Influenza Virus Transmission Is Dependent
on Relative Humidity and Temperature
Anice C. Lowen1*, Samira Mubareka1, John Steel1, Peter Palese1,2*
1 Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America, 2 Department of Medicine, Mount Sinai School of Medicine,
New York, New York, United States of America

Using the guinea pig as a model host, we show that aerosol spread of influenza virus is dependent upon both ambient
relative humidity and temperature. Twenty experiments performed at relative humidities from 20% to 80% and 5 8C,
20 8C, or 30 8C indicated that both cold and dry conditions favor transmission. The relationship between transmission
via aerosols and relative humidity at 20 8C is similar to that previously reported for the stability of influenza viruses(except at high relative humidity, 80%), implying that the effects of humidity act largely at the level of the virus
particle. For infected guinea pigs housed at 5 8C, the duration of peak shedding was approximately 40 h longer thanthat of animals housed at 20 8C; this increased shedding likely accounts for the enhanced transmission seen at 5 8C. To
investigate the mechanism permitting prolonged viral growth, expression levels in the upper respiratory tract ofseveral innate immune mediators were determined. Innate responses proved to be comparable between animals
housed at 5 8C and 20 8C, suggesting that cold temperature (5 8C) does not impair the innate immune response in thissystem. Although the seasonal epidemiology of influenza is well characterized, the underlying reasons for
predominant wintertime spread are not clear. We provide direct, experimental evidence to support the role ofweather conditions in the dynamics of influenza and thereby address a long-standing question fundamental to theunderstanding of influenza epidemiology and evolution.
Citation: Lowen AC, Mubareka S, Steel J, Palese P (2007) Influenza virus transmission is dependent on relative humidity and temperature. PLoS Pathog 3(10): e151. doi:10.
1371/journal.ppat.0030151

experiments performed in the winter months yielded atransmission rate of 58.2%; in contrast, a rate of only
34.1% was observed in the summer months [10]. While thesedata suggested that the seasonal inﬂuences acting on humans
also affect laboratory mice, no mechanism to explain theobservations was identiﬁed.
Herein, we directly tested the hypotheses that ambient air
temperature and RH impact the efﬁciency with whichinﬂuenza virus is spread. As a mammalian animal model weused Hartley strain guinea pigs, which we have recently shown
to be highly susceptible to infection with human inﬂuenza
viruses [11]. Importantly, we also found that naı̈ve guinea pigs
readily become infected when exposed to inoculated guinea
pigs, unlike mice, which do not efﬁciently transmit inﬂuenza
virus [11]. Thus, by housing infected and naı̈ve guinea pigs
together in an environmental chamber, we were able to assess
the efﬁciency of transmission under conditions of controlled
RH and temperature. Our data show that both RH andtemperature do indeed affect the frequency of inﬂuenza virustransmission among guinea pigs, although via apparently
differing mechanisms.

Introduction
Inﬂuenza A virus, of the family Orthomyxoviridae, carries
an RNA genome consisting of eight segments of negativestranded RNA. This genome encodes one or two nonstructural proteins and nine structural proteins, which,
together with a host cell–derived lipid envelope, comprisethe inﬂuenza virus particle. Inﬂuenza virus causes widespread
morbidity and mortality among human populations worldwide: in the United States alone, an average of 41,400 deaths
and 1.68 million hospitalizations [1] are attributed toinﬂuenza each year. In temperate regions like the UnitedStates, this impact is felt predominantly during the wintermonths; that is, epidemics recur with a highly predictableseasonal pattern. In northern latitudes, inﬂuenza viruses
circulate from November to March, while in the southernhemisphere inﬂuenza occurs primarily from May to September [2]. Tropical regions, by contrast, experience inﬂuenza
throughout the year, although increased incidence has beencorrelated with rainy seasons [2,3]. Despite extensive documentation of the seasonal cycles of inﬂuenza and curiosity
as to their causes, little concrete data is available to indicate
why inﬂuenza virus infections peak in the wintertime.
Theories to explain the seasonal variation of inﬂuenza havetherefore proliferated over the years (reviewed in [4]).
Current hypotheses include ﬂuctuations in host immune
competence mediated by seasonal factors such as melatonin[5] and vitamin D [6] levels; seasonal changes in host behavior,such as school attendance, air travel [7], and indoor crowding
during cold or rainy weather; and environmental factors,
including temperature [8], relative humidity (RH), and the
direction of air movement in the upper atmosphere [9]. In
early studies using mouse-adapted strains of inﬂuenza virus,
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Editor: Ralph S. Baric, University of North Carolina, United States of America
Received July 11, 2007; Accepted September 5, 2007; Published October 19, 2007
Copyright: Ó 2007 Lowen et al. This is an open-access article distributed under theterms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original authorand source are credited.
Abbreviations: Ct, cycle threshold; p.i., post-inoculation; PFU, plaque-forming
units; RH, relative humidity* To whom correspondence should be addressed. E-mail: anice.lowen@mssm.edu(ACL), peter.palese@mssm.edu (PP)1470

