text:
Charting the host adaptation of influenza viruses.
ABSTRACT Four influenza pandemics have struck the human population during the last 100 years causing substantial morbidity and mortality. The pandemics were caused by the introduction of a new virus into the human population from an avian or swine host or through the mixing of virus segments from an animal host with a human virus to create a new reassortant subtype virus. Understanding which changes have contributed to the adaptation of the virus to the human host is essential in assessing the pandemic potential of current and future animal viruses. Here, we develop a measure of the level of adaptation of a given virus strain to a particular host. We show that adaptation to the human host has been gradual with a timescale of decades and that none of the virus proteins have yet achieved full adaptation to the selective constraints. When the measure is applied to historical data, our results indicate that the 1918 influenza virus had undergone a period of preadaptation prior to the 1918 pandemic. Yet, ancestral reconstruction of the avian virus that founded the classical swine and 1918 human influenza lineages shows no evidence that this virus was exceptionally preadapted to humans. These results indicate that adaptation to humans occurred following the initial host shift from birds to mammals, including a significant amount prior to 1918. The 2009 pandemic virus seems to have undergone preadaptation to human-like selective constraints during its period of circulation in swine. Ancestral reconstruction along the human virus tree indicates that mutations that have increased the adaptation of the virus have occurred preferentially along the trunk of the tree. The method should be helpful in assessing the potential of current viruses to found future epidemics or pandemics.
- SourceAvailable from: A. Romero[Show abstract] [Hide abstract]
ABSTRACT: Influenza A is a highly contagious respiratory virus in constant evolution and represents a threat to both veterinary and human public health. IA viruses (IAVs) originate in avian reservoirs but may adapt to humans, either directly or through the spillover to another mammalian species, to the point of becoming pandemic. IAVs must successfully be able to (i) transmit from animal to human, (ii) interact with host cells, and (iii) transmit from human to human. The mechanisms by which viruses evolve, cause zoonotic infections, and adapt to a new host species are indeed complex and appear to be a heterogeneous collection of viral evolutionary events rather than a single phenomenon. Progress has been made in identifying some of the genetic markers mainly associated with virulence and transmission; this achievement has improved our knowledge of how to manage a pandemic event and of how to identify IAVs with pandemic potential. Early evidence of emerging viruses and surveillance of animal IAVs is made possible only by strengthening the collaboration between the public and veterinary health sectors.Influenza and Other Respiratory Viruses 09/2013; 7(s2). · 1.47 Impact Factor
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ABSTRACT: The sociological and biological factors which gave rise to the three pandemic waves of Spanish influenza in England during 1918-19 are still poorly understood. Symptom reporting data available for a limited set of locations in England indicates that reinfection in multiple waves occurred, suggesting a role for loss of infection-acquired immunity. Here we explore the role that changes in host immunity, driven by a combination of within-host factors and viral evolution, may play in explaining weekly mortality data and wave-by-wave symptomatic attack-rates available for a subset of English cities. Our results indicate that changes in the phenotype of the pandemic virus are likely required to explain the closely spaced waves of infection, but distinguishing between the detailed contributions of viral evolution and changing adaptive immune responses to transmission rates is difficult given the dearth of sero-epidemiological and virological data available even for more contemporary pandemics. We find that a dynamical model in which pre-pandemic protection in older "influenza-experienced" cohorts is lost rapidly prior to the second wave provides the best fit to the mortality and symptom reporting data. Best fitting parameter estimates for such a model indicate that post-infection protection lasted of order months, while other statistical analyses indicate that population-age was inversely correlated with overall mortality during the herald wave. Our results suggest that severe secondary waves of pandemic influenza may be triggered by viral escape from pre-pandemic immunity, and thus that understanding the role of heterosubtypic or cross-protective immune responses to pandemic influenza may be key to controlling the severity of future influenza pandemics.Epidemics 09/2014; 8C:18-27. · 2.26 Impact Factor
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ABSTRACT: Please see later in the article for the Editors' Summary.PLoS Medicine 05/2014; 11(5):e1001646. · 15.25 Impact Factor
Page 1
Charting the Host Adaptation of Influenza Viruses
Mario dos Reis,?,1Asif U. Tamuri,?,1Alan J. Hay,2and Richard A. Goldstein*,1
1Division of Mathematical Biology, National Institute for Medical Research, London, United Kingdom
2Division of Virology, National Institute for Medical Research, London, United Kingdom
?These authors contributed equally to this work.
*Corresponding author: E-mail: email@example.com.
Associate editor: Alexei Drummond
Four influenza pandemics have struck the human population during the last 100 years causing substantial morbidity and
mortality. The pandemics were caused by the introduction of a new virus into the human population from an avian or
swine host or through the mixing of virus segments from an animal host with a human virus to create a new reassortant
subtype virus. Understanding which changes have contributed to the adaptation of the virus to the human host is essential
in assessing the pandemic potential of current and future animal viruses. Here, we develop a measure of the level of
adaptation of a given virus strain to a particular host. We show that adaptation to the human host has been gradual with
a timescale of decades and that none of the virus proteins have yet achieved full adaptation to the selective constraints.
When the measure is applied to historical data, our results indicate that the 1918 influenza virus had undergone a period
of preadaptation prior to the 1918 pandemic. Yet, ancestral reconstruction of the avian virus that founded the classical
swine and 1918 human influenza lineages shows no evidence that this virus was exceptionally preadapted to humans.
These results indicate that adaptation to humans occurred following the initial host shift from birds to mammals,
including a significant amount prior to 1918. The 2009 pandemic virus seems to have undergone preadaptation to
human-like selective constraints during its period of circulation in swine. Ancestral reconstruction along the human virus
tree indicates that mutations that have increased the adaptation of the virus have occurred preferentially along the trunk
of the tree. The method should be helpful in assessing the potential of current viruses to found future epidemics or
Key words: adaptive evolution, antigenic shift, virus evolution, H1N1, selective constraints, Spanish flu.
Influenza A is a negative-strand RNA virus with a genome
composed of eight genetic segments encoding 11 proteins.
Influenza A viruses are categorized by the antigenicity
of the two surface glycoproteins, the hemagglutinin
(HA) and neuraminidase, which are differentiated as 16
(H1–H16) and 9 (N1–N9) subtypes, respectively. Although
the natural reservoir of influenza A is waterfowl, where the
virus propagates causing little or no disease (Webster et al.
1992), viruses periodically transmit to other host species
resulting infrequently in a stable lineage in, for example,
poultry, swine, and most significantly, humans. These
host-shift events can result from the transfer of a complete
virus from one host to another or from genetic reassort-
ment, where a chimera is formed by the mixing of genetic
segments from a virus of a different host with genetic seg-
ments of a virus already circulating in the ''new'' host.
Over the last century, four influenza pandemics have re-
sulted from the establishment of a new virus in humans as
a result of the introduction of genetic elements from an
animal virus either in toto or through reassortment causing
substantial morbidity and mortality. In 1918–1919, an influ-
enza A H1N1 virus pandemic struck thehuman population,
infecting a third of the world population and killing 2.5% of
those infected (Taubenberger and Morens 2006), causing
over 50 million deaths (Johnson and Mueller 2002). Around
the same time, a panzootic was observed in swine, which is
thought to have been the origin of the ''classical swine''
lineage observed especially in North America. The timing
and nature of the host-shift events that caused the
near simultaneous human and swine epidemics have been
2005; Antonovics et al. 2006; Gibbs M and Gibbs A 2006;
Taubenberger 2006; dos Reis et al. 2009; Smith, Bahl,
et al. 2009). Reassortment, whereby circulating human vi-
ruses acquired novel avian-like gene segments, resulted in
two further pandemics in 1957 (H2N2) and 1968 (H3N2)
subtype. In 1977, an H1N1 virus reappeared in the human
population and cocirculated with H3N2 until 2009. The re-
emerging virus closely resembled the H1N1 viruses that had
circulated approximately 25 years earlier (Nakajima et al.
1978; dos Reis et al. 2009), suggesting that the virus was
a member of the 1957 lineage and hadbeen held inartificial
evolutionary stasis during this time (Palese 2004).
lineage resulted from a direct transmission from an avian
© The Author(s) 2010. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Open Access
Mol. Biol. Evol. 28(6):1755–1767. 2011doi:10.1093/molbev/msq317Advance Access publication November 25, 20101755
Research article
Page 2
reassortant viruses appeared in pigs in North America that
initially combined genetic elements from human H3N2
(PB1, H3, and N2) with classical swine viruses followed by
the introduction of genetic elements from avian influenza
(PA and PB2) (Zhou et al. 1999). This ''triple-reassortant''
strain then underwent various reassortments acquiring ge-
(N1 and MP) before undergoing a host shift to humans, re-
H1N1 2009). First identified in April 2009 (Centers for Dis-
ease Control and Prevention 2009; Dawood et al. 2009;
Smith, Vijaykrishna, et al. 2009), it quickly spread through-
relevance to humans are listed in table 1.
