Re: Don't Blame Birds for 1918 Flu
The new (13Jul09) PNAS paper: (I couldn't get the charts, maybe someone can do that)
http://www.pnas.org/content/early/2009/07/10/0904991106.full.pdf
Dating the emergence of pandemic influenza viruses
Gavin J. D. Smitha,b,1, Justin Bahla,b,1, Dhanasekaran Vijaykrishnaa,b,1, Jinxia Zhanga,b, Leo L. M. Poona, Honglin Chena,b,
Robert G. Webstera,c,2, J. S. Malik Peirisa,d, and Yi Guana,b,2
aState Key Laboratory of Emerging Infectious Diseases & Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong,
Pokfulam, Hong Kong SAR, China; bInternational Institute of Infection and Immunity, Shantou University, Shantou, Guangdong 515031, China; cVirology
Division, Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38015; and dHKU-Pasteur Research Centre, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
Contributed by Robert G. Webster, May 26, 2009 (sent for review March 31, 2009)
Pandemic influenza viruses cause significant mortality in humans.
In the 20th century, 3 influenza viruses caused major pandemics:
the 1918 H1N1 virus, the 1957 H2N2 virus, and the 1968 H3N2 virus.
These pandemics were initiated by the introduction and successful
adaptation of a novel hemagglutinin subtype to humans from an
animal source, resulting in antigenic shift. Despite global concern
regarding a new pandemic influenza, the emergence pathway of
pandemic strains remains unknown. Here we estimated the evolutionary
history and inferred date of introduction to humans of
each of the genes for all 20th century pandemic influenza strains.
Our results indicate that genetic components of the 1918 H1N1
pandemic virus circulated in mammalian hosts, i.e., swine and
humans, as early as 1911 and was not likely to be a recently
introduced avian virus. Phylogenetic relationships suggest that the
A/Brevig Mission/1/1918 virus (BM/1918) was generated by reassortment
between mammalian viruses and a previously circulating
human strain, either in swine or, possibly, in humans. Furthermore,
seasonal and classic swine H1N1 viruses were not derived directly
from BM/1918, but their precursors co-circulated during the pandemic.
Mean estimates of the time of most recent common ancestor
also suggest that the H2N2 and H3N2 pandemic strains may
have been generated through reassortment events in unknown
mammalian hosts and involved multiple avian viruses preceding
pandemic recognition. The possible generation of pandemic strains
through a series of reassortment events in mammals over a period
of years before pandemic recognition suggests that appropriate
surveillance strategies for detection of precursor viruses may abort
future pandemics.
H1N1 influenza A swine virus evolution molecular clock
Pandemic influenza outbreaks pose a significant threat to
public health worldwide as highlighted by the recent introduction
of swine-derived H1N1 virus into humans (1). In the
20th century, 3 influenza viruses caused major pandemics: the
1918 H1N1 virus, the 1957 H2N2 virus (H2N2/1957), and the
1968 H3N2 virus (H3N2/1968) (2, 3). These pandemics were
initiated by the introduction and successful adaptation of a novel
hemagglutinin subtype to humans from an animal source, resulting
in antigenic shift (4, 5). A number of hypotheses have
been proposed for the development of pandemicity of the
influenza virus, including direct introduction into humans from
an avian origin and reassortment between avian and previously
circulating human viruses, either directly in humans or through
an intermediate mammalian host (6–9).
Based on studies of amino acid similarities of all 8 gene
segments of A/Brevig Mission/1/1918 virus (BM/1918), it was
concluded that this virus most likely was derived directly from an
avian precursor that was introduced to humans shortly before the
pandemic (10, 11). This interpretation is controversial because
of variant gene phylogenies that either conflict with this theory
or remain ambiguous because of a lack of contemporaneous
viruses (12–14). Analysis of sequences generated from the
H2N2/1957 and H3N2/1968 strains showed that these pandemics
were caused by genetic reassortment between avian and preexisting
human viruses (8). The H2N2/1957 pandemic strain
contained introduced hemagglutinin, neuraminidase, and PB1
genes, whereas the H3N2/1968 pandemic strain incorporated
avian HA and PB1 genes (2).