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Seasonal Factors Affecting Spread of Influenza

Author Summary
In temperate regions influenza epidemics recur with marked
seasonality: in the northern hemisphere the influenza season spans
November to March, while in the southern hemisphere epidemics
last from May until September. Although seasonality is one of themost familiar features of influenza, it is also one of the least
understood. Indoor crowding during cold weather, seasonal
fluctuations in host immune responses, and environmental factors,
including relative humidity, temperature, and UV radiation have all
been suggested to account for this phenomenon, but none of thesehypotheses has been tested directly. Using the guinea pig model,
we have evaluated the effects of temperature and relative humidity
on influenza virus spread. By housing infected and naı̈ve guinea pigs
together in an environmental chamber, we carried out transmission
experiments under conditions of controlled temperature andhumidity. We found that low relative humidities of 20%–35% weremost favorable, while transmission was completely blocked at a highrelative humidity of 80%. Furthermore, when guinea pigs were kept
at 5 8C, transmission occurred with greater frequency than at 20 8C,
while at 30 8C, no transmission was detected. Our data implicate low
relative humidities produced by indoor heating and cold temperatures as features of winter that favor influenza virus spread.

Results
Twenty replicate experiments were performed in which all
factors remained constant except for the RH and/or temperature inside the environmental chamber. Each experiment
involved eight guinea pigs, and transmission under each set of
conditions was assessed in duplicate. The arrangement ofanimals in the environmental chamber is illustrated in Figure
1. Virus contained in nasal wash samples collected onalternating days post-inoculation (p.i.) was titrated by plaqueassay to determine the infection status of each animal. Serumsamples were collected from each animal prior to infection
and on day 17 p.i., and seroconversion was assessed byhemagglutination inhibition assay (results in Table S1).
In general, the behavior (level of activity, food and water
consumption, symptoms of infection) of guinea pigs was notobserved to change with the ambient relative humidity.
Likewise, animals housed at 5 8C behaved in a similar mannerto those housed at 20 8C. Guinea pigs kept at 30 8C consumed
more water than those housed under cooler conditions, andappeared lethargic. Consistent with our previous observations [11], inﬂuenza virus–infected guinea pigs did not display
detectable symptoms of disease (e.g., weight loss, fever,sneezing, coughing) during the experiments described.

Figure 1. Arrangement of Infected and Exposed Guinea Pigs inEnvironmental Chamber
In each experiment, eight animals were housed in a Caron 6030
environmental chamber. Each guinea pig was placed in its own cage,
and two cages were positioned on each shelf. Naı̈ve animals were placed
behind infected animals, such that the direction of airflow was toward
the naı̈ve animals. The cages used were open to airflow through the top
and one side, both of which were covered by wire mesh. Althoughinfected and exposed guinea pigs were placed in pairs, air flowed freely
between shelves, allowing transmission to occur from any infected to
any naı̈ve animal.
doi:10.1371/journal.ppat.0030151.g001

we believe this variation is due to the stochastic nature of
infection. However, while most infection events were theproduct of primary transmission from an inoculated animal,
others could be the result of secondary transmission from apreviously infected, exposed guinea pig. With the exceptionof the lack of transmission at 80% RH, the observed
relationship between transmission and RH is similar to thatbetween inﬂuenza virus stability in an aerosol and RH [12],suggesting that at 20 8C the sensitivity of transmission tohumidity is due largely to virus stability.