Although sporadic cross-species transmissions are com-
mon, transmissions leading to the establishment of new
mammalian lineages have been relatively rare. For a virus
to infect, replicate, and transmit efficiently in a different
host species, it must undergo a series of host-specific adap-
tations. It must have a HA protein that can efficiently bind
to and infect the host cell (Rogers et al. 1983; Nobusawa
et al. 1991; Connor et al. 1994; Vines et al. 1998; Matroso-
vich et al. 2000). It also requires a polymerase and nucle-
oprotein complex that can actively replicate the virus
genome within the targeted host cells. This involves the
polymerase and ribonucleoprotein complex interacting
can take place inside the nucleus. The polymerase complex
should also efficiently replicate the virus RNA at the host-
specific body temperature (for reviews, see Naffakh et al.
2008; Ruigrok et al. 2010). The virus must also confront
the various host-specific immune responses. The mecha-
nisms of adaptation are poorly understood, and all virus
proteins may potentially play some role. Host shifts occur
due to a mixture of factors, including virus potential, host
susceptibility, and possibly chance occurrences. Although
a comprehensive understanding of host shifts would re-
quire addressing all these aspects, assessing the degree
of adaptation of the virus to the new host is an important
factor in assessing the potential pandemic risk of new
We recently developed a maximum likelihood phy-
logenetic method to detect and characterize amino acid
locations in influenza virus proteins that evolve under
host-specific constraints (Tamuri et al. 2009). In this paper,
we describe howwe can usethesemeasures to characterize
how well any given virus sequence is adapted to the selec-
tive constraints imposed by avian or human hosts. We fo-
cus on the host shift that led to the 1918 H1N1 pandemic
and the process of adaptation of the viral proteins during
the approximately 70 years that the viruses have circulated
in the human population. We address such questions as
the rate of host adaptation for the individual proteins,
the degree of human adaptation found in currently circu-
lating strains, and how the avian viruses that initiate hu-
man pandemics compare with other avian viruses.
Materials and Methods
Host Adaptation Measure
In addition to identifying locations in influenza proteins
where there is a change in selective constraints following
a host shift from birds to humans, our previous work also
provided us with the expected equilibrium frequency of
amino acid Aiat identified location k evolving in host h,
frequencies to constructa measureof host adaptation. Con-
sider that we have identified N locations in a given protein
where there is a difference in selective constraints in human
and avian hosts. If we assume that the selective constraints
act at the protein level, we can, following Yang and Nielsen
(2008), express the equilibrium frequencies ph
terms of the ''fitness parameters'' for those amino acids
where p?
for the nucleotide found in position l of codon I, and the
sum is over all codons that code for amino acid Ai. With this
expression, we can write Fh
K(Ai) represents the nucleotide biases and the proportionality
constant. Assuming that the fitness effects of the differ-
ent locations are additive, we can create a measure of host
adaptation hhðfSkgÞ of a virus with amino acid sequence
kðAiÞ (Tamuri et al. 2009). We can use these equilibrium
kðAiÞ in
Ilrepresents the background equilibrium frequency
kðAiÞ5KðAiÞ þ logðph
kðAiÞÞ, where
Table 1. Significant Events of Relevance to Recent Human Pandemics.
Late 1970s
Late 1990s
Late 1990s
Event Segments
Resulting Pandemic/
Panzootic Lineage
Classical swine (H1N1)
Spanish flu (H1N1)
Asian flu (H2N2)
Hong Kong flu (H3N2)
Russian flu (H1N1)
Eurasian swine (H1N1)
Reassortant swine (H3N2)
Triple-reassortant swine (H3N2)
Host shift: ? to swine
Host shift: ? to human
Host shift: avian to human
Host shift: avian to human
Reintroduction of human H1N1 virus
Avian to swine
Host shift: human to swine
Host shift: avian to swine
Mixing between swine
All segments
All segments
H3, N2, PB1 from human
H1 from classical swine; N1,
M from Eurasian swine; NS, NP, PA,
PB1, PB2 from Triple-reassortant
All segments2009Swine to humanPandemic H1N1 2009
dos Reis et al. · doi:10.1093/molbev/msq317
Page 3
fSkg, where Skis the amino acid found at identified location k:
kðSkÞÞ þ KðSkÞ?
kðSkÞÞ? þ N?K;
of K(Ai), N times?K, which is only a function of the background
distribution of nucleotides and should not vary significantly
from one sequence to another.
Fully adapted proteins that had equilibrated with the
selective constraints would have amino acid frequencies
at the various sites given by the equilibrium frequencies
acid frequencies at each location given by p0ðAiÞ, the fre-
quency of amino acid Ai averaged over our influenza
sequence database. For convenience, we scale hhðfSkgÞ
so that an ensemble of random proteins have an average
proteins have an average host adaptedness of 1 by
kðAiÞ. We can model random proteins as having amino
Hh5hhðfSkgÞ ? ÆhhæRandom
hhðfSkgÞ for an ensemble of random and adapted sequences,
i51p0ðAiÞ logðph
kðAiÞÞ þ N?K;
kðAiÞ logðph
kðAiÞÞ þ N? K:
Note that N?K drops out of equation (3) and does not
need to be computed. Our results and conclusions were
negligibly affected by our choice of p0ðAiÞ, which was only
used to scale the adaptedness values. We call Hhthe
''human adaptedness'' when the host h is human and
the ''avian adaptedness'' when the host is avian. Further
discussion of this measure as well as a numerical example
is provided in the Appendix.
Individual sequences can have host adaptedness values
less than zero or greater than one if the sequences have
frequency ph
sequences or a greater number of favorable (high equilib-
rium frequency ph
ted sequences.
The maximum likelihood estimate ˆ ph
zero for all amino acids not present at identified
location k. In order to avoid logarithms ofzero in equations
(2) and (4), we incorporated pseudocounts into the calcu-
kðAiÞ) residues compared with random
kðAiÞ) residues compared with fully adap-
kðAiÞ of ph
kðAiÞ is
lation of ph
kðAiÞ5ˆ ph
kðAiÞ þ d
1 þ 20d
where d was set equal to 10?6. Varying d did not appreciably
change the results.
Sequence Data and Analysis
The data collection and analysis were performed as de-
scribed previously (Tamuri et al. 2009). Briefly, sequences
for human and avian influenza A viruses (excluding labo-
Resource Database (Bao et al. 2008). The sequence data
were culled to eliminate near-identical sequences and
the result of sporadic transmissions between hosts. The se-
quences were aligned at the amino acid level (MUSCLE;
(PAL2NAL; Suyama et al. 2006); the nucleotide data were
segment using PhyML (Guindon and Gascuel 2003; HKY85
model; Hasegawa et al. 1985; gamma-distributed rates).
Branch lengths representing amino acid evolutionary dis-
tances were then optimized for this fixed-tree topology us-
ing the corresponding amino acid data (PAML; Yang 1997,
2007; WAG substitution matrix; Whelan and Goldman
2001; gamma-distributed rates). The number of sequences
used (with the exception of the PB1, M1, and M2; see be-
low) is listed in table 2. The phylogenetic trees are shown in
figure 1.
We then identified locations with significant support
for having undergone changes in selective constraints ac-
companying the host-shift event from birds to humans,
using the analysis described previously (Tamuri et al.
2009), related to the approach described by Forsberg
and Christiansen (2003). In this method, locations in the
influenza genome were analyzed under two models. The
first model assumes that the pattern of evolution for
thegivenlocationishost independent,andthesite-specific
amino acid composition is estimated. The second model
depends on the particular host (avian or human), and
two sets of site-specific amino acid compositions (one
for each host) are estimated. The likelihoods of the two
models were compared, and the statistical significance
of the host-specific model was obtained for the given
location. The procedure was then repeated for all polymor-
phic locations in all proteins, and a false discovery rate
(FDR) approach (Benjamini and Hochberg 1995) was used
Table 2. Protein Sequences Used in the Analysis.
Number of
Human Sequences
Number of
Avian Sequences
Adaptation of Influenza Viruses · doi:10.1093/molbev/msq317
Page 4
to correct for multiple testing, resulting in a set of locations
that differ in the two hosts.
Using a FDR cutoff of 0.20, we identified 294 sites on six
different proteins as described in the supplementary
material, Supplementary Material online (H1: 84 sites,
N1: 68 sites, NS1: 28 sites, NP: 48 sites, PA: 27 sites, and
PB2: 39 sites). (M1, M2, and PB1 have relatively few loca-
tions undergoing changes in selective constraints and thus
do not have sufficiently robust statistics for computing hu-
man and avian adaptedness.) We used these 294 sites to
calculate host adaptedness for the various human and
avian virus sequences as well as for the pandemic H1N1
2009 virus and selected classical swine and Eurasian swine
virus sequences, using equations (2–4) described above.
The strains used in the analysis and their host adaptedness
tary Material online. Varying the FDR threshold between
5% and 20% or random resampling of included sites results
in different magnitudes of change in adaptedness but has
little effect on the qualitative results.