However, the evolutionary history of these 3 pandemic viruses
remains unclear, and that lack of understanding hinders the
recognition of and preparedness for future influenza pandemics.
We therefore investigated evolutionary mechanisms of pandemic
emergence by conducting comparative genetic analyses of
all available viruses associated with the emergence of the 1918,
1957, and 1968 pandemics.
Bayesian relaxed molecular clock phylogenetic methods, as
implemented in BEAST, use flexible evolutionary models to
infer the timing of evolutionary events, so that the evolutionary
rate can vary among branches on the tree and uncertainty caused
by missing data and unknown evolutionary rates can be incorporated
(15). In the case of influenza, the times of most recent
common ancestor (TMRCA) provide an estimate of when virus
genes emerged in a given host that allows the time of interspecies
transmission to be inferred.
Here we estimated the evolutionary history to investigate the
possible date of introduction to humans of each of the genes for
all 20th century pandemic influenza strains. Mean TMRCA
estimates of each gene segment of H1N1 viruses shows that the
components of the 1918 pandemic strain were circulating in
mammalian hosts, i.e., swine and humans, at least 2 to 15 years
before pandemic occurrence. Phylogenetic analyses suggest that
the 1918 H1N1 pandemic virus most likely was generated by
reassortment between mammalian viruses and a previous human
strain and was not a pure avian virus. We also show that seasonal
and classic swine H1N1 viruses were not derived directly from
BM/1918; rather, their precursors co-circulated during the pandemic.
MeanTMRCAestimates also suggest that the avian-derived
genes of the H2N2 and H3N2 pandemic strains may have been
introduced to humans on multiple occasions over a number of years.
Results and Discussion
Evolutionary Inferences on the Origin of BM/1918, Human and Swine
H1N1 Viruses. To establish when H1N1 virus genes were introduced
to mammals, we co-estimated phylogenies and TMRCA
for all known mammalian, i.e., swine and human, H1N1 virus
genes (supporting information (SI) Table S1). For each of the 8
genes, mammalian H1N1 viruses (BM/1918, seasonal H1N1, and
classic swine H1N1) formed monophyletic clades (node 1 in Fig.
1 and Figs. S1–S8 and Table 1). For 6 genes (H1, N1, PB2, NP,
M, and NS), avian viruses formed distant monophyletic groups
to mammalian H1N1 genes (Fig. 1 A and B and Figs. S1–S3 and
S6–S8). In the PB1 and PA genes, a small clade of avian viruses
formed a group more closely related to mammalian H1N1
viruses, providing direct phylogenetic evidence of a more recent
avian source (Fig. 1 C and D and Figs. S4 and S5). The TMRCA
estimates for node 1 ranged from 1881 [95% Bayesian credible
interval (BCI) 1813–1912] for the PB2 gene to 1907 (BCI
1892–1918) for the N1 gene (Table 1). Ages of the H1 and NP
genes at node 1 could not be calculated because of uncertainty
in the phylogenies (Fig. 1A and Figs. S1 and S6). Importantly,
TMRCAs at node 1 indicate that the PB2 andMgene precursors
of all human H1N1 viruses were present in mammalian hosts
(e.g., swine) at least 6 years before the 1918 pandemic (Table 1).
Preliminary phylogenetic analysis showed that the BM/1918
virus H1, N1, PB1, PA, and NP genes clustered with human
H1N1 influenza A viruses, whereas its PB2, M, and NS genes
clustered with swine. These relationships have high statistical
support (bootstrap support 80%) except for the placement of
BM/1918 virus in the M gene phylogeny. Estimates for BM/1918
virus TMRCAs (node 2) ranged from 1903 (BCI 1867–1918) for
the PB2 gene to 1916 (BCI 1910–1918) for the HA gene (Table
1). These mean TMRCA estimates suggest that the BM/1918
virus genes were present in swine or human hosts 2 to 15 years
before the pandemic. The TMRCA of the M gene at node 2
could not be estimated, but the TMRCA at node 1 indicated that
the BM/1918 M gene precursor probably was present in mammalian
hosts before 1911 (Fig. S7 and Table 1).