Transmission Efficiency Is Dependent on Relative
Humidity
The results of transmission experiments performed at 20
8C and ﬁve different RHs (20%, 35%, 50%, 65%, and 80%)
indicated that the efﬁciency of aerosol spread of inﬂuenza
virus varied with RH. Transmission was highly efﬁcient(occurred to three or four of four exposed guinea pigs) at
low RH values of 20% or 35%. At an intermediate RH of
50%, however, only one of four naı̈ve animals contracted
infection. Three of four exposed guinea pigs were infected at
65% RH, while no transmission was observed at a high RH of
80% (Figure 2). Where transmission was observed, thekinetics with which infection was detected in each exposed
animal varied between and within experiments. To an extent,
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Transmission Efficiency Is Inversely Correlated with
Temperature
To test whether cold temperatures would increase transmission, the ambient temperature in the chamber was
lowered to 5 8C and experiments were performed at 35%–
80% RH. Overall, transmission was more efﬁcient at 5 8C:
75%–100% transmission occurred at 35% and 50% RH, and1471

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Seasonal Factors Affecting Spread of Influenza

Figure 2. Transmission of Influenza Virus from Guinea Pig to Guinea Pig Is Dependent on Relative Humidity
Titers of influenza virus in nasal wash samples are plotted as a function of day p.i. Overall transmission rate and the RH and temperature conditions ofeach experiment are stated underneath the graph. Titers from intranasally inoculated guinea pigs are represented as dashed lines; titers from exposedguinea pigs are shown with solid lines. Virus titrations were performed by plaque assay on Madin Darby canine kidney cells.
doi:10.1371/journal.ppat.0030151.g002

Innate Immune Response Is Not Affected by Low Ambient
Temperature

50% transmission was observed at 65% and 80% RH (Figure
3A–3H). The statistical signiﬁcance of differences in transmission rates at 5 8C compared to 20 8C was assessed using the
Fisher’s exact test. While at 35% and 65% RH the difference
was not found to be signiﬁcant, at both 50% and 80% RH,
transmissibility at 5 8C was found to be greater than that at 20
8C (p , 0.05). Conversely, when the ambient temperature wasincreased to 30 8C and transmission experiments carried out
at a low RH of 35%, no transmission was observed (Figure 3I
and 3J).

The observed differences in viral titers between animals
housed at 20 8C and 5 8C suggested that cold temperature has
a negative impact on host defenses early in infection. We
therefore used real-time PCR to assess innate immune
function in guinea pigs housed at both temperatures. The
nasal turbinates were removed from three animals on each ofdays 1, 2, 3, 5, and 7 p.i. RNA extracted from these tissues was
subjected to reverse transcription with an oligo dT primer
followed by quantitative, real-time PCR with target-speciﬁcprimers. Mx1, TLR3, MDA-5, IRF7, STAT1, RANTES, MCP1,
MCP3, and IL-1b were found to be upregulated in response toinfection in animals housed under both conditions (Figure 5).
Peak expression was seen at day 3 p.i. for all of these genes
except RANTES, which increased steadily up to day 7 p.i., andMDA-5 and STAT1, which showed similarly high levels of
expression on days 2 and 3 p.i. While RANTES expression waselevated more in animals at 5 8C than in animals at 20 8C (p ¼
0.09), peak levels of IL-1b and MDA-5 were higher in guinea
pigs at 20 8C (p , 0.05). TLR3, MCP1, MCP3, and MDA-5 were
all expressed to signiﬁcantly higher levels in animals at 5 8C
than at 20 8C late in infection (day 5 and/or 7 p.i.), an
observation that may simply reﬂect the levels of virus present
at these time points. No signiﬁcant upregulation wasobserved in either group of guinea pigs for TNFa, TBK1,
IRF5, or IFNc (Figure 5). Overall, these data indicate that
innate immunity was not greatly impaired in guinea pigs
housed at 5 8C relative to those at 20 8C. These ﬁndings argue
against the idea that increased physiological stress experi-