FIG. 1. Phylogenetic trees of the various genes in the analysis, with host and lineage indicated by branch colors: avian (black), human (red),
classical swine (blue), and Eurasian swine (cyan). For PA and PB2, we additionally include triple-reassortant swine sequences (green). The
location of the host shift used in the calculations is indicated by an orange dot. Shifting the location of the host-shift event did not appreciably
change the results. 1918 and pandemic H1N1 2009 sequences are labeled. Horizontal line represents a branch length corresponding to 0.1
nucleotide substitutions per site. Only the avian and human sequences were used in determining the significant locations, the equilibrium
amino acid frequencies, and the ancestral reconstructions as explained in the text.
dos Reis et al. · doi:10.1093/molbev/msq317
Page 5
Reconstructing the Host-Shift Sequence
We are also interested in studying the host adaptedness of
the ancestor of the 1918 pandemic virus. The host shift was
assumed to have occurred at the midpoint of the branch
connecting the parent node of the 1918 human H1N1 se-
quence with its parent as shown in figure 1. (Moving the
host shift along this branch did not appreciably affect the
results of the calculation.) Using the maximum likelihood
of our site-wise nonhomogenous model, we calculated the
posterior probability of every amino acid for every site at
the host-shift event (Koshi and Goldstein 1996). We sam-
pled sequences from the posteriors 1,000 times, calculating
the host adaptedness for each reconstruction. The mean
and 95% confidence intervals (CIs) of the human adapted-
ness and avian adaptedness measures were constructed
based on this sampling.
Reconstructing the Pattern of Sequence Changes
We performed a reconstruction of the most likely set of-
substitutions for each protein using the joint recon-
struction method of Pupko (Pupko et al. 2000) under
the WAG amino acid substitution model (Whelan and
Goldman 2001) with site-optimized rates. We then calcu-
lated human adaptedness measures for each node of the
phylogenetic tree following the avian-to-human host shift.
By traversing the phylogenetic tree for the protein starting
at the host-shift node down through the human lineage,
we calculated the change in human adaptedness along
the trunk of tree, leaf nodes, and the remaining internal
Fits to Host Adaptedness Data
To study the change in host adaptedness with time, we fit
the host adaptedness of human virus sequences (ignoring
sporadic H5N1 infections) as a function of isolation date to
two possible functional forms: (a) an exponential decay to
baseline equal to 1.0, where HðtÞ51 ? Aexpð ? t=sÞ, and
(b) an exponential decay to an adjustable baseline, where
HðtÞ5B ? Aexpð ? t=sÞ. The adjustable parameters are, as
appropriate, the amplitude of change A, the adaptation
time s, and the asymptotic value B. We subtracted 25 years
from the isolation date of post-1977 human H1N1 viruses
corresponding to the time that these viruses were in arti-
ficial evolutionary stasis (dos Reis et al. 2009). We used the
likelihood ratio test (P , 0.05) to test whether model (a)
can be rejected in favor of (b) as outlined in the Appendix.
For the chosen model, we calculated CIs for the parameters
and thetime whenthefitmatchesthehuman adaptedness
at the host-shift sequence through bootstrapping by sam-
pling the residuals.
Figure 2 shows the host adaptedness (human or avian) val-
ues computed for the H1, N1, NS1, NP, PA, and PB2 pro-
representing the human pandemic viruses of 1918 and
2009 are indicated. In addition, we represent the position
of the reconstructed virus at the host-shift event that gave
rise to the 1918 pandemic. This figure highlights that the
avian sequences are at equilibrium, clustering around 1.0,
FIG. 2. Host adaptedness values for a series of different virus sequences, including avian (black), human (red), classical swine (blue), Eurasian
swine (cyan), and the host-shift sequence (orange). Open red circles represent post-1977 human H1N1 viruses whose isolation times were
corrected as described in the text. Error bars for the host-shift sequence represent the 95% CI indicating the uncertainty in the ancestral
reconstruction. For PA and PB2, we additionally include triple-reassortant swine sequences (green). 1918 and pandemic H1N1 2009 sequences
are labeled. Human sequences inside the distribution of avian sequences represent sporadic H5N1 infections.
Adaptation of Influenza Viruses · doi:10.1093/molbev/msq317
Page 6
whereas mammalian viruses are spread out, suggesting an
ongoing adaptation process.
To evaluate whether the virus at the pre-1918 host-shift
the fraction of avian viruses that were less well adapted to
avian and human hosts compared with the host-shift virus.
As shown in figure 3, the avian adaptedness and human
adaptedness of the host-shift virus are generally within
the distribution of values obtained for other avian viruses,
although, interestingly, the polymerase proteins (PA and
PB2) have relatively high avian adaptedness. This suggests
that the host-shift virus was not exceptionally preadapted
to humans. Figure 3 also shows how the pandemic H1N1
2009 virus proteins compared with the corresponding pro-
teins of the lineage from which the genetic element came
(i.e., the human adaptedness and avian adaptedness values
for the H1, NS1, and NP proteins are compared with those
from classical swine viruses, those for PA and PB2 are com-
pared with avian virus proteins, and N1 is compared with
the corresponding protein of Eurasian swine viruses). The
pandemic H1N1 2009 virus proteins, with the exception
of N1, seem to be more adapted to humans than might
be expected. In particular, the human adaptedness of the
pandemic H1N1 2009 PA protein is larger than 99% of
the corresponding proteins from avian viruses. The N1 pro-
Eurasian swine N1 proteins, with a human adaptedness
value more typical of avian sequences; the latter results
H1N1 2009 PA and PB2 proteins have high human adapt-
edness, even relative to the distribution found in the
swine triple reassortants. Contributing to this are the PB2
A684S and PA K356R substitutions that have occurred in
these two proteins prior to the 2009 pandemic (Tamuri
et al. 2009).
Figure 4 shows the changing avian adaptedness and hu-
man adaptednessvaluesas afunction ofisolationyear.Wa-
terfowl virus proteins show an average avian adaptedness
close to one, agreeing with the notion that waterfowl is the
natural reservoir of influenza A. Conversely, human viruses
show a trend toward increasing human adaptedness and
decreasing avian adaptedness with time of isolation. Inter-
estingly, the 1918 human virus shows intermediate values
for both avian adaptedness and human adaptedness,
especially for the H1 segment.
Also included in figure 4 is a least-squares fit of an ex-
ponential to the human adaptedness data for the human
virus lineage, performed as described in the Materials and
Methods section. Fitting parameters are shown in table 3.
Best fits were obtained with a timescale for adaptation
(s, the time necessary for 63.2% of the adaptation to oc-
cur) on the order of 30–70 years, fastest for H1, N1, NP,
and PB2 and slowest for NS1. We would expect that the
asymptotic human adaptedness values for these extrapo-
lations should equal 1.0. In fact, significantly better fits
were obtained for four of the proteins when the asymp-
totic values are larger (H1 and N1) or smaller (NP and
PB2) than 1.0. Extrapolation of these fits to the human
adaptedness at the host-shift event can provide an esti-
mate of the timing of this host shift. We performed a boot-
strap analysis by sampling on the residuals. The estimated
host-shift timings are all consistent with previous esti-
mates (1883–1912) based on nucleotide evolution (dos
Reis et al. 2009).
In addition to reconstructing the virus at the time of the
host shift, we also performed an optimal reconstruction of
the various substitutions that occurred in the human lin-
eage following the host-shift event. We separated these in-
to changes that occurred in the ''trunk'' of the tree
connecting the host-shift event directly with recent virus
sequences, other interior branches, and exterior branches
ending at isolates. As shown in figure 5, we found signifi-
cant differences in the nature of the sequence changes that
occuralongthesedifferentsetsofbranches; branchesalong
the trunk of the tree are characterized by a much higher
likelihood of an increase in human adaptedness compared
withother branchesinthetree. Thiswasobservedforevery
gene considered separately.
Properties, Limitations, and Approximations of the
We previously developed a method for identifying changes
in selective constraints acting on influenza virus proteins
FIG. 3. Comparison of various proteins from the pre-1918 host-shift
reconstruction and pandemic H1N1 2009 virus with those of the
host viruses from which they emerged. Points in blue show the
percentage of avian virus protein sequences that have avian and
human adaptedness values lower than that of the pre-1918 host-
shift reconstruction. Points in red show the percentage of avian (PA
and PB2), Eurasian swine (N1), or classical swine (H1, NP, and NS1)
virus sequences with human adaptedness or avian adaptedness
values lower than the pandemic H1N1 2009 sequences. The human
adaptedness values for the pre-1918 host-shift proteins are well
within the distribution expected for avian sequences, suggesting
that the host-shift virus was not exceptional, whereas the pandemic
H1N1 2009 virus proteins, with the exception of N1, have greater
than average human adaptedness, indicating preadaptation to the
new human host.
dos Reis et al. · doi:10.1093/molbev/msq317
Page 7
FIG. 4. Human adaptedness and avian adaptedness values for a series of different virus sequences as a function of time. Color coding is as in
figure 1, including avian (black), human (red), classical swine (blue), Eurasian swine (cyan), triple-reassortant (green), and the host-shift
sequence (orange). Open red circles represent post-1977 human H1N1 viruses whose isolation times were corrected as described in the text.
Human sequences inside the distribution of avian sequences represent sporadic H5N1 infections. Abscissa error bars for the host-shift sequence
represent 95% CIs for the timing of this event as determined from an analysis of nucleotide evolution (dos Reis et al. 2009), whereas ordinate
error bars indicate the uncertainty of the ancestral reconstruction. Least-squares fits to the human adaptedness of the human virus sequences,
as described in the text, are included as a solid line, whereas the extrapolation to the host-shift event is shown as a dashed line.
