surface genes to ferrets results in
viruses with more efficient transmission profiles, similar upper
respiratory tract pathology, and increased lung pathology. These
advantage in more than one virus background.
These viruses differ only in the origin of their NA segment. The
1WF10 (pH1N1 NA) shows reduced titer (Fig. 1) compared with
the 2WF10 (H9N2 NA). Additionally, the mismatched surface
proteins of the 1WF10 caused a phenotypic change in respiratory
transmission (Fig. 3). This finding indicates that the 2WF10 virus
surface genes of mixed origin. However, and perhaps more im-
portantly, because the 1P10 (H9N1) virus showed the fastest
amino acid changes (P10 mutations T189A in HA1 and G192R
in HA2 compared with WF10), provide the necessary balance
between HA and NA activities to improve viral fitness and trans-
mission. This phenomenon has been seen in pandemic viruses
before. The 1957 H2N2 pandemic arose from a reassortment in
genes; however, the H3 HA of avian origin was perfectly com-
patible with the N2 NA from the human strain in the 1968 H3N2
pandemic (22, 23).
These results also support the idea that the TRIG cassette
confers an infection/transmission advantage to a virus regardless
of surface genes. The H9N2 with seasonal H3N2 internal genes
could not transmit to respiratory contacts. However, the same
surface genes with the TRIG cassette transmitted via respiratory
contact. These results are consistent with the fact that, after 80 y
as the dominant subtype in swine population in North America,
the classic swine H1N1 was replaced in a few years by an as-
sortment of TRIG viruses.
Pathologically, these viruses are similar to the seasonal strains
of human influenza. The infected lungs show mild-to-moderate
bronchointerstitial pneumonia similar to what is seen with sea-
sonal H3N2 (24), seasonal H1N1, and pH1N1 (25, 26) infections
in ferrets. Tracheal pathology was again similar to pathology
seen in typical human virus infections (24–26). Recently, Sun
et al. determined the growth characteristics and pathology as-
sociated with 127 H9 containing H9N2:pH1N1 reassortants in
mice (27). Although ∼25% of these ressortants showed similar
pathology to either parental virus, about 10% showed pathoge-
nicity higher than either virus. These results and ours highlight
the potential for a H9:pH1N1 reassortant virus with increased
pathogenicity and respiratory transmissibility.
Although amino acid changes were identified in RC contact
ferrets for some of these viruses, it is unlikely that these changes
were determinants for transmission because they were not found
in nasal washes from DI or DC ferrets. We do not know if these
four artificially created reassortants would be created during
a coinfection in the field. It is unknown if these reassortants would
be the most fit or if some other combination would be more
suitable. It needs to be determined if these or some other reas-
sortant virus would out-compete either parental virus in a naive
host and what effects preexisting immunity would have. These
experiments were performed in ferrets, a model of human in-
fection, but other intermediate species, such as swine or poultry,
might act as better hosts for reassortment. Our results highlight
the need to develop better H9N2 surveillance in both swine and
humans as these viruses continue to show features consistent with
with 1 × 106TCID50of either 1P10, 2P10, 1WF10, or 2WF10 and tissues were
collected at 5 dpi. Samples were cut into 5-μm thick sections and stained
using a standard H&E protocol by Histoserv Inc. (Magnification: 200×.)
Pathology produced by H9N2:pH1N1 viruses. Ferrets were inoculated
| www.pnas.org/cgi/doi/10.1073/pnas.1108058108 Kimble et al.
Materials and Methods
Viruses and Cells. The reverse genetic systems for WF10 (H9N2), P10 (H9N2),
and pH1N1 viruses have been previously described (28). The plasmid set for
pH1N1 was kindly provided by Ron A. Fouchier, Erasmus Medical Center,
Rotterdam, The Netherlands. The viruses were generated by reverse genetics
as previously reported (28). Virus stocks were produced in MDCK cells. Full-
length sequencing of viral stocks was performed to verify gene combina-
tions and later for mutation analysis of respiratory droplet contact samples.
Sequences were generated using the Big Dye Terminator v3.1 Cycle Se-
quencing kit 1 in a 3500 Genetic Analyzer (Applied Biosystems).