Viral Shedding Is Increased in Animals at 5 8C
We also observed that, although changes in RH did notaffect the kinetics of viral shedding in inoculated guinea pigs,changes in temperature did. At 5 8C and all RHs tested, theintranasally inoculated guinea pigs shed higher titers of virus
on days 4, 6, and 8 post-infection; most notably, peak
shedding was extended in these animals by 2 d relative toguinea pigs housed at 20 8C (Figure 4). We tested whether the
peak duration of viral shedding was statistically longer at 5 8C
than at 20 8C by comparing the last day on which the nasal
wash titer was  106 plaque-forming units (PFU)/ml for eachguinea pig housed at 5 8C and at 20 8C. The average of this lastday value was 3.93 6 0.63 d for animals at 5 8C and 2.21 6
0.61 d for animals at 20 8C. By the Student’s t-test for two
independent samples, this value was signiﬁcantly higher foranimals housed at 5 8C than those housed at 20 8C (p ,0.0005). The increased duration of peak shedding at 5 8C is
most likely the cause of increased transmission under cold
conditions.
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Seasonal Factors Affecting Spread of Influenza

Figure 3. Transmission of Influenza Virus from Guinea Pig to Guinea Pig Is Highly Efficient at 5 8C and Blocked at 30 8C
Titers of influenza virus in nasal wash samples are plotted as a function of day p.i. Overall transmission rate and the RH and temperature conditions ofeach experiment are stated underneath the graph. Titers from intranasally inoculated guinea pigs are represented as dashed lines; titers from exposedguinea pigs are shown with solid lines.
doi:10.1371/journal.ppat.0030151.g003

Mechanism of Variation in Transmission with Relative
Humidity

enced under cold conditions leads to a weakening of theimmune response [13].

Three mechanisms could potentially explain the observed
inﬂuence of RH on transmission. The ﬁrst acts at the level ofthe host: breathing dry air could cause desiccation of thenasal mucosa, leading to epithelial damage and/or reduced
mucociliary clearance, which would in turn render the hostmore susceptible to respiratory virus infections. Long-term
exposure to dry air is likely to affect inﬂuenza virus growth in
the upper respiratory tract, and may indeed play a role ininﬂuenza seasonality. Nevertheless, based on the brevity ofthe exposure of naı̈ve guinea pigs to dry air before becoming
infected (less than 72 h), we do not believe that thismechanism played a signiﬁcant role in the observed effects.
The second mechanism acts at the level of the virus particle.
The stability of inﬂuenza virions in an aerosol has beenreported to vary with RH [12,14,15]. The most recent of thesereports [12] shows viral stability to be maximal at low RH(20%–40%), minimal at intermediate RH (50%), and high at
elevated RH (60%–80%). The similarity between these dataon stability and our own on transmission is striking, andsuggests that the stability of the virus in aerosols is a key
determinant of inﬂuenza virus transmission (with the
exception of the absence of transmission at high RH). The
third mechanism acts at the level of the vehicle, therespiratory droplet. At low RH, evaporation of water fromexhaled bioaerosols would occur rapidly, leading to theformation of droplet nuclei; conversely, at high RH, small
respiratory droplets would take on water, increase in size andsettle more quickly out of the air [16]. Droplet nuclei are lessthan 5 lm in diameter and, unlike larger droplets, they

Discussion
We have shown that the transmission of a human strain of
inﬂuenza virus between guinea pigs, a highly susceptible
mammalian species, is acutely sensitive to conditions of RH
and temperature. Our data suggest that these two environmental factors could contribute to the seasonality of
epidemic inﬂuenza.

Figure 4. Guinea Pigs Housed at 5 8C Shed Influenza Virus at Higher
Titers on Days 4, 6, and 8 p.i. Than Guinea Pigs Housed at 20 8C
Average viral titers in nasal wash samples collected from animals housed
at either 5 8C or 20 8C and all RH tested are plotted as function of timepost-infection. Error bars indicate standard deviation; * indicatesstatistically significant difference between titers at 5 8C and 20 8C, withp  0.005.
doi:10.1371/journal.ppat.0030151.g004
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Figure 5. Antiviral and Pro-Inflammatory Responses Are Similar between Guinea Pigs Housed at 5 8C and 20 8C
Levels of the indicated mRNA transcripts present in nasal turbinates of infected guinea pigs housed at 5 8C (black bars) or 20 8C (grey bars) were
quantified by real-time PCR of reverse transcribed mRNA. RNA levels were normalized to b-actin and are expressed as fold-induction over mock-infectedguinea pig. Error bars represent standard deviation. * indicates a statistically significant (p , 0.05) difference between 5 8C and 20 8C.
doi:10.1371/journal.ppat.0030151.g005