Adaptation of Influenza Viruses · doi:10.1093/molbev/msq317
Page 8
corresponding to a change in host (Tamuri et al. 2009). In
contrast to previous methods that ignore the phylogenetic
relationship of the isolates and treat each sequence as an
2007; Miotto et al. 2008) resulting in both lack of sensitivity
and exaggerated estimation of statistical significance, we
analyzed the nature of the substitutions that occur during
the evolutionary process and identified when there is sta-
tistical support that these substitution patterns are host
dependent. In this way, we were able to both identify lo-
cations where selective constraints differ and characterize
the nature of these differences.
In particular, rather than calculating the observed fre-
quencies of the amino acids found in different positions,
our analysis provides the equilibrium amino acid frequen-
cies, given the estimated substitution rates. Observed fre-
quencies are biased by similarities between evolutionarily
related viruses and are time dependent as the viruses adapt
to the new host following the host-shift event. In contrast,
equilibrium frequencies represent the asymptotic value for
an ensemble of adapted viruses at equilibrium with the
constraints. We have used these equilibrium frequencies to
develop a measure of how well any virus protein matches
corresponding host adaptedness of the viruses to the two
hosts. We can then visualize the process of adaptation to
the new host following a host shift and provide insight into
what might have occurred both prior to and following the
host-shift event.
Our evolutionary model assumes that fitness effects at
each location are additive and constant within each host
these assumptions are not strictly valid. Selective con-
straints can change as the proteins evolve within a host,
(Blackburne et al. 2008). Adaptation to humans can occur
through different sets of substitutions, indicating that the
acids found at other locations. This is clearly seen in HA,
where significant differences in structure are reflected in
different characteristic substitutions necessary for recogni-
tion of receptors on the target human cells (Rogers et al.
1983; Nobusawa et al. 1991; Connor et al. 1994; Vines et al.
1998; Matrosovich et al. 2000). Different substitutions in
response to host shifts to human are not confined to these
membrane proteins as is clear from considering PB2 627;
E627K was experimentally identified as an important sub-
stitution necessary for the virus to replicate and spread
in mammals (Subbarao et al. 1993; Hatta et al. 2001;
Tarendeau et al. 2008; Steel et al. 2009). The pandemic
H1N1 2009 virus maintains an glutamic acid at this loca-
tion, and it appears that a basic amino acid (E) at position
591 compensatesfor the absence ofthe basicamino acid at
position 627 (Yamada et al. 2010).
Such violations might explain the asymptotic values for
the exponential fits to the human adaptedness with isola-
tion time. According to our model, we would expect this
asymptotic value to be 1.0, which is the average adapted-
ness of viruses at equilibrium with the human selective
adaptednessvaluewasnot 1.0,suggesting thattheselective
constraints on the individual locations might be changing
either because of changes in the immunity of the host pop-
ulation or because of interaction between the various lo-
cations in the protein. Herd immunity dynamics would
tend to increase the asymptotic values over 1.0 as there
would be a need for the virus to continue to adapt to
the new constraints represented by the adapting host im-
mune response. Correspondingly, H1 and N1, the surface
glycoproteins most involved in antigenic recognition, have
we might expect that there were a number of different
ways that a protein could adapt to its host, and adaptation
in some locations might lessen the pressure to adapt in
others (as in the example of the complementarity of the
basic amino acids at positions 591 and 627 of PB2 as men-
tioned above) in violation of our assumption of additivity.
In this case, we would expect asymptotic values less than
1.0 as is observed for PB2 (0.84) and NP (0.98).
The magnitudes of the changes in host adaptedness are
different for the different proteins, representing the variety
FIG. 5. Relative fraction of ''trunk'' branches (red), other interior
branches (green), or exterior branches to isolates (blue) that are
characterized by a negative, neutral, or positive change in human
adaptedness, following the shift from avian-to-human host prior to
1918. Error bars represent standard error based on the number of
observations. All genes show a similar distribution.
Table 3. Curve-Fitting Parameters with 95% CIs.
Time t (years)
(33.21, 35.42)
(31.10, 35.64)
(62.88, 84.03)
(23.58, 43.29)
(42.44, 61.76)
(24.70, 50.09)
Equilibrium Value
(if different from 1.0)
1.08 (1.08, 1.09)
(1906.1, 1907.8)
(1903.4, 1906.8)
(1891.5, 1903.5)
(1883.0, 1904.9)
(1872.5, 1898.0)
(1894.2, 1911.6)
dos Reis et al. · doi:10.1093/molbev/msq317
Page 9
of degrees of difference in selective constraints in the two
hosts. Locations that undergo a relaxation in selective con-
small change in human adaptedness (avian virus sequences
are compatible with the human constraints) but a larger
change in avian adaptedness (many human viruses will
not be compatible with the avian constraints). The oppo-
site relationship would hold for a tightening of selective
constraints. The amount of scatter in host adaptedness
values for the various proteins mostly reflects the number
of significant sites considered, which range from 27 sites in
PA to 84 locations in H1.
Ourexponentialfittohuman adaptedness,extrapolated
to the host-shift event, is in rough agreement with the es-
timate of 1883–1912 obtained through the analysis of nu-
cleotide composition changes (dos Reis et al. 2009). These
extrapolated values, however, should be treated with cau-
tion as they assume that adaptation to the human host
occurred in a similar manner prior to and following
1918. If the intermediate host prior to the 1918 pandemic
was swine, it is likely that the rate of adaptation was
slower before 1918 and the host shift occurred earlier than
indicated by the extrapolations. The extrapolation also as-
sumes thatthe functional form oftheadaptation process is
correct and that the changing human adaptedness can
be represented by an exponential with a single timescale.
It might be conjectured that the adaptation was faster im-
event. This can be modeled as a mixture of exponentials
with different adaptation times; the locations with the
shortest adaptation times would equilibrate fastest, leaving
locations with longer adaptation times to equilibrate lon-
ger after the host-shift event. To test this possibility, the
human adaptedness data were fit to an ensemble of expo-
nentials with a Gaussian distribution of adaptation rates.
This more complicated model could not be justified by
the data, but this does not indicate that some mixture
of substitution rates would not give an improved fit.
It is clear that the mathematical model developed here
still leaves much unknown about evolution of influenza
and host shifts. Our current model should be considered
as a basic framework onto which more complete models
can be developed. Particularly, modeling variation in se-
lective constraints along time and within hosts could pro-
vide a better understanding of the adaptation process.
Our assumption of additiveness can also be relaxed, and
models that consider interactions among locations could
be developed.
How Typical Was the Host-Shift Virus?
It is not clear why a particular virus undergoes a host-shift
event. One possibility is that chance mutations result in
a ''preadapted'' virus particularly fit for the new host prior
to the host transfer event. The other possibility is that the
virus is not distinctive, and the host transfer of a particular
virus is simply a chance occurrence. The answer to this
question has important consequences for our ability to
characterize the pandemic potential of zoonotic viruses.
To distinguish between these two possibilities, we recon-
structed the ancestral sequence of the virus that under-
went the shift to humans prior to the 1918 pandemic as
well as analyzing the 2009 pandemic virus.
We observed that the avian-like pre-1918 host-shift vi-
rus, as best shown in figure 3, has human adaptedness val-
ues within the distribution of what would be expected for
an avian virus, which suggests that the identity of the virus
tunity.In contrast,thepandemicH1N1 2009virus proteins,
with the exception of N1, were more adapted to humans
than would be expected, given their origin. The most in-
teresting examples of such preadaptation are in PB2 and
PA; in both proteins, there was an initial host shift from
birds to swine, presumably around 1998, followed by the
host shift to humans in 2009. While circulating in swine,
both experienced substitutions identified with increasing
human adaptedness (e.g., PB2 A684S and PA K356R) prior
to the shift to humans (Tamuri et al. 2009). The resulting
increase in human adaptedness for PA is especially large
as there are comparatively fewer host-specific locations
in this protein compared with PB2. N1 of the 2009 pan-
demic virus was not as well adapted to humans as N1 from
other Eurasian influenza viruses, although it is about as well
adapted as a typical avian virus. The relatively lower adapt-
edness for this particular gene may represent a random
fluctuation that is compensated for by the greater adapt-
edness of the other genes.
Changing Adaptedness in the Phylogenetic Tree
We note that adaptation to the new host has occurred
preferentially along the ''trunk'' of the phylogenetic tree,
whereas other branches where the adaptation does not oc-
cur as quickly tend to represent evolutionary ''dead ends.''
This would be expected if such sequence changes increase
the fitness of these sequences in the new host relative to
those viruses experiencing alternative substitutions. This
points to the possibility that measures, such as human
adaptedness, can be used to provide insight into why cer-
tain lineages persisted and others did not.
Ancestral Reconstruction Methods
Analyses of both the host-shift viruses and the changes
along the tree required reconstruction of the evolutionary
trajectories. We used marginal reconstruction for the
ancestral sequences (Koshi and Goldstein 1996) and joint
reconstruction (Pupko et al. 2000) for the historical
The reconstruction of the ancestral sequence relies on
an accurate model of the substitution process, which
we observe to depend upon the host, especially for the
substitution models is especially important for examining
the evidence for preadaptation in the host-shift virus
as some changes that might reflect the adaptation of
the virus to the new host may, with an inappropriate
host-independent evolutionary model, appear to be prior
Adaptation of Influenza Viruses · doi:10.1093/molbev/msq317
Page 10
to the host shift. We were specifically interested in identi-
fying evidence for preadaptation that cannot be explained
use of host-dependent models and the exclusion of viruses
from other than avian and human hosts. Although it is
standard, especially for experimental work, to consider
the most likely sequence, we generated an ensemble of
sequences by sampling from the posterior probabilities
of the reconstruction, allowing us to determine unbiased
statistical properties of this ensemble (Williams et al.