Interestingly, the TMRCA distributions of the PB2, NP, and
NS genes of the BM/1918 virus (Table 1) suggest those genes
have circulated in humans since the 1889 H3 influenza pandemic
(16, 17). Earlier estimates of mutation rates of the NP gene
suggested that human H1N1 and classic swine influenza viruses
emerged from the avian source around 1912 or 1913 (18).
However, our results suggest that the same NP gene lineage has
been circulating in human influenza A viruses since the 19th
century, consistent with the report by Gammelin et al. (19).
Extensive arguments, based primarily on similarity between
consensus amino acid sequences, have been made that the
BM/1918 virus was derived directly from an avian progenitor,
contrary to phylogenetic evidence (10–13, 19, 20). These residue
similarities may help explain the avian-like phenotype of the
BM/1918 virus, particularly its high virulence in mammals (21).
Taken together, our results indicate that it is unlikely that the
BM/1918 virus could have resulted from adaptation of an entire
avian virus introduced directly into humans shortly before the
pandemic. More likely, it was generated by reassortment between
previously circulating swine and human strains and introduced
avian viruses over a period of years.
It generally has been assumed that after the pandemic the
BM/1918 virus established in humans to form the seasonal H1N1
influenza lineage (e.g., 2, 3, 9, 20). However, our phylogenetic
analysis shows that only the PB1, PA, NP, and N1 genes of seasonal
H1N1 were derived from BM/1918 (Fig. 1 and Figs. S1–S8).
Comparisons of TMRCA estimates of the HA for the BM/1918
virus (node 2, TMRCA 1916, BCI 1910–1918) and seasonal H1N1
lineage (node 3, TMRCA 1913, BCI 1895–1925) indicate that these
H1 lineages diverged (node 1a,TMRCA1905, BCI 1887–1917) and
co-circulated during the 1918 pandemic (Fig. 1A).
Phylogenetic relationships between BM/1918 and classic swine
H1N1 virus PB2, M, and NS genes also indicate that classic swine
H1N1 is a reassortant between BM/1918 and an unknown virus.
As such, classic swine H1N1 is derived partially from BM/1918
and is not a precursor of the 1918 pandemic virus (Fig. 1 and
Figs. S1–S8) (9).
It therefore appears that at least 3 reassortant H1N1 variants
co-circulated: BM/1918 and the precursors of seasonal and
classic swine H1N1 viruses. The co-circulation of the BM/1918
and seasonal H1N1 viruses may explain reports of influenza
outbreaks of varying severity during the 1918 pandemic (22, 23).
Here we provide the first evidence that seasonal H1N1 viruses
were not derived directly from BM/1918 but co-circulated during
the pandemic. This evidence may be relevant to the current
emergence and potential pandemicity of swine-derived H1N1
viruses in humans (1).
Phylogenetic analyses of the re-emergent H1N1/1977 virus
confirmed that each of 8 genes was directly derived from those
H1N1 viruses circulating in the 1950s (Fig. 1 and Figs. S1–S8).
Dating the time of emergence of each gene segment showed
similar TMRCAs with a mean of 2 to 3 years before the
detection of the viruses (Table 1 and Table S2). These results
support the hypothesis that the re-emergence of H1N1/1977 most
likely resulted from accidental laboratory re-introduction (2).
Emergence of H2N2 and H3N2 Pandemic Viruses. Phylogenies confirmed
that the H2N2/1957 was a genetic reassortant between
previously circulating human and avian viruses, with the novel
H2, N2, and PB1 genes derived from Eurasian avian sources
(Fig. 1 A, C, and D, Figs. S2–S6 and S8–S11). The mean TMRCA
estimates of the introduced genes of the H2N2 pandemic suggest
that the introduction of these 3 genes into human populations
occurred 2 to 6 years before the pandemic.
Ages of the novel H3 (TMRCA 1968, BCI 1967–1968) and
PB1 (TMRCA 1967, BCI 1966–1968) genes indicated that the
introduction occurred between 1966 and 1968 (Table 1). The
remaining genes of the H3N2/1968 virus came from the previous
human H2N2 virus. The upper BCI estimates of the human
H3N2 PB1 and HA TMRCAs indicate that this virus may have
circulated in humans as early as 1966; the last record of H2N2
in the human population was from 1968, indicating that H2N2
and H3N2 viruses co-circulated in humans for approximately 1
to 3 years (Table 1). This observation is consistent with the
phylogenies of the shared genes, with the exception of the NS
gene, in which late H2N2 and early H3N2 do not form separate
monophyletic lineages (e.g., blue boxes in Fig. 1 B and D).