improved transmission at low temperature could be due to aneffect on the host. This effect may act at the level of primary,
physical barriers to infection. Cooling of the nasal mucosa isthought to increase the viscosity of the mucous layer andreduce the frequency of cilia beats [8]. In this way, breathing
cold air would slow mucociliary clearance and thereby
encourage viral spread within the respiratory tract. An
alternative possibility is that, also due to cooling of themucosal layer, virus residing in the upper airways is morestable when infected animals are kept at 5 8C. Improved
persistence of released virus would increase the amount of
viable virus shed, and would furthermore augment ampliﬁ-

remain airborne for an extended period of time, thereby
increasing the opportunity for transmission of pathogens
they carry [17]. Our data suggest that, in this model system,
the formation of droplet nuclei is important to transmission;
we propose that at high RH (80%) exhaled respiratory
droplets settle too rapidly to contribute to inﬂuenza virus
spread. Based on our data, we present a model in Figure 6 of
how transmission is affected by changes in RH.

Mechanism of Improved Transmission at 5 8C
Despite the apparent ﬁtness of animals housed at 5 8C,
increased viral shedding under these conditions suggests that
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transmission among guinea pigs housed in the same cage (i.e.,
direct contact or fomite-mediated infection) is affected by
RH and temperature. If transmission in this setting is not
impaired at 30 8C, this may suggest that contact-based spread
predominates in the tropics, whereas aerosol transmission
plays a larger role in temperate climates.
Using the guinea pig model, we report a systematic analysisof the effects of RH and temperature on inﬂuenza virustransmission in a controlled setting. These data provide
valuable insight into the seasonality of inﬂuenza and will aid
further research into both local and global patterns ofinﬂuenza virus spread within and between human populations. Our ﬁndings furthermore suggest a novel means ofinfection control for an important human pathogen. Inﬂuenza virus transmission indoors could potentially be curtailed
by simply maintaining room air at warm temperatures (.208C) and either intermediate (50%) or high (80%) RHs.

Materials and Methods
Virus. Inﬂuenza A/Panama/2007/99 virus (Pan/99; H3N2) was kindly
supplied by Adolfo Garcı́a-Sastre and was propagated in Madin Darby
canine kidney cells.
Animals. Female Hartley strain guinea pigs weighing 300–350 g
were obtained from Charles River Laboratories. Animals wereallowed free access to food and water and kept on a 12-h light/darkcycle. Guinea pigs were anesthetized for the collection of blood andof nasal wash samples, using a mixture of ketamine (30 mg/kg) andxylazine (2 mg/kg), administered intramuscularly. All procedures were
performed in accordance with the Institutional Animal Care andUsed Committee guidelines. During guinea pig transmission experiments, strict measures were followed to prevent aberrant crosscontamination between cages: sentinel animals were handled before
inoculated animals, gloves were changed between cages, and work
surfaces were sanitized between guinea pigs.
Transmission experiments. The term ‘‘aerosol’’ is used herein todescribe respiratory droplets of all sizes. The term ‘‘droplet nuclei’’ isused to refer to droplets that remain airborne (typically less than 5
lm in diameter).
Each transmission experiment involved eight guinea pigs. On day
0, four of the eight guinea pigs were inoculated intranasally with 103
PFU of inﬂuenza A/Panama/2007/99 virus (150 ll per nostril in
phosphate buffered saline [PBS] supplemented with 0.3% bovine
serum albumin [BSA]) and housed in a separate room from theremaining animals. At 24 h p.i., each of the eight guinea pigs wasplaced in a ‘‘transmission cage’’, a standard rat cage (Ancare R20
series) with an open wire top, which has been modiﬁed by replacing
one side panel with a wire grid. The transmission cages were thenplaced into the environmental chamber (Caron model 6030) with two
cages per shelf, such that the wire grids opposed each other (Figure1). In this arrangement, the guinea pigs cannot come into physicalcontact with each other. Each infected animal was paired on a shelfwith a naı̈ve animal. The guinea pigs were housed in this way for 7 d,after which they were removed from the chamber and separated. On
day 2 p.i. (day 1 post-exposure) and every second day thereafter up to
day 12 p.i., nasal wash samples were collected from anesthetizedguinea pigs by instilling 1 ml of PBS-BSA into the nostrils andcollecting the wash in a Petri dish. Titers in nasal wash samples were
determined by plaque assay of 10-fold serial dilutions on Madin
Darby canine kidney cells. Serum samples were collected from eachanimal prior to infection and on day 17 post-infection, andseroconversion was assessed by hemagglutination inhibition assay.
All transmission experiments reported herein were performed
between September 2006 and April 2007.
Analysis of expression levels of mediators of innate immunity.
Guinea pigs were inoculated with 103 PFU of Pan/99 virus intranasallyand immediately housed under the appropriate conditions (5 8C or 20
8C and 35% RH). At days 1, 2, 3, 5, and 7 post-infection, three guinea
pigs were killed and their nasal turbinates removed. Tissues wereplaced immediately in RNAlater reagent (Qiagen), and stored at 4 8C
for 1 to 5 d. RNA was extracted from equivalent masses of tissue using
the RNAeasy Protect Mini kit (Qiagen) and subjected to DNAsetreatment (Qiagen). One microgram of RNA was subjected to reverse
transcription using MMLV reverse transcriptase (Roche). One