2006). For this paper, we recreated an ensemble of sequen-
ces representing the virus at the point of host transfer.
In this way, we were able to obtain the mean and CIs
for the human adaptedness and avian adaptedness at
this point.
Moreaccurate ancestral reconstruction could havebeen
achieved by modeling selective constraints in swine. Iden-
tification of three sets of selective constraints per location
provides computational and statistical challenges. Particu-
larly, with three sets of constraints, alternative models are
not nested, and the likelihood ratio test cannot be used.
We are currently working on updating our models to in-
cluded swine, and we are exploring computationally inten-
sive Monte Carlo methods to construct suitable null
distributions for hypothesis testing. For this reason, in
our joint reconstruction, we used a more standard method
with substitution models that did not depend on either
host or location.
The History of the 1918 Pandemic
As is clear in figures 2 and 4, significant adaptation to hu-
man selective constraints had occurred prior to the 1918
pandemic. This is in seeming contrast to the conclusions
made by Taubenberger et al. (2005), who concluded that
the 1918 virus sequences more closely resemble avian than
human virus sequences. The difference in conclusions be-
tween earlier work and this work can be explained by a dif-
ference in focus; previous work considered all the amino
acid changes that had occurred in the virus proteins,
whereas our methods allow us to focus on locations in-
volved in host adaptation.
The degree of human adaptation prior to the 1918
pandemic can be explained in three ways: (a) The virus
had ''preadapted'' to humans in its avian host, presumably
as a result of stochastic fluctuations, perhaps explaining
virus as exerted by a human host. (a) seems unlikely as the
the range of observed avian viruses. In addition, our recon-
preadaptation. Although we cannot rule out the possibility
icant period of time prior to the subsequent pandemic, the
similarity of avian and porcine cell receptors, the observed
successful avian-to-swine host shift in 1979 compared with
the lack of precedent for a successful avian-to-human shift,
and the difficulty in the virus existing undetected for so
et al. 2009).
Adaptation to humans during virus evolution in swine is
possible if there are similarities in the selective constraints
imposed on viruses in these two species. In fact, human
adaptedness values for H1, NP, PA, and PB2 are higher
in the classical swine lineage than in avian isolates. The in-
creasing human adaptedness ofthe Eurasian swine H1 after
the initial host shift in 1979 is clear in figure 4. If the evo-
lution of the human virus prior to 1918 occurred mostly in
swine, we would expect the human adaptedness values for
the 1918 human virus to resemble the human adaptedness
values of classical swine. This is true for most proteins, al-
though the 1918 virus N1 and NP proteins have signifi-
cantly higher human adaptedness than is observed in
later classical swine viruses. Resolution of this issue will re-
quire greater availability of early influenza viruses or more
sophisticated evolutionary models. We also note that the
2009 virus seems to have preadapted to humans during its
circulation in swine. This again highlights the ability of
swine to preadapt viruses to human hosts, suggesting
a potentially similar role for swine in facilitating the
1918 and 2009 human pandemics.
The results described above seem to suggest that, al-
though the virus that underwent the first host-shift event
from birds to mammals before the 1918 pandemic seems
unexceptional, the virus had substantially adapted to hu-
mans prior to the subsequent pandemic. Similarly, we can
detect substantial adaptation to humans in five of the
virus genes in the triple reassortant prior to the 2009 pan-
demic. Although the causes of a pandemic are complex,
involving a mixture of virus properties, host susceptibilities,
and historical contingencies, these results indicate that the
degree of human adaptation of the virus plays an impor-
tant role in host shifts to humans.
Supplementary Material
Supplementary material is available at Molecular Biology
and Evolution online (http://www.mbe.oxfordjournals.
We would like to thank John McCauley for helpful discus-
Medical Research (Medical Research Council, UK), the
Wellcome Trust, and the European Union FP6 FLUPOL
contract (grant number SPSB-CT-2007-044263).
dos Reis et al. · doi:10.1093/molbev/msq317
Page 11
Example of Adaptedness Calculation
Consider an aligned set of protein sequences of length 2
where two different residues, A and B, are observed. Imag-
ine our analysis indicates that A is strongly favored in hu-
entire viral genome, both residue types are found equally
out at the end of the calculation), we can express the raw
of the equilibrium frequencies:
B50:5).IgnoringtheeffectofN?K (whichdrops
hHumanðAAÞ5logð0:7Þ þ logð0:7Þ5 ? 0:71;
hHumanðABÞ5logð0:7Þ þ logð0:3Þ5 ? 1:56;
hHumanðBAÞ5logð0:3Þ þ logð0:7Þ5 ? 1:56;
hHumanðBBÞ5logð0:3Þ þ logð0:3Þ5 ? 2:41:
An ensemble of random sequences, where each possible
sequence is equally likely, would have an average hHuman
ÆhHumanæRandom50:25 ? ð ? 0:71Þ þ 0:5 ? ð ? 1:56Þþ
0:25 ? ð ? 2:41Þ5 ? 1:56. In an ensemble of fully adapted
sequences, where the proportion of As and Bs at each loca-
tion matches the equilibrium frequencies, we would expect
to find 49% AA, 21% AB, 21% BA, and 9% BB. Such an en-
semble would have an average hHumanof ÆhHumanæAdapted5
0:49? ð ? 0:71Þ þ 0:42? ð ? 1:56Þ þ 0:09 ? ð ? 2:41Þ5
?1:22.Wescalethe humanadaptednessvaluesbysubtract-
ing the average value of the random ensemble and dividing
by the difference between the average of the adapted and
random ensembles to yield
?1:22?ð?1:56Þ5 ? 2:50:
As desired, our random ensemble of sequences (with equal
mixtures of AA, AB, BA, and BB) would have an average
human adaptedness value of 0, whereas our adapted en-
semble would have an average human adaptedness value
of 0:49 ? 2:5 þ 0:42 ? 0 þ 0:09 ? ð ? 2:5Þ51.
In this case, BB has an adaptedness value less than 0,
whereas AA has an adaptedness value greater than 1. This
is because BB is less adapted than the average of a random
ensemble, 75% of which have at least one more favored A;
conversely, AA is better adapted than the average of an
ensemble of adapted proteins, 51% of which have at least
one less favored B.
As we sum over an increasingly large number of locations
with a similar degree of selective constraints, both random
tures of more favorable and less favorable residues. As a re-
sult, random sequences would have adaptedness values
approximately normally distributed around 0, whereas
adapted proteins would have adaptedness values approx-
imately normally distributed around 1. For example, imag-
ine we had N identical independent locations such as the
one described above, with a favorable residue A (equilib-
rium frequency in humans pHuman
51 ? pHuman
both residues are equally represented (p0
this case, the human adaptedness is equal to
) and less favorable res-
). Imaginethatover theentiregenome,
B5 0.5). In
2nA? 1
? 1;
where nAis the fraction of residues in the sequence that are A.
In this simplemodel,the distributionof nAinrandomsequen-
ces would follow a binomial distribution with nA50:5±
resulting in a distribution of human adaptedness values with
mean 0 and standard deviation (SD)
p ,
p . Conversely,
nomial distribution with nA5pHuman
ing in a distribution of human adaptedness values with mean
Fits to Adaptedness Data
As described in the text, we fit the host adaptedness of
human viral sequences as a function of isolation date
H(t) to two possible functional forms:
, result-
1 and SD
ðaÞ ˆHðtÞ51 ? Aexpð ? t=sÞ
ðbÞ ˆHðtÞ5B ? Aexpð ? t=sÞ:
Assuming that the errors are normally distributed with var-
iance d2, the likelihood function K for the fit can be rep-
resented as
5 ? Nlogð
where v25P
when d5
, where
of (b). Computed P values were
dexpð ?ðHðtiÞ?ˆHðtiÞÞ2
Þ ? NlogðdÞ ?
i51ðHðtiÞ ?ˆHðtiÞÞ2. This function is a maximum
ffiffiffi ffi
K5 ? Nlogð
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Adaptation of Influenza Viruses · doi:10.1093/molbev/msq317


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Date(s): 11/14/14 - 11/30/15
From: Cedarville Lodge
Phone: (800) 222-2949
Stay three or more nights and receive 20% off your nightly room rate. Free continental breakfast, free WiFi, heated indoor pool, and hot tub. Special cannot be combined with any other offers or discounts.
Date(s): 10/17/14 - 12/31/14
From: Detroit Marriott at the Renaissance Center
Phone: (800) 228-9290
Located at the top of the Detroit Marriott at the Renaissance Center, this eclectic fine dining restaurant features incomparable food, great service and a world-class wine list to accompany a panoramic view of the Detroit and Canadian...
Date(s): 10/14/14 - 12/31/14
From: Detroit Marriott Troy
Laugh the night away at a great rate with our Comedy Package! Package includes deluxe guestroom accommodations, two tickets to Mark Ridley's Comedy Castle, and breakfast for two at 200 West and access to hotel amenities, including the...