Differences in the TMRCA estimates raise the possibility that
the introduced genes of the H2N2 and H3N2 pandemic strains
may have been introduced sequentially from multiple sources
over a number of years. Because of a lack of sequence data for
swine influenza from these periods, the involvement of swine in
the generation of these pandemic strains cannot be precluded.
Conclusions
The results of our study have provided fresh insights into
pandemic emergence by raising the possibility that all 3 pandemic
influenza strains of the 20th century may have been
generated through a series of multiple reassortment events and
emerged over a period of years before pandemic recognition.
Furthermore, results indicate that each of these strains was
produced by reassortment between the previously circulating
human virus and at least 1 virus of animal origin. The novel gene
segments for the H2N2/1957 and H3N2/1968 pandemics seem to
have originated from avian hosts, but the zoonotic sources of the
introduced viral gene segments for the 1918 pandemic remain
ambiguous. However, evidence suggests that, over a number of
years, avian gene virus segments have entered mammalian
populations where the viruses may have undergone reassortment
with the prevailing human virus. Given the frequent interspecies
transmission of influenza viruses between swine and humans, it
is most likely that such reassortment events occurred in swine
before pandemic emergence.
Interestingly, our analyses suggest that in the 1918 and 1957
pandemics novel NA and internal genes may have been introduced
into the prevailing human virus strains before the acquisition
of the novel pandemic HA. Frequent detection of seasonal
human influenza strains in swine indicates that pandemic precursor
viruses probably have circulated in either swine or human
Table 1. Times of most recent common ancestors of human pandemic influenza viruses and related lineages populations. The hypothetical precursors to the H2N2 and H3N2 pandemics have not been detected, probably because they originated in Asia where little or no surveillance was conducted at that time (2).
If future pandemics arise in this manner, this interval may
provide the best opportunity for health authorities to intervene
to mitigate the effects of a pandemic or even to abort its
emergence. However, our findings argue the need for highthroughput
characterization of all 8 gene segments of human
virus isolates, even those that have unremarkable HA antigens,
particularly of human viruses isolated in hotspots for zoonotic
infections with avian influenza viruses. At present, global influenza
surveillance in humans focuses attention primarily on
hemagglutinin. Although this focus will continue to be required
for strain selection for seasonal influenza vaccines, our findings
argue that this surveillance will not suffice for early warning of
an incipient pandemic.
Methods
Preliminary Phylogenetic Analyses and Data Preparation. Provisional phylogenetic analyses were carried out for all available influenza gene sequences using the neighbor-joining method in PAUP* 4b10 (24) with a best-fit nucleotide substitution model (25) and an appropriate outgroup (Table S1). The purpose of these large-scale phylogenetic analyses was to identify relationships between pandemic strains and all other sequences. These lineages (in particular, avian, swine, and human) were identified in each tree as monophyletic clades with bootstrap support of 80% or higher.
Based on these preliminary analyses, 11 datasets were compiled for human
influenza viruses: the hemagglutinin (HA: H1, H2, H3), neuraminidase (NA: N1, N2), and the 6 internal gene segments (PB2, PB1, PA, NP, M, and NS allele A), together with genes from representative influenza viruses isolated from other hosts (birds, swine, horses, and other mammals). Full details of the final datasets that were used for all subsequent analyses are given in Table S1. Phylogenetic Inference, Estimation of Nucleotide Substitution Rates and Times of Divergence. To estimate divergence times and rates of nucleotide substitutions in influenza A viruses, we applied a relaxed-clock Bayesian Markov chain Monte Carlo method as implemented in BEAST v1.4.8 (26). This method allows variable nucleotide substitution rates among lineages and also incorporates phylogenetic uncertainty by sampling phylogenies and parameter
estimates in proportion to their posterior probability (26). The marginal
likelihoods of 3 different clock models, strict clock, uncorrelated exponential
clock (uced), and uncorrelated log-normal clock (ucld), were compared using
a Bayes factor test for best fit (15, 27, 28). This test revealed that for all genes
the uced model, which allows evolutionary substitution rates to vary within an
exponential distribution along branches, was the best fit for the sequence
data (Table S2). The outgroup sequences were not included in the BEAST
analyses; rather, the relationships based on the tree topologies from the
preliminary analyses described earlier were enforced as prior assumptions for
the Bayesian analyses. Trees generated from the BEAST analyses were rooted
by fixing basal node relationships of the major lineages (avian, swine, and
human) in all phylogenies (Table S1).
