Journal of General Virology (2014), 95, 1969–1982
DOI 10.1099/vir.0.065227-0
Dating the origin of the genus Flavivirus in the light
of Beringian biogeography
John H.-O. Pettersson and Omar Fiz-Palacios
Correspondence
John H.-O. Pettersson
[email protected] or
[email protected]
Received 28 February 2014
Accepted 4 June 2014
Department of Systematic Biology, Evolutionary Biology Centre, Uppsala University,
Uppsala, Sweden
The genus Flavivirus includes some of the most important human viral pathogens, and its
members are found in all parts of the populated world. The temporal origin of diversification of the
genus has long been debated due to the inherent problems with dating deep RNA virus evolution.
A generally accepted hypothesis suggests that Flavivirus emerged within the last 10 000 years.
However, it has been argued that the tick-borne Powassan flavivirus was introduced into North
America some time between the opening and closing of the Beringian land bridge that connected
Asia and North America 15 000–11 000 years ago, indicating an even older origin for Flavivirus.
To determine the temporal origin of Flavivirus, we performed Bayesian relaxed molecular clock
dating on a dataset with high coverage of the presently available Flavivirus diversity by combining
tip date calibrations and internal node calibration, based on the Powassan virus and Beringian
land bridge biogeographical event. Our analysis suggested that Flavivirus originated ~85 000
(64 000–110 000) or 120 000 (87 000–159 000) years ago, depending on the circumscription
of the genus. This is significantly older than estimated previously. In light of our results, we
propose that it is likely that modern humans came in contact with several members of the genus
Flavivirus much earlier than suggested previously, and that it is possible that the spread of several
flaviviruses coincided with, and was facilitated by, the migration and population expansion of
modern humans out of Africa.
INTRODUCTION
The genus Flavivirus, within the family Flaviviridae, currently
consists of .70 virus species distributed all over the globe
(Gould et al., 2003; Gubler et al., 2007; Lindenbach et al.,
2007). It includes numerous viruses of major human health
concern, such as Dengue virus (DENV), Japanese encephalitis virus, West Nile virus and the type species for
flaviviruses, yellow fever virus (flavus: ‘yellow’), giving the
family and the genus its name (Gould & Solomon, 2008;
Mackenzie et al., 2004). The genome of flaviviruses consists
of a positive-sense ssRNA molecule of ~11 kbp. One single
ORF encodes three structural proteins (capsid, pre-membrane and envelope) and seven non-structural proteins (NS1,
NS2A, NS2B, NS3, NS4A, NS4B and NS5) flanked by
untranslated regions (Chambers et al., 1990; Lindenbach &
Rice, 2003).
The genus Flavivirus has been divided into four main
groups based on ecological characteristics, molecular phylogenetic analyses, vector specificity and virus performance in
host cells (Gaunt et al., 2001; Gould et al., 2001; Kuno,
2007). Most of the recognized flaviviruses belong to the
mosquito-borne flaviviruses (MBFs) that are commonly, but
Three supplementary figures and three supplementary tables are
available with the online version of this paper.
065227 G 2014 The Authors
not exclusively, vectored by either Aedes or Culex mosquitoes. The second group, tick-borne flaviviruses (TBFs), the
only monophyletic group of Flavivirus, are vectored by
ixodid ticks, and are further subdivided into a mammal and
a seabird group based on their host specificity (Grard et al.,
2007; Gritsun et al., 2003). The no-known vector flaviviruses
(NKVFs) are associated with either bats or rodents and
infect vertebrates without an apparent arthropod vector
transmitting the viruses (Porterfield, 1980). The majority of
the known flaviviruses are zoonotic, i.e. pathogenic viruses
that can be transmitted between humans and other animals.
However, the fourth group, the insect-specific flaviviruses
(ISFs; flaviviruses that are only capable of replicating in
insect cells) (Kuno, 2007), is most likely an undersampled
and very diverse group (Cook et al., 2012).
The idea of a molecular clock has been used to address
many hypotheses in the study of emerging viral diseases,
especially for diseases caused by RNA viruses (Bromham &
Penny, 2003), e.g. to reject the hypothesis of a potential
spread of human immunodeficiency virus (HIV) in the
1950s through a contaminated polio vaccine (Korber et al.,
2000). However, dating the origin of viruses is a complex
and challenging task. For viruses with high rates of evolution, the original phylogenetic signal is difficult to deduce
even with complex evolutionary models because the signal
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Printed in Great Britain
1969
J. H.-O Pettersson and O. Fiz-Palacios
diminishes with time due to repeated substitutions at the
same site (Holmes, 2003a). The extremely high substitution
rate observed for RNA viruses is mostly due to their errorprone replication and repair machinery (Bromham &
Penny, 2003; Duffy et al., 2008). Unequal substitution rates
among lineages of the same genus further adds to the
complexity of accurately estimating the time to the most
recent common ancestor (tMRCA) (Duffy et al., 2008;
Holmes, 2003a; Sanjuán, 2012).