Date(s): 08/22/14 - 12/31/14
From: Courtyard By Marriott - Auburn Hills
Phone: (248) 373-4100
Calling all concert-goers! Are you ready to rock? Rock on with our convenient concert fever transportation package at the newly renovated Courtyard Detroit Auburn Hills! Concert Fever Transportation Package includes overnight...
Date(s): 10/01/14 - 06/08/15
From: Hampton Inn - Marshall
Phone: (269) 789-0131
Book your stay at Hampton Inn Marshall and have a fun night out at the dinner theater at Cornwell's Dinner Theater in Marshall, Michigan. Cornwell's features other events and attractions for you to enjoy when you attend! Receive two...
From: Courtyard by Marriott - Detroit Downtown
Enjoy time with your favorite person while we help make this a stay to remember at the Courtyard Detroit Downtown. Rates from: $184-$254 Valid Thursday–Sunday through December 31, 2014. After enjoying a romantic evening on the town,...
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StartUpHire is the place to find Product-Designer jobs in and near . Review the list below of Product-Designer jobs. These Product-Designer jobs represent some of the most rewarding careers available. You can refine your job search for Product-Designer jobs in by adding a job title, company name, or keyword to the search box above … or click the "Advanced Search" link for more options. Don't see the employment opportunity you're looking for? Check back soon for more Product-Designer jobs in , or sign up for an email alert (link in upper left) to receive periodic updates on new Product-Designer jobs in , .
Location: Boulder, CO Alteryx is the leader in data blending and advanced analytics software. Alteryx Analytics provides analysts with an intuitive workflow for data blending and advanced analytics that leads to deeper insights in hours, not the weeks typical of traditional10/18/2014 Boulder, CO Save This Job
Good design and experience is at the core of Empirix product vision and the UI/UX Lead is a key member of the Product and Engineering organization. He/She will be responsible for collaborating with and leading a team of world-class UI/UX developers. This position will work in10/18/2014 Billerica, MA Save This Job
We?re looking for a dynamic, self-starter to join our Trade team, working remotely in New York City. Candidates for this role will maintain and grow an existing book of business, while prospecting and delivering new business for the Brand in a defined territory. To be successful11/5/2014 Sausalito, CA Save This Job
August is seeking an Intern UX/UI Designer to be involved in the design of our mobile and web applications. The Intern UX/UI Designer will work with August's design team to create/edit visual design specs and prototypes, and assist in conducting user studies and user interviews.10/28/2014 San Francisco, CA Save This Job
Department: Content Strategy & Behavior Design Department Reports to: Creative Director What is RedBrick Health? RedBrick is a health engagement technology company that helps employers reinvigorate their health & wellness programs. We combine financial accountability, clinical10/20/2014 Minneapolis, MN Save This Job
Merrimack is a biopharmaceutical company discovering, developing and preparing to commercialize innovative medicines paired with companion diagnostics for the treatment of cancer. Merrimack seeks to gain a deeper understanding of underlying cancer biology through its systems11/26/2014 Cambridge, MA Save This Job
Working independently, the Sr. Producer, Mobile Games oversees the creation and operation of mobile games by external developers through Kongregate?s mobile publishing program. The Senior Producer oversees the work 3rd party game developers and also serves as an individual11/24/2014 San Francisco, CA Save This Job
The Art Director is responsible for establishing and communicating that artistic vision of the project to the art staff. The Art Director is also responsible for managing the art team towards the execution of the vision while considering the production, design and technicalSan Rafael, CA Save This Job
at Houzz Palo Alto Upgrade your career at one of the hottest startups in Silicon Valley! If you are great at what you do, thrive in a highly collaborative environment, and don?t mind the occasional meeting in the bathroom (don?t worry, it?s just one of our conference rooms), we11/21/2014 Palo Alto, CA Save This Job
Achronix Hardware Engineers (Core Team) are responsible for researching, designing and developing electronic component and systems for semiconductor manufacturing company?s FPGAs. The Core Technology team at Achronix owns the reconfigurable ?fabric? (look-up tables, routing,11/20/2014 Santa Clara, CA Save This Job
About Agility Agility Fuel Systems is the leading designer and producer of alternative fuel storage and delivery systems for heavy duty trucks, buses and specialty vehicles. Our systems can be fueled by compressed and liquid natural gas, as well as hydrogen and other11/18/2014 Charlotte, NC Save This Job
About Agility Agility Fuel Systems is the leading designer and producer of alternative fuel storage and delivery systems for heavy duty trucks, buses and specialty vehicles. The result of two industry-leading companies, Agility combines FAB?s world-class expertise in compressed11/18/2014 Santa Ana, CA Save This Job
Company Overview Ruckus Wireless is a pioneer in the wireless infrastructure market, enabling carriers and enterprises to stay ahead of the exploding demand for high-bandwidth applications and services. The Ruckus Smart Wi-Fi technology redefines what?s possible in wireless11/12/2014 Sunnyvale, CA Save This Job
Tracking Code 289-621 Job Description GetWellNetwork, Inc. provides patient engagement solutions that help health care providers engage, educate and empower patients along the care continuum. Our patient-centered platform, delivered across multiple technology platforms including11/12/2014 Bethesda, MD Save This Job
Purposes: Fast-growing Analog Semiconductor pre-IPO company that?s focused on Analog Power & Programmable Analog ICs is looking for a highly motivated, hands-on, and experienced Senior Digital Design Engineer. The Designer will be responsible for creating world class designs for10/19/2014 Dallas, TX Save This Job
Purposes: Fast-growing Analog Semiconductor pre-IPO company that?s focused on Analog Power & Programmable Analog ICs is looking for a highly motivated, hands-on, and experienced Senior Analog Design Engineer. The Designer will be responsible for creating world class designs for10/19/2014 Dallas, TX Save This Job
As Principle Hardware Engineer you will lead design and development of our next generation wireless audio and related products. You will work closely with our ME, Embedded S/W, DSP, acoustics and product marketing teams to architect and implement high quality, cost effective10/19/2014 Sunnyvale, CA Save This Job
Company / Job Description DICOM Grid is a healthcare SaaS company that provides secure and simple access to medical images "in the cloud," reducing unnecessary and redundant tests, lowering radiation exposure to patients, and making medical images immediately available to10/14/2014 Phoenix, AZ Save This Job
Job Description The candidate will manage three design teams and work at all levels of design including supervising memory architect to analyze topology tradeoffs and generate detailed specifications, working in team to propose circuit level implementations, reviewing10/11/2014 Santa Clara, CA Save This Job
Job Description Position Summary: The copywriter will be responsible for researching and interviewing as required, and creating compelling content to be used in all marketing collateral and campaigns, including but not limited to: Product briefs Brochures Website Product spec10/11/2014 Columbus, OH Save This Job


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OutKast's Andre 3000 is continuing to influence rap's current generation of hip-hop stars' albums, as he's agreed to appear on Grand Hustle rapper B.o.B.'s forthcoming Strange Clouds sophomore LP.
According to Grand Hustle boss T.I., fans can expect to hear Andre and Bobby Ray sharing microphone time.
Tip told XXLMag.com that the two ATLiens collaborated on a track called, "Play the Guitar" off of Bobby Ray's forthcoming sophomore album, Strange Clouds. Bob plays guitar on the cut, which also includes vocals from the recently freed King of the South. "They sampled that verse on [Drake's] 'Fancy' where I say, "She play n*ggas like B.o.B play the guitar." (XXL Mag)
Strange Clouds is not the only sophomore LP Dre is attached to, as recent reports have previously suggested that he will appear on rap star Drake's Take Care.
Back in the control room, there's a pillowy mid-tempo beat on loop, with muffled drum sounds and a whale-like synth bassline that [producer 40] is improvising on a small keyboard. It's a two-track instrumental from December 2009, which Drake had asked Shebib to try to find. The night before, Drake received a call from Andre 3000, who said he wanted to get on the rapper's next LP–and, specifically, on a beat made by 40. The original name of the instrumental was "Good Enough for the Both of Us." "What a sh*tty title!" Drake says. Suddenly they switch to boardroom mode. Drake, 40, and Drake's DJ, Future the Prince, discuss Andre 3000 in frank, mathematical terms: the timbre of his voice, the cadences he prefers. They recall his obscure "Walk it Out" freestyle and the run of features since then, and talk about how their instrumental could be modified to better suit Andre's delivery. (The FADER)
In early October, B.o.B. discussed busting rhymes on his second solo effort.
"For me I feel like I been going through phases. And I feel like I'ma always go through phases," he explained in an interview. "When I first started the first song I ever released was 'Cloud 9.' And that was like a smoker's anthem. The first whole half of the song I was just singing on it — I kinda went through that phase. Went through [an] alternative phase where all I wanted to do was make rock music. Now I just feel like rapping. I feel like naturally I'ma sing, throw a melody here and there but I'm in emcee mode now….Man we gonna get that [out] I'ma say, March. I'm aiming for early March. I don't wanna say too much." ("The Breakfast Club")
Last month, the Atlanta rapper revealed his new album's title.
Fresh off performing in front of President Obama at a fundraiser in Los Angeles, Bobby Ray caught a flight back to ATL. He appeared on V103's "The Ryan Cameron Show" on Tuesday, where he revealed Strange Clouds as the title. "'Strange Clouds' is the first single off of my sophomore album, actually titled Strange Clouds," he announced. The project, which serves as the follow-up to his Grammy-nominated debut B.o.B Presents: The Adventures of Bobby Ray, is preceded by the single of the same name featuring Lil Wayne. The song went to iTunes on Tuesday and has already cracked the top 5. (Rap-Up)
A Strange Clouds release date has not yet been announced.