In analyzing protein-coding sequences, we used the SRD06 codon position
model to partition the data (29). The first partition unifies the first plus second codon positions, and the second partition describes the third codon position.
Because each dataset included multiple non-mixing populations, a constant
population coalescent tree prior over the unknown tree space and relatively
uninformative priors over the remaining model parameter space were assumed
for each dataset (15).Wecarried out 3 independent analyses for 20–60
million generations sampled to produce at least 10,000 trees for each data set
to ensure adequate sample size of all analysis parameters including the
posterior, prior, nucleotide substitution rates, and likelihoods (effective sample size 200). The mean substitution rates, mean TMRCAs, and maximum clade credibility phylogenetic trees then were calculated after the removal of an appropriate burn-in (10%–15% of the samples in most cases, with 1 exception, in which 20% was removed for analyses of the PB1 gene) following visual inspection in TRACER version 1.4 (30).
TMRCA estimates of introduced genes of pandemic viruses were used to
infer the time of incorporation of these genes to form the pandemic virus
particle. For example, the H2N2/1957 and the H3N2/1968 pandemic strains
were generated by known reassortment events between previously circulating
human strains and introduced avian genes. The TMRCA estimates with
BCIs of these introduced genes provide an estimated timeline for the generation of the pandemic strain.
Furthermore, because we are dealing with interspecies transmission events
from a natural (avian) gene pool to other species, with very limited subtypes
of influenza virus present, we believe it is reasonable to interpret TMRCAs as
providing an estimate of time-bounds of interspecies transmission events. It
has been well described that avian viruses rarely transmit to mammalian hosts,
and in talking about initial transmission events, we have been very careful to
indicate the uncertainty as to which mammalian host is involved. Likewise,
transmission of influenza virus from swine to humans also is a rare event. The
high level of host restriction between avian and mammalian hosts and hostadapted influenza viruses also supports our interpretation.
ACKNOWLEDGMENTS.
This study was supported by the Area of Excellence
Scheme of the University Grants Committee (Grant AoE/M-12/06) of the Hong
Kong SAR Government, the National Institutes of Health [National Institute of
Allergy and Infectious Disease (NIAID) contract HHSN266200700005C], and
the Li Ka Shing Foundation. G.J.D.S. is supported by a career development
award under NIAID contract HHSN266200700005C.
1. Anonymous (2009) Update: Infections with a swine-origin influenza A (H1N1) virus -
United States and other countries, April 28, 2009. Morb Mortal Wkly Rep 58:431–433.
2. Kilbourne ED (2006) Influenza pandemics of the 20th century. Emerging Infectious
Diseases 12:9–14.
3. Taubenberger JK, Hultin JV, Morens DM (2007) Discovery and characterization of the
1918 pandemic influenza virus in historical context. Antiviral Therapy 12:581–591.
4. Gething MJ, Bye J, Skehel J, Waterfield M (1980). Cloning and DNA sequence of
double-stranded copies of haemagglutinin genes from H2 and H3 strains elucidates
antigenic shift and drift in human influenza virus. Nature 287:301–306.
5. Fang R, Jou WM, Huylebroeck D, Devos R, Fiers W (1981) Complete structure of
A/duck/Ukraine/63 influenza hemagglutinin gene: Animal virus as progenitor of human
H3 Hong Kong 1968 influenza hemagglutinin. Cell 25:315–323.
6. Scholtissek C, Rohde W, von Hoyningen V, Rott R (1978) On the origin of the human
influenza virus subtype H2N2 and H3N2. Virology 87:13–20.