Figure 6. Variation of Transmission Efficiency with Relative Humidity: A
Model
At 20 8C (dashed line), transmission efficiency is highest at low RH, wheninfluenza virions in an aerosol are relatively stable, and desiccation of
exhaled respiratory droplets produces droplet nuclei. Transmission isdiminished at intermediate RH when virus particles are relatively
unstable, but improves in parallel with influenza virus stability at higher
humidities. At high RH, evaporation from exhaled particles is limited,
respiratory droplets settle out of the air, and transmission is blocked. At 5
8C (solid line), transmission is more efficient than at 20 8C, but is reduced
to a rate of 50% at higher humidities.
doi:10.1371/journal.ppat.0030151.g006

cation of virus in the nasal passages through re-infection. The
block in transmission at 30 8C and 35% RH could be
explained by the opposite effect: warming of the nasal
mucosa may lead to more rapid inactivation of virus particles.

Implications for Influenza Seasonality
To our knowledge, we demonstrate for the ﬁrst time thatcold temperatures and low relative humidity are favorable tothe spread of inﬂuenza virus. Although other factors likely
contribute to the periodicity of inﬂuenza epidemics, it is clearthat air temperature and RH could play an important role.
Mathematical modeling indicates that only a small seasonal
forcing is required to produce oscillations in infection rate ofhigh amplitude [18]; accordingly, it is possible that theextended exposure of a small proportion of the population
to outdoor winter conditions would comprise a sufﬁcient
force to create seasonal epidemics. Although the effect may
therefore be small and difﬁcult to detect, the importance of
RH and temperature in the epidemiology of human inﬂuenza
could be veriﬁed based on surveillance data. To this end,
surveillance data with greater spatial resolution (i.e., on aregional rather than national scale) and concurrent meteorological data is needed.
The observed lack of transmission among animals housed
at 30 8C raises the question of how, if our model isrepresentative of human infection, the spread of inﬂuenza
viruses occurs in tropical climates. Experiments to moreclosely examine this question are underway. Our preliminary
data suggest that transmission at 30 8C is not improved at RHs
higher than 35%. It will be interesting to test whether
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Supporting Information