Check out a recent B.o.B. interview down below:


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Watch_Dogs Gameplay: Hacking Trailer Explains What The Heck is CtOS?! (video)
As companies, neighborhoods and whole cities go digital–living off the virtual lifeblood of a single interconnected overly complex piece of software, one can only assume the inevitable. The inevitable has become commonplace ubiquity for regular gamers. I'm talking about hacking. Virtually all of LA and a couple other cities have hotwired the totality of their towns traffic lights, cross walks and such to work off a centrally governed network. If I were a hacker… That just sounds like an amusement park filled with mayhem-laden possibilities. In fact theirs an app for that.
It's called Watch_Dogs. It may not allow you to hack a cities unified network of information and governing systems… Oh wait! That's exactly what this game is all about, albeit safe and tame in the virtual world. This latest bit of cyber crumpet is all about the insane amount of control you have of the the cities ctOS. What is ctOS, you ask? Well watch the latest set of moving pictures from Ubisoft.
Shawn Sanders
Shawn loves gadgets, literature, history and games. For 10yrs+ he's straddled both the comic book & video game industries, as a writer, editor, marketing officer & producer. Shawn got his start in tech & games as an editor & Hardware Director for GameRevolution.com. More notable accomplishments include Executive Producer on mobile games Geometry Wars: Retro Evolved & The Shroud.
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1 Comment to Watch_Dogs Gameplay: Hacking Trailer Explains What The Heck is CtOS?! (video)
The depth of this game continues to amaze me.


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Teamson Design - Crackled Rose - Toy Boxes & ChestProduct Id: 186317 Phone Code: ACE9-4228
Regular: $198.00 Sale: $148.80 (You Save 25%)
Usually ships in 1 to 3 Days
Product Description
What a great way to organize those toys! Here is a hand painted toy chest that you little ones will love!
- Hand Painted
- Material: MDF
- Some assembly may required
Note: Some Assembly May Be Required.
Teamson products are tested in third party certified labs to meet or exceed current safety regulation, including lead level requirements


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- Shopping Bag ( 0 items )
At the publisher's request, this title is being sold without Digital Rights Management software (DRM) applied.
"Estleman brings high style to his writing, the sentences are things of beauty in and of themselves. He has brought poetry, historical truth, and great wisdom to the genre."—Tulsa World
Posted December 3, 2009
I Also Recommend:
Estleman has nailed the western novel. His characters are well developed relative to many writers in the genre and the story line is always believable. No superman here just solid action with a touch of suspense.
1 out of 1 people found this review helpful.Was this review helpful? Yes NoThank you for your feedback. Report this reviewThank you, this review has been flagged.


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November 28, 2014
Roger Larson, a Lincoln community leader who helped support various organizations and programs over several decades, has died at 87.
Southwood Lutheran Church says Larson died Tuesday. A cause of death was not released.
Larson had a 40-year career at Lincoln radio station KFOR. For years, he ended radio editorials with, "I am Roger Larson, and that is the way I see it."
Larson also held leadership roles for more than 40 organizations. He is credited with raising millions of dollars for community projects.
Mayor Chris Beutler said in a statement that Larson laid down the groundwork for Lincoln's current success.
Larson's memorial is scheduled Friday morning at Southwood Lutheran Church.
Click Here to access the online Public Inspection File
Viewers with disabilities can get assistance accessing this station's FCC Public Inspection File by contacting the station with the information listed below. Questions or concerns relating to the accessibility of the FCC's online public file system should be directed to the FCC at 888-225-5322, 888-835-5322 (TTY), or firstname.lastname@example.com.


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Junior Member
Hi all I'm looking on getting nocturne or kog'maw as they're both good AD carry's (well, not nocturne) any1 know how to be aggressive with them?
In situations such as;
Enemies are wasting away your tower
A team fight started without u
You are ganking an enemy
Early game you are going to your lane casually
The Tinkerer
Senior Member
You can only successfully be aggressive with any champ if you know what you're doing. Just keep your distance with kog and you'll do fine.
© 2014 Riot Games, Inc. All rights reserved. Riot Games, League of Legends and PvP.net are trademarks, services marks, or registered trademarks of Riot Games, Inc.


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Jay Hillman, 89, retired Northwestern faculty member, author, amateur musician
Jordan Jay Hillman, 89, died on April 8, 2014 after an eight-and-a-half-year journey with Alzheimer's. Born in Waukegan in 1924, he was known to friends and colleagues as "Jay." He served during World War II as an Army Air Corps navigator in the Eighth Air Force Unit and liked to reminisce about
flying. He earned a Master of Arts degree in Political Science, and a Juris Doctor from the University of Chicago and later received a Doctor of Juridical Science from Northwestern University.
He was vice-president and general counsel for the Chicago
and North Western Railway and joined the law school faculty at Northwestern in 1966. In 1975 he took a temporary leave from teaching and served as general counsel for the U.S. Railway Association.
He pursued many social causes beyond his chosen profession and served on the Evanston school board as well as the CTA board and various boards related to the arts. He was strongly committed to humanitarian endeavors, expressing his deepest convictions and principles through both philanthropy and service. He was a lover of nature, animals, children, walking, and classical music, played the clarinet, and after retirement, joined a chamber group.
During the last five years of his life, he lived in Oak Park and was surrounded by his loving family, friends, and caregivers. His book, The Torah and Its God: A Humanist Inquiry was published in 2001 and embodies his deep respect for scholarship, humanism, and Judaic history.
Jay Hillman is survived by his wife, Karen G. Hillman; his two daughters from a former marriage, Deborah Jay Hillman and Amy Elizabeth Stearns; his grandchildren, Benjamin Johnson and Rachel Johnson; his great-grandson, Cooper Jay Johnson; his son-in-law, Kenneth Johnson (Amy Stearns); and by Jane Ellen Birdsong (Benjamin Johnson).
A memorial service will be held in Oak Park in June.
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This is another custom estate find. The Pearls are 8mm's and have nice luster and color. The setting is 14K yellow gold and weights 8 grams.
Size 7 1/2.
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Latest News
LaCrosse Talk
Information about and from LaCrosse Talk
WI's Do Not Call List or call (866) 966-2255
MN's Do Not Call List or call (800) 921-4110
Federal Do Not Call List or call (888) 382-1222
Charges Faced by Former Municipal Prosecutor Pete Kisken
Superintendent Hopeful Of Budget Repair Bill
Later today the Onalaska School District, like many districts around the state, will find out how much of hit they could take after Governor Scott Walker releases details on his budget repair bill.
Superintendent John Burnett says if what he's heard is true about the bill, the district could take a $1.5 million reduction in state aid. But he says there is a bright side......possibly.Some relief may be ins tore to help districts balance their budgets.
Governor Scott Walker says the next state budget will have over a billion-dollars less in state aid to local governments and schools. The Republican governor will unveil his fiscal plans for the next two years at 4pm.
Vet Manager Back In Court
An office manager at a Sparta Veterinary Clinic, who is accused of ordering and stealing almost six thousand hydrocodone pills and using company money to pay for them, was in a Monroe County courtroom today.
36 year old Katherine Watters, of rural Sparta, is charged in Monroe County Circuit Court with 11 counts of obtaining a controlled substance by fraud and five counts of forgery.
She is accused of ordering the pills with $4500 of vet clinic monies, then taking them home to support a habit. She also gave some to her boyfriend who then sold them.
Watters plead not guilty and will be back in court in March.
Sparta Thugs
Sparta Police are searching for two armed men that broke into a home yesterday afternoon.
A 22 year old Sparta woman told police that two white men wearing ski masks and armed with semi automatic handguns entered her home in the 100 block of North Water st. and searched for something they thought was in the home.
After the pair fled on foot with the loot they were after, they were seen getting into a maroon 4 door sedan.
Police are looking for a 6'2" slim guy and a 5'9" heavy set guy.
Monroe County Fund Shot Down
A possible one million dollar fund that would have taken excess Monroe County department funds and put them towards construction of a justice center, will not see the light of day. Last week, the resolution fell short of the two-thirds requirement when supervisors voted 12-10 in favor of it.
The account would have set aside $1 million of unused 2010 funds for future construction of a Justice Center. Supervisors voted to eliminate $348 thousand that would have been taken out of the solid waste fund. Also cut was monies from the original $119 thousand of the unused 2010 balance of the Board of Prisoners.
Board chairman Rick Irwin says a similar resolution may be brought before the board in the future.
Boys and Girls Club Wall of Fame Announced
The Boys and Girls Club of Greater Lacrosse has announced the date forthe 26th Annual Wall of Fame Recognition Banquet. This year's event will take place on the evening of Friday, April 29th at the Waterfront's Cargill Room at 5:30 p.m. This year's award recipients are: Wall of Fame Inductees: Barb Binsfeld & Don Weber, President's Award: Pat Smith, Adult & Youth Award: Dick & Sheila Wieser, Distinguished Service Award: Mary Patros and the Corporate Award: Kwik Trip


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I corresponded with Linda Mossmayer a few years ago. No luck there either, but thanks for taking the time to send leads my way. Acc. to Mom, the "Living Bigham", she did not know her stepfather wasn't her father until she was an adult, so he must have skipped out pretty soon after the marriage.