Reconstruction of divergence times requires a temporal
reference to convert branch lengths of a phylogenetic tree
into time. This temporal reference is usually in the form of
a fossil or biogeographical event (i.e. internal node calibration), as in most eukaryote studies, or isolation dates (i.e.
tip date calibration), as with bacteria and virus studies.
From the biogeographical events, fossils or isolation dates,
the ages of internal nodes can be estimated. The match
between virus and host phylogenies has led to the suggestion that some virus lineages originated millions of years
ago. However, this has been strongly rejected by molecular
clock studies indicating a virus origin of only a few thousand
years ago (Holmes, 2003a; Worobey et al., 2010). This
controversy has sparked a debate regarding the validity and
utility of molecular clock reconstructions using tip dates
versus internal (deep) calibration to study and infer the
temporal scale of virus evolution (Sharp & Simmonds,
2011).
Previous studies based on molecular clocks have suggested
that the Flavivirus clade originated ~10 000 years ago in
Africa (Gould et al., 2001) from a non-vectored mammalian virus ancestor (Gould et al., 2003), followed by the
radiation of TBFs and MBFs during the last 5000 and 3000
years, respectively (Zanotto et al., 1996), and ISFs ~3000
years ago (Crochu et al., 2004). Other studies have also
dated small clades of individual flaviviruses using tip dates
as calibration points. These estimates are broadly congruent
with the notion of a Flavivirus origin within the last 10 000
years (e.g. Dunham & Holmes 2007; May et al., 2011; Pan
et al., 2011; Twiddy et al., 2003). However, recent analyses
on TBFs, based on complete genomes analysed with a
relaxed molecular clock and Bayesian methods, have estimated the age for the TBF clade to be at least 16 000 years
(Heinze et al., 2012), indicating that the age for the
Flavivirus genus as a whole should be significantly older
than suggested previously.
Within the TBFs, a close relationship between the biogeography of the Beringian land bridge, the historical
landmass that connected north-east Siberia with Alaska and
north-west Canada (Hultén, 1937), and the evolution of the
Powassan virus (POWV) clade, the only TBFs present in
North America, has been pointed out recently (Ebel, 2010;
Heinze et al., 2012). Geological studies estimate that the land
bridge was open for mammal land migration before the end
of the last glaciation between 15 000 and 11 000 years ago
(Elias, 2001; Elias et al., 1996; Kelly, 2003; Mandryk et al.,
2001). The absence of tick-borne encephalitis virus (TBEV)
1970
and Russian POWV lineages in North America is consistent
with the idea that the colonization of POWV into North
America happened under a single event by a land route, also
supported by the lack of evidence for continuous movement
of POWV and TBEV between Russia and North America by
either seabirds or mosquitoes (Heinze et al., 2012).
Therefore, it is unlikely that POWV emerged in North
America before the land bridge became accessible again after
the last glacial maximum. Consequentially, it is improbable
that POWV emerged after the Bering Strait was formed. The
most probable explanation, given the current knowledge and
present distribution of the POWV lineages, is that during the
course of TBF evolution POWV diverged from other TBF
lineages in a single introduction event by the land route
during the presence of the Beringian land bridge 15 000–
11 000 years ago (Heinze et al., 2012).
POWV could have been introduced into North America
by either humans or other mammals that colonized the
Americas during the existence of the Beringian land
bridge via one of two routes: the interior route or coastal
route. (i) The interior route became accessible when the
process of deglaciation created a corridor between the
Laurentide and Cordilleran glaciers. The corridor did not
exist during the last glacial maximum. The corridor
started to open ~15 000 years ago (Dixon, 2013; Dyke,
2004), and became habitable for humans and other
mammals ~13 500 years ago (Dixon, 2013). (ii) Towards
the end of the last glacial maximum, the glaciers bordering to the south-west coast of Alaska through western
Canada started to melt. Around 16 000 years ago, the
glaciers had receded to the extent that the coastal habitats
could support human populations (Dixon, 2013 and
references therein). The coastal route is also likely to be
the route by which humans first entered the Americas
(Achilli et al., 2013; Bodner et al., 2012; Fagundes et al.,
2008; Goebel et al., 2008; Schurr, 2004).