7. Scholtissek C, Bu¨ rger H, Kistner O, Shortridge KF (1985) The nucleoprotein as a possible
factor in determining host specificity of influenza H3N2 viruses. Virology 147:287–294.
8. Kawaoka Y, Krauss S, Webster RG (1989) Avian-to-human transmission of the PB1 gene
of influenza A viruses in the 1957 and 1968 pandemics. J Virol 63:4603–4608.
9. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y (1992) Evolution and
ecology of influenza A viruses. Microbiol Rev 56:192–179.
10. Taubenberger JK, et al. (2005) Characterization of the 1918 influenza virus polymerase
genes. Nature 437:889–893.
11. Taubenberger JK, Morens DM (2006) 1918 influenza: The mother of all pandemics.
Emerging Infectious Diseases 12:15–22.
12. Gibbs MJ, Gibbs AJ (2006) Molecular virology: Was the 1918 pandemic caused by a bird
flu? Nature 440:E8.
13. Antonovics JH, Hood ME, Baker CH (2006) Molecular virology: Was the 1918 flu avian
in origin? Nature 440:E9.
14. Taubenberger JK, et al. (2006) Molecular virology: Was the 1918 pandemic caused by
a bird flu? (Reply). Nature 440:E9–E10.
15. Drummond AJ, Ho SYW, Phillips MJ, Rambaut A (2006) Relaxed phylogenetics and
dating with confidence. PLoS Biol 4:e88.
16. Fedson DS, Huber MA, Kasel JA, Webster RG (1972) Presence of A-Equi-2 hemagglutinin
and neuraminidase antibodies in man. Proc Soc Exp Biol Med 139:825–826.
17. Masurel N, Marine WM (1973) Recycling of Asian and Hong Kong influenza A virus
hemagglutinins in man. Am J Epidemiol 97:44–49.
18. Gorman OT, et al. (1991) Evolution of influenza A virus nucleoprotein genes: Implications
for the origins of H1N1 human and classical swine viruses J Virol 65:3704–3714.
19. Gammelin M, et al. (1990) Phylogenetic analysis of nucleoproteins suggests that
human influenza A viruses emerged from a 19th-century avian ancestor. Mol Biol Evol
7:194–200.
20. Reid AH, Fannin TG, Janczewski TA, Lourens RM, Taubenberger JK (2004) Novel origin
of the 1918 pandemic influenza virus nucleoprotein gene. J Virol 78:12462–12470.
21. Tumpey TM, et al. (2005) Characterization of the reconstructed 1918 Spanish influenza
pandemic virus. Science 310:77–80.
22. Andreasen V, Viboud C, Simonsen L (2008) Epidemiologic characterization of the 1918
influenza pandemic summer wave in Copenhagen: Implications for pandemic control
strategies. J Infect Dis 197:270–278.
23. Barry JM, Viboud C, Simonsen L (2008) Cross-protection between successive waves of
the 1918–1919 influenza pandemic: Epidemiological evidence from US Army camps
and from Britain. J Infect Dis 198:1427–1434.
24. Swofford DL (2001) PAUP*: Phylogenetic analysis using parsimony (and other methods)
4.0 Beta. Sunderland, MA: Sinauer Associates.
25. Posada D, Crandall KA (1998) MODELTEST: Testing the model of DNA substitution.
Bioinformatics 14:817–818.
26. Drummond AJ, Rambaut (2007) A BEAST: Bayesian evolutionary analysis by sampling
trees. BMC Evolutionary Biology 7:214.
27. Kass RE, Raftery AE (1995) Bayes factors. J Am Stat Assoc 90:773–795.
28. Suchard MA, Weiss RE, Sinsheimer JS (2001) Bayesian selection of continuous-time
Markov chain evolutionary models. Mol Biol Evol 18:1001–1013.
29. Shapiro B, Rambaut A, Drummond AJ (2006) Choosing appropriate substitution models
for the phylogenetic analysis of protein-coding sequences. Mol Biol Evol 23:7–9.
30. Rambaut A, Drummond AJ (2007) Tracer v1.4: MCMC trace analyses tool. Available at:
http://beast.bio.ed.ac.uk/Tracer. Accessed June 20, 2008.