Four reassortant viruses were generated: the 1WF10 virus encodes the HA
from WF10 and the remaining seven genes from pH1N1; the 2WF10 virus
contains the HA and NA from WF10 and the remaining six genes of pH1N1;
the 1P10 virus contains the HA from (P10) and the other seven genes from
pH1N1, whereas the 2P10 contains the HA and NA genes from P10 and the
remaining six genes from pH1N1. The median TCID50of each virus as well as
titers for the growth curve experiments was determined in MDCK cells.
Plaque Assays. Briefly, confluent MDCK cell monolayers in six-well plates were
infected with 10-fold dilutions of virus for 1 h at 37 °C. Cells were washed
twice with PBS and covered with an overlay of modified Eagle’s medium
containing 0.9% agar, 0.02% BSA, 1% glutamine, and 1 μg/mL trypsin. After
3 d of incubation at 37 °C, 5% CO2, the overlays were removed and the cells
were stained with Crystal violet.
Infection and Transmission in Ferrets. Infection and transmission were carried
out as described previously (7). Complete description of the experimental
approach in ferrets is provided in SI Materials and Methods. Briefly, 3- to
site in which a mutation was observed during peak shedding compared with the wild-type virus. Solid lines indicate days in which sequences were performed.
Dotted lines indicated days in which viruses were detected but no sequences were generated (PB2 T58I DI and DC 2P10). Amino acids are indicated in color.
A/V, T/I, and S/N indicate mixed virus population on the date shown.
Amino acid mutations during transmission of H9N2:pH1N1 viruses. Sequences from virus-positive nasal washes were generated for every ferret at the
dpi, one inoculated ferret was moved to a clean isolator with a naive ferret in direct contact (DC, blue lines). Additionally, another naive ferret was placed in
the same isolator in a manner such that no direct contact was possible, only respiratory droplet contact (RC, green lines). Nasal washes were collected daily
and titered in MDCK cells. Each virus was tested in duplicate.
Nasal wash titers from DI, DC, and RC ferrets. Ferrets (red lines) were infected with 1× 106TCID50of 1P10 (A), 2P10 (B), 1WF10 (C), or 2WF10 (D). At 1
Kimble et al. PNAS
| July 19, 2011
| vol. 108
| no. 29
7-mo-old ferrets were used in which a temperature transponder was in- Download full-text
troduced. Transmission studies were performed in an ABSL3+facility in wire
cages inside HEPA-filtered isolators (7). Animal studies were approved by the
Animal Care and Use Committee of the University of Maryland (protocol RO-
09-93). Each experiment consisted of three ferrets in duplicate for each virus.
One ferret was inoculated intranasally (DI) with 106TCID50of virus. At 1 dpi,
two naive ferrets were added to the cage. One ferret was added in direct
contact (DC) with the DI ferret, and the second naive ferret was added to the
other half of the cage separated by two layers of thin wire mesh allowing
only respiratory contact (RC). Body weight and temperature were measured
daily and nasal washes were collected for 14 dpi (except where noted). Nasal
washes were collected as described (7) and tested for virus by FluDetect
(Synbiotics Corp.), aliquoted, and stored at −80 °C until use. Seroconversion
was detected at 14 dpi. Two additional ferrets were infected with each virus
for pathology and virus localization at 3 and 5 dpi.
ACKNOWLEDGMENTS. We thank Troy Sutton, Lindomar Pena, and Yonas
Araya for their help with animal studies; Jianqiang Ye for assistance with the
ELISA assays; Amy Vincent for her assistance with the immunohistochemistry
protocol; and Theresa Wolter-Marth and Andrea Ferrero for coordination of
Services and 2007-04981 from the National Institute of Food and Agriculture-
US Department of Agriculture, and Contract HHSN266200700010C from the
National Institute of Allergy and Infectious Diseases-National Institutes of
Health, and a Cooperative Agreement from the US Department of Agricul-
ture-Agricultural Research Service.
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| www.pnas.org/cgi/doi/10.1073/pnas.1108058108Kimble et al.