Presently, there are coding nucleotide genomes available from
.70 unique strains of the genus Flavivirus, including viruses
recognized by the International Committee on Taxonomy of
Viruses (http://www.ictvonline.org/virusTaxonomy.asp?version=
2013). Here, we combine this information together with
relaxed molecular clock methods implementing a Bayesian
approach, again to estimate and shed some light on divergence times of the genus Flavivirus and groups within. Aided
by biogeographical calibration, we report the first study, to
the best of our knowledge, estimating the age and rates of
substitution of the genus Flavivirus as a whole, including its
major groups, using complete coding nucleotide genomes.
We explore multiple internal calibration points using
Bayesian methods in combination with a relaxed molecular
clock to reconstruct divergence times. We compare our
results with previous studies reconstructing divergence
times for different clades within the genus Flavivirus. We
also discuss the incongruence between virus-dating studies
using molecular clocks and RNA virus evolution. Finally, we
propose a temporal and biogeographical scenario for the
evolution of the genus Flavivirus.
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Journal of General Virology 95
Origin of the genus Flavivirus
RESULTS AND DISCUSSION
Phylogenetic reconstruction of the genus
Flavivirus
The phylogeny of Flavivirus and its major groups was
inferred using a genus-wide sampling approach including 86 complete coding flavivirus genomes with known
isolation dates (Table 1) analysed by both Bayesian and
maximum-likelihood methodologies. The overall tree
topology from all of the different analyses was in agreement with previous published phylogenies based on the
NS3 gene (Billoir et al., 2000; Cook & Holmes, 2006;
Grard et al., 2007), multiple genes (Medeiros et al., 2007)
and complete genomes (Cook & Holmes, 2006; Cook et al.,
2012; Grard et al., 2007, 2010; Kuno & Chang, 2006; Lobo
et al., 2009). Our results were consistent with what is
commonly referred to as an NS3-like topology (Fig. 1).
Two datasets were analysed, a nucleotide and an amino
acid sequence alignment, using Bayesian and maximumlikelihood approaches. In our Bayesian phylogenetic analysis, the nucleotide and amino acid 50 % majority rule
consensus trees from MrBayes (Figs S1 and S2, available in
the online Supplementary Material) and the maximum
clade credibility tree from BEAST (Figs 1 and S3), all rooted
with bovine viral diarrhea virus 1 (BVDV-1), produced
topologies with strong support for the focal nodes (A–O).
The Bayesian trees were also supported by the topology of
the maximum-likelihood trees, although the sister relationships between Tamana bat virus (TABV) and all other
groups were unresolved in the maximum-likelihood trees
(data not shown, available upon request).
There was maximum posterior probability (1.0 pp) for the
four major clades roughly corresponding to the conventional grouping of Flavivirus (Gould et al., 2001, 2003);
the TBF clade (node M), the NKVF clade (NKVFa, node
N), a MBF-dominated group (MBFdom, node F) and the
ISF clade (ISFa, node E), sister to the other three groups
(Fig. 1). The MBFdom group (node F) includes (i) the
Aedes MBF clade (MBFAedes, node H), (ii) a NKVF clade
(NKVFb), sister to the MBFAedes clade, (iii) a grade of
likely insect-specific flaviviruses consisting of Chaoyang
virus, Donggang virus, Lammi virus and Nounane virus
(nodes I and J) (Huhtamo et al., 2009; Kolodziejek et al.,
2013; Lee et al., 2013) and (iv) the Culex MBF clade
(MBFCulex, node K). The present study supports the fact
that the majority of the members of the genus Flavivirus
show a very strong pattern of host–vector association
(Gould et al., 2001, 2003), as all groups showed distinct
clustering depending on vector association. It is possible
that the positioning of the NKVFb clade is a consequence
of secondary loss of vector capability (Gould et al., 2001,
2003). Likewise, it is possible that the grade of ISFs (nodes
I and J) within the MBFdom group is also due to secondary
loss of vector capability.
The main disagreement between our findings and other
studies relates to the position of TABV. TABV has
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previously been suggested to be the sister group to the
ISFa clade (node E) based on complete coding amino acid
sequence analysis (Cook et al., 2012). However, in the
present study, TABV appears to have diverged before the
ISFa clade (node B, 1.0 pp, based on Bayesian nucleotide
and amino acid phylogenetic analyses), which instead is
sister to the TBFs, NKVFs and MBFs (Figs 1, S1 and S2).