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Posted: 11/21/2014
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After a relatively quiet year following 2012′s Anna Karenina, Keira Knightley has been hitting the big screens in a big […]
First of all, don't scoff. You may think you know how to talk about things that should apparent to everyone. […]
British people get the majority of their complaining done in private, hours after the offending incident took place. Registering even […]
Since 2007, the NFL has exported regular season games to London where quarterbacks, wide receivers and tight ends have thrilled […]
The 'Doctor Who' cast
Brace yourselves, stateside Whovians: BBC America has just announced that cast and crew from Doctor Who will arrive in New York this week to promote the upcoming season premiering April 23. Matt Smith, Karen Gillan, and Arthur Darvill will kick off the press tour with a signing at the Fifth Avenue Barnes & Noble store in Manhattan on Friday, April 8.
And on Monday, April 11 at 7 pm EST, BBC America is holding a free U.S. premiere screening of the two-part season opener at the Village East Cinema in Manhattan. The screening will be followed by a Q&A with Smith, Gillan, Arthur Darvill, Alex Kingston, AND showrunner Steven Moffat. Nerdist's Chris Hardwick will moderate, and the Q&A will be recorded live for a special Nerdist podcast.
Note: the screening is first-come, first-served, and an RSVP does not guarantee admission. Turnout to the Season 5 premiere screening at Village East Cinema wrapped around a city block last year, and while there will be additional seating this year, you might want to show up a bit earlier than 6:59 pm. Just a word to the wise.
Kevin Wicks founded BBCAmerica.com's Anglophenia blog back in 2005 and has been translating British culture for an American audience ever since. While not British himself - he was born and raised in St. Louis, Missouri - he once received inordinate hospitality in London for sharing the name of a dead but beloved EastEnders character. His Anglophilia stems from a high school love of Morrissey, whom he calls his "gateway drug" into British culture.
Chef James Briscione's career began humbly; he was a football star and dishwasher at a brunch spot in his adolescence. […]
Although we don't grow up with Thanksgiving, Brits in the U.S. are often charged with the task of "doing" Thanksgiving […]
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Medical Marijuana Dispensaries May Return to Michigan
Posted: 07/14/2014 - Senate meetings this week could lead to the return of medical marijuana dispensaries.
Posted: 07/14/2014 - Senate meetings this week could lead to the return of medical marijuana dispensaries.
Posted: 07/14/2014 - Supporters of marijuana decriminalization are looking to the Detroit suburb of Grosse Pointe Park as a possible tipping point in the debate over the drug in Michigan.
Posted: 07/14/2014 - The Lansing City Council is voting, tonight, on the acceptance of a grant to help fund a pilot program to help fight domestic violence.
Updated: 07/14/2014 - Do you have an unexplained rash that won't seem to go away? You may be having an allergic reaction to nickel in a personal electronic device like an iPad.
Updated: 07/14/2014 - The Michigan legislature is planning an active summer session. They may vote as soon as Wednesday on marijuana measures.
Posted: 07/13/2014 - The Michigan Film Office says it has awarded a $1.5 million film incentive to the "Ask Dr. Nandi" talk show.
Posted: 07/13/2014 - Israel is warning residents of northern Gaza to evacuate their homes as it widens its offensive.
Posted: 07/13/2014 - An appeals court won't stop the delivery of mail at the Livingston County jail from the American Civil Liberties Union
Posted: 07/13/2014 - A stabbing investigation led Lansing police to a home on the 1300 block of Roosevelt Avenue, Sunday morning
Posted: 07/12/2014 - Man taken to local hospital from house fire.
Posted: 07/12/2014 - The Lansing Color Run supports both the Sparrow Children's Center and Kelleigh's Cause, an organization that raises awareness for AVM.
Updated: 07/12/2014 - Lansing police are searching for an armed man suspected of breaking into a home on Reo Road.
Updated: 07/11/2014 - Old Town's economic revitalization is moving east as entrepreneurs fill vacant buildings.
Updated: 07/12/2014 - Sources telling Detroit newspapers more than two-thirds voted to pass the "Grand Bargain."
Updated: 07/12/2014 - Toll will stay at $4.00 per car.
Updated: 07/12/2014 - Police say they found burglary tools and stolen property in suspects' car.
Updated: 07/12/2014 - It's Saturday morning in Adado Riverfront Park.
Updated: 07/11/2014 - Good Samaritans asking to help with projects around your home? Police in Bath Township say it's too good to be true, at least it was for two homeowners.
Updated: 07/12/2014 - An afternoon police chase from Clinton County into some Lansing neighborhoods was caught on the dash cam of a state police car. The man responsible has been arraigned.
Posted: 07/11/2014 - No signs of air or water contamination.
Updated: 07/11/2014 - Authorities say two toddlers have drowned in a Michigan pond.
Updated: 07/11/2014 - The Grand Valley State University Board of Trustees has voted to increase tuition by $149 per semester.
Updated: 07/11/2014 - Hudsonville and Pure Michigan want you to vote on the next Pure Michigan ice cream flavor.
Updated: 07/11/2014 - Police in Bath Township are warning residents of a scam that happened Thursday where a suspect distracted a homeowner while other man entered the home.
Updated: 07/11/2014 - The dash cam from a Michigan State Police Officer's vehicle caught all of yesterday's car chase.


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Enjoy full use of the Spa for 2 hours and the following treatments:-
Luxury Hot Stone Massage
Decléor Aromatherapy Facial – allow your therapist to assist you in choosing the facial most suited for your particular skin type.
Two course Luncheon in the Club Room
Jessica GELeration Luxury Manicure - includes hand exfoliation, massage, cuticle care, nail shape and finished with Jessica GELeration gel polish.
Jessica GELeration Luxury Pedicure - includes foot exfoliation, massage, cuticle care, nail shape and finish with Jessica GELeration gel polich.
A day of luxury and top to toe pure pampering - £220.00
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Use of the Spa for 2 hours and your choice of either:
A Decléor Aroma Body Massage
Decléor Aromatherapy Facial – includes a cleanse, massage, mask and moisturise to suit the specific needs of your skin
Jessica Luxury Manicure – a luxury Jessica Manicure to condition hands and nails, includes a Jessica Midi Nail Polish
Jessica Luxury Pedicure – a luxury Jessica pedicure to soothe tired feet, includes a Jessica Midi Nail Polish
Refreshing Lemon Tea
A half day of heavenly pampering for - £75.00
Alternatively, you may enjoy TWO of the above treatments of your choice for - £105.00
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Use of the Spa for 2 hours and the following treatments:
Decléor Aromatherapy Body & Scalp Massage – a heavenly massage offering the incredibly soothing sensation of a warm aromatic balm combined with the expert touch of your therapist's hands, followed by a deeply relaxing scalp massage.
Two-course Luncheon in the Club Room
Decléor Aroma Expert Facial – allow your therapist to assist you in choosing the facial most suited to your particular skin type. This also includes a hand and arm massage.
A Luxury Manicure – will nourish and condition the hands and nails, and includes a Jessica Midi Nail Polish.
A heavenly day of pampering for - £170.00
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(9.30am to 4.00pm approximately)
Use of swimming pool and relaxation area for 2 hours.
Luxury Face & Body Treatment - this nurturing treatment will help to preserve the tone of your skin, reducing thre risk of stretchmarks, lighten your legs and brighten your complexion.
Two-course Luncheon in the Club Room
A Luxury Manicure & Pedicure - to revitalise and condition both hands and feet. Also includes a Jessica midi nail colour.
A gentle, nurturing pampering - £170.00
Use of the Spa for 2 hours including gymnasium, sauna, steam room, spa bath and relaxation by the pool.
Intensive Energising Body Treatment.
A revitalising treatment for a pure sensation of energy and deep relaxation.
Two-course Luncheon in the Club Room
Decléor Cleansing Back Treatment.
This complete treatment acts like a facial. Deeply purifying essential oils will decongest to leave the skin looking cleaner, brighter and healthier.
Intensive Energising Face Treatment.
Detoxifying treatment for a clear, pure, fresh skin and a rebalanced, de-stressed complexion.
Price – £170.00
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text:
POSTED: Thursday, October 11, 2012 - 4:00pm
UPDATED: Thursday, October 11, 2012 - 4:04pm
NBC NATIONAL NEWS — Trying to stay afloat in the digital age?
How about a "like" vest.
It's a concept linked to Facebook, making the rounds online.
Say someone likes something on your Facebook page?
A special vest you wear inflates and gives you a virtual hug.
The technology was reportedly developed by students for a class project at MIT and Harvard.
"I guess I could use more hugs in my life, so it's pretty cool," said Su Vora, an MIT student.
Mateo Williams sees it as a concept for inflating egos.
"It would feel weird to have hugs all the time for things that you really don't deserve," Williams said.
And getting all puffed up isn't exactly the connection everyone craves.
"It's like someone is surprise hugging. It would just be without notice, I guess, which would kind of be weird, Frances Liu, an MIT students.
For others, it's an idea to "like;" one that might pave the way for more technology to wear.
"I think the technology is interesting in terms of other applications," said Michael Walsh, an MIT student.