microlitre of the resultant product was used as the template in a
SYBR green (Invitrogen) real-time PCR assay (Roche Light Cycler
480) with Ampli-taq Gold polymerase (Perkin-Elmer). Primers usedwere as follows:b- a c t i n f A A A C T G G A A C G G T G A A G G T G ; b- a c t i n r
CTTCCTCTGTGGAGGAGTGG; Mx1 f CATCCCYTTGrTCATCCAGT;
Mx1 r CATCCCyTTGRTCATCCAGT; MDA-5 f GAGCCAGAGCTGATGARAGC; MDA-5 r TCTTATGWGCATACTCCTCTGG; IL-1b f GAAGAAGAGCCCATCGTCTG; IL-1b r CATGGGTCAGACAACACCAG;RANTES f GCAATGCTAGCAGCTTCTCC; RANTES r TTGCCTTGAAAGATGTGCTG; TLR3 f TAACCACGCACTCTGTTTGC;TLR3 r ACAGTATTGCGGGATCCAAG; TNFa f TTCCGGGCAGATCTACTTTG; TNFa r TGAACCAGGAGAAGGTGAGG; MCP-1 f ATTGCCAAACTGGACCAGAG; MCP-1 r CTACGGTTCTTGGGGTCTTG;
MCP-3 f TCATTGCAGTCCTTCTGTGC; MCP-3 r TAGTCTCTGCACCCGAATCC; IFNc f GACCTGAGCAAGACCCTGAG; IFNc r
TGGCTCAGAATGCAGAGATG; STAT1 f AAGGGGCCATCACATTCAC; STAT1 r GCTTCCTTTGGCCTGGAG; TBK1 f CAAGAAACTyTGCCwCAGAAA; TBK1 r AGGCCACCATCCAykGTTA; IRF5 f
CAAACCCCGaGAGAAGAAG; IRF5 r CTGCTGGGACtGCCAGA; IRF7
f TGCAAGGTGTACTGGGAGGT; IRF7 r TCACCAGGATCAGGGTCTTC (where R ¼ A or G, Y ¼ C or T, W ¼ A or T, K ¼ T or
G). Primer sequences were based either on guinea pig mRNA sequences
available in GenBank (MCP1, MCP3, IL-1b, IFNc, RANTES, TLR3, TNFa,
and b-actin), or on the consensus sequence of all species available in
GenBank (Mx1, MDA-5, IRF5, IRF7, STAT1, and TBK1). Sequencing ofeach PCR product indicated that all primer pairs were speciﬁc for the
expected transcript. Reactions were performed in duplicate andnormalized by dividing the mean value of the cycle threshold (Ct) of bactin expressed as an exponent of 2 (2Ct) by the mean value of 2Ct for thetarget gene. The fold-induction over the mock-infected was thencalculated by dividing the normalized value by the normalized mock
value. Data is represented in Figure 5 as the mean of three like samples(nasal turbinates harvested on the same day p.i. from three guinea pigs) 6
standard deviation.
Statistical analyses. Statistical analyses were performed using
GraphPad Prism 5 software.

Table S1. Seroconversion of Inoculated and Exposed Guinea Pigs
Results of hemagglutination inhibition tests for each transmission
experiment are shown.
Found at doi:10.1371/journal.ppat.0030151.st001 (58 KB DOC).
Accession Numbers
The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) accession numbers of guinea pig genes used for primer design are as
follows: b-actin (AF508792.1); IFNc (AY151287.1); IL-1b (AF119622);
MCP-1 (L04985); MCP-3 (AB014340); RANTES (CPU77037); TLR3
(DQ415679.1); and TNFa (CPU77036).

Acknowledgments
We would like to thank Adolfo Garcı́a-Sastre, Thomas Moran, andChris Basler for helpful discussion, Christopher Narbus for excellent
technical assistance, and Charalampos Valmas for help with statisticalanalyses.
Author contributions. ACL and PP conceived and designed theexperiments. ACL, SM, and JS performed the experiments. All
authors analyzed the data. ACL wrote the paper.
Funding. This work was supported by grants from the W. M. Keck
Foundation, the Centers for Disease Control R21 (U01CI000354–01),
and the Center for Investigating Viral Immunity and Antagonism (1UC19 AI062623–023) (to PP). ACL is a Parker B. Francis Fellow in
Pulmonary Research. SM was supported by Sunnybrook Health
Sciences Centre, Toronto, Canada, and a Ruth L. Kirschstein
Physician Scientist Research Training in Pathogenesis of Viral
Diseases Award (Mary Klotman, P.I.). PP is a senior fellow of the
Ellison Medical Foundation.
Competing interests. The authors have declared that no competinginterests exist.

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