Our results are consistent with the topology of previous
complete genome and gene-based phylogenetic studies (de
Lamballerie et al., 2002; Hoshino et al., 2009; Lobo et al.,
2009).
Divergence times of the genus Flavivirus
Viruses, and RNA viruses in particular, provide an excellent opportunity to study evolutionary change because of
their relatively high rate of substitution that allows for
evolution to be observed within human timescales (Bromham
& Penny, 2003; Drummond et al., 2003; Duffy et al., 2008;
Holmes, 2003a). Many studies have used molecular clocks to
date recent divergences (hundred-year scale; see: Kumar et al.,
2010; Mehla et al., 2009; Mohammed et al., 2011; Patil et al.,
2011; Ramı́rez et al., 2010; Weidmann et al., 2013), but here
we show the utility of using internal calibration that allows for
the reconstruction of deeper divergence times.
The divergence times under different calibration schemes
are summarized in Tables 2 and S1. All BEAST runs were
performed with an uncorrelated log-normal relaxed
molecular clock, as the null hypothesis of a strict global
substitution rate was rejected by the maximum-likelihood
molecular clock test (data not shown).
Divergence times of the genus Flavivirus:
calibration schemes compared
To examine the temporal origin of Flavivirus, we performed
several analyses in BEAST as outlined in Methods. Using only
tip date calibration resulted in 12–14 % older mean divergence times for nodes A–O than using the internal Beringian
calibration (node O calibrated to 15 000–11 000 years ago)
together with tip dates. For example, the age of the root
(node A) varied between 265 000 [95 % highest posterior
density (HPD): 25 600–2 768 000] and 230 500 (156 100–
322 700) years ago, respectively (Fig. 1, Table 2). When using
TBF internal calibration (node M, calibrated to 16 100–
44 929 years ago) based on the results from Heinze et al.
(2012) using only tip dates as calibration, 17–18 % younger
mean divergence times were recovered for nodes A–O compared with using tip dates only (Table 2). Both Beringian
and TBF calibration recovered similar mean divergence
times, although TBF calibration recovered 4–5 % younger
mean estimates for nodes A–O than Beringian calibration
(Table 2). Also, allowing for a wider range of the Beringian
calibration, i.e. 16 000–10 000 years, following Dixon (2013),
TBF recovered similar ages for the split of POWV and its
sister (node O) at 12 500 (10 000–15 600) years ago and for
the root (node A) at 228 500 (142 900–332 500) years ago as
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1971
Vector groups include insect-specific (IS), mosquito-borne (MB), no-known vector (NKV) and tick-borne (TB) flaviviruses.
Virus
Virus abbreviation
Strain
Journal of General Virology 95
Aedes virus
Alfuy virus
Alkhurma virus
Apoi virus
Bagaza virus
Bagaza virus
Baiyangdian virus
Banzi virus
AEFV
ALFV
AHFV
APOIV
BAGV
BAGV
BYDV
BANV
AEFV_SPFLD_MO_2011_MP6
MRM3929
1176
ApMAR-Kitaoka
Spain H/2010
DakAr-B209
BYD-1
SAH-336
Bouboui virus
Bovine viral diarrhoea virus 1
Bussuquara virus
Cell fusing agent virus
Chaoyang virus
Culex flavivirus
Deer tick virus
Dengue virus type 1
Dengue virus type 2
Dengue virus type 3
Dengue virus type 4
Donggang virus
Edge Hill virus
Entebbe bat virus
Gadgets Gully virus
Greek goat encephalitis virus
Hanko virus
Iguape virus
Ilheus virus
Japanese encephalitis virus
Jugra virus
Kadam virus
Kamiti River virus
Karshi virus
Kedougou virus
Kokobera virus
Koutango virus
Kunjin virus
Kyasanur forest disease virus
Lammi virus
BOUV
BVDV-1
BSQV
CFAV
CHAOV
CxFV
DTV
DENV-1
DENV-2
DENV-3
DENV-4
DGV
EHV
ENTV
GGYV
GGEV
HANKV
IGUV
ILHV
JEV
JUGV
KADV
KRV
KSIV
KEDV
KOKV
KOUV
KUNV
KFDV
LAMV
DAK-AR-B490
NADL
BeAn-4073
ROK144
Tokyo
CTB30-CT95
45AZ5
New-Guinea-C
H87
H241
DG0909
YMP-48
UgIL-30
CSIRO122
Vergina
SPAn-71686
Original
JaOArS982
P9-314
AMP6640
SR-82
LEIV_2247
DakAar-D1470
AusMRM-32
DakAr-D-5443
MRM61C
G11338
Source of isolate
Vector
group
Year
GenBank
accession no.
Aedes albopictus
Centropus phasianius
Homo sapiens
Apodemus spp.
Alectoris rufa
Mixed Culex
Duck
Homo sapiens, Culex, Mansonia
africana
Anopheles paludis
IS
MB
TB
NKV
MB
MB
MB
MB
2011
1966
1995
1954
2010
1966
2010
1956
KC181923.1
AY898809.1
AF331718.1
AF160193.1
HQ644143.1
AY632545.2
JF312912.1
DQ859056.1
MB
Alouatta beelzebul
Aedes spp., Aedes egypti
Aedes vexans nipponii
Culex pipiens
Ixodes dammini
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Aedes spp.
Aedes vigilax
Tadarida limbata
Ixodes uriae
Capra aegagrus hircus
Mosquitoes
Sentinel mouse
Aedes spp. and Psorophora spp.
Mosquito?
Aedes spp.
Rhipicephalus pravus
Aedes macintoshi
Ornithodoros papillipes
Aedes minutus
Culex annulirostris
Tatera kempi
Culex annulirostris
Haemaphysalis spinigera
Mosquitoes
MB
IS
IS
IS
TB
MB
MB
MB
MB
IS
MB
NKV
TB
TB
IS
MB
MB
MB
MB
TB
IS
TB
MB
MB
MB
MB
TB
IS
1967
1963
1956
1975
2003
2003
1995
1974
1944
1956
1956
2009
1969
1957
1975
1969
2005
1979
1944
1982
1968
1967
1999
1972
1972
1960
1968
1960
1957
2004
DQ859057.1
NC_001461.1
NC_009026.2
NC_001564.1
JQ068102.1
AB262759.2
NC_003218.1
U88536.1
AF038403.1
M93130.1
M14931.2
NC_016997.1
DQ859060.1
DQ837641.1
DQ235145.1
DQ235153.1
JQ268258.1
AY632538.4
AY632539.4
M18370.1
DQ859066.1
DQ235146.1
NC_005064.1
NC_006947.1
AY632540.2
NC_009029.2
EU082200.1
D00246.1
JF416959.1
FJ606789.1
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J. H.-O Pettersson and O. Fiz-Palacios
1972
Table 1. Flaviviruses used in this study
http://vir.sgmjournals.org
Table 1. cont.
Virus
LGTV
LIV
MEAV
MODV
MMLV
MVEV
NAKV
NMV
NOUV
NTAV
OHFV
OHFV
PCV
POTV
POWV
QBV
RBV
ROCV
RFV
SABV
SREV
SEPV
STWV
SSEV
SPOV
SLEV
TABV
TBEV-Sib
TBEV-FE
TBEV-Eu
TBEV-FE
TBEV-Eu
TBEV-Sib
TBEV
TBEV
TMUV
TSEV
TYUV
UGSV
USUV
Strain
TP21
369/T2
T70
M544
ATCC-VR-537
MVE-1-51
Uganda08
CY1014
IPDIA
Kubrin
Guriev
56
IBAN-10069
LB
VN180
RiMAR
SPH-34675
Eg-Art-371
DakAR-D4600
CSIRO-4
MK7148
87/2617
SM-6V-1
MSI-7
Tr127154
Vasilchenko
Sofjin-HO
Salem
Oshima-5-10
Neudoerfl
Est54
886-84
178-79
ZJ GH-2
TTE80
6017
ORIGINAL
SAAR-1776
Source of isolate
Vector
group
Year
GenBank
accession no.
Ixodes granulatus Supino
Ixodes ricinus
Ornithodoros maritimus
Peromyscus maniculatus
Myotis lucifugus
Homo sapiens
Mansonia africana nigerrima H4A1
Culex annulirostris
Uranotaenia mashonaensis
Mosquitoes
Homo sapiens
Homo sapiens
Coquillettidia xanthogaster
Cricetomys gambianus
Homo sapiens
Culex tritaeniorhynchus
Tadarida brasiliensis mexicana
Homo sapiens
Argas hermanni
Tatera kempi
Ornithodoros capensis
Mansonia septempunctata
Broiler chicken
TB
TB
TB
NKV
NKV
MB
IS
MB
IS
MB
TB
TB
IS
MB
TB
IS
NKV
MB
TB
MB
TB
MB
MB
TBF
MB
MB
NKV
TB
TB
TB
TB
TB
TB
TB
TB
MB
TB
TB
MB
MB
1956
1963
1981
1958
1958
1956
2008
1998
2004
1966
1947
1947
2010
1989
1958
2002
1954
1975
1968
1968
1974
1966
2000
1987
1955
1975
1973
1969
1937
2006
1995
1971
2000
1984
1979
2010
1969
1971
1947
1958
AF253419.1
NC_001809.1
DQ235144.1
AJ242984.1
AJ299445.1
AF161266.1
GQ165809.2
KC788512.1
FJ711167.1
JX236040.1
AY438626.1
AB507800.1
KC505248.1
DQ859067.1
L06436.1
NC_012671.1
AF144692.1
AY632542.4
DQ235149.1
DQ859062.1
DQ235150.1
DQ859063.1
JX477686.1
DQ235152.1
DQ859064.1
DQ359217.1
NC_3996.1
AF069066.1
AB062064.1
FJ572210.1
AB062063.2
TEU27495
GU183384.1
EF469662.1
EF469661.1
JQ314465.1
DQ235151.1
DQ235148.1
DQ859065.1
AY453412.1
Mansonia spp.?
Pteronotus parnellii
Homo sapiens
Homo sapiens
Macaca sylvanus
Dog
Ixodes ricinus
Ixodes persulcatus
Clethrionomys rufocanus
Ixodes persulcatus
Goose
Aedes spp.
Culex neavei
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Origin of the genus Flavivirus
1973
Langat virus
Louping ill virus
Meaban virus
Modoc virus
Montana myotis leukoencephalitis virus
Murray Valley encephalitis
Nakiwogo virus
New Mapoon Virus
Nounane virus
Ntaya virus
Omsk hemorrhagic fever virus
Omsk hemorrhagic fever virus
Palm Creek Virus
Potiskum virus
Powassan virus
Quang Binh virus
Rio Bravo virus
Rocio virus
Royal Farm virus
Saboya virus
Saumarez Reef virus
Sepik virus
Sitiawan virus
Spanish sheep encephalitis virus
Spondweni virus
St. Louis encephalitis virus
Tamana bat virus
Tick-borne encephalitis virus
Tick-borne encephalitis virus
Tick-borne encephalitis virus
Tick-borne encephalitis virus
Tick-borne encephalitis virus
Tick-borne encephalitis virus
Tick-borne encephalitis virus
Tick-borne encephalitis virus
Tembusu virus
Turkish sheep encephalitis virus
Tyuleniy virus
Uganda S virus
Usutu virus
Virus abbreviation
DQ859058.1
AF196835.2
NC_1563.2
AY765264.1
EU082199.1
AF094612.1
AB114858.1
DQ859059.1
1955
1999
1937
1997
1968
1979
1971
1947
MB
MB
MB
MB
MB
MB
NKV
MB
Wesselsbron virus
West Nile virus
West Nile virus
West Nile virus
Yaounde virus
Yellow fever virus
Yokose virus
Zika virus
WESSV
WNV
WNV
WNV
YAOV
YFV
YOKV
ZIKV
SAH-177-99871-2
NY99-flamingo382-99
B956
Rabensburg-97-103
DakAr-Y276
Trinidad-79A-788379
Oita-36
MR-766
Merino sheep
Phoenicopterus chilensis
Homo sapiens
Culex pipiens
Culex nebulosis
Haemagogus spegazzini
Miniopterus fuliginosus
Rhesus monkey
Year
Source of isolate
Strain
Virus abbreviation
Virus
Table 1. cont.
1974
compared with the more narrow Beringian calibration
(Table S1).
Vector
group
GenBank
accession no.
J. H.-O Pettersson and O. Fiz-Palacios
The use of the Yang96 model with tip dates as calibration
recovered older ages and broader intervals (95 % HPD)
compared with using the SRD06 model (Table S1).
However, both SRD06 and Yang96 model analyses under
Beringian calibration recovered similar ranges for the 95 %
HPD intervals (Table S1). The difference in estimates seen
between SRD06, allowing for the third codon position to
vary independently of the linked first and second codon
positions, and Yang96, allowing for all three codon
positions to vary, is an indication that divergence time
estimates become biased towards the present, with higher
substitution rates per nucleotide (Worobey et al., 2010).
The differences seen between the calibration schemes are
explained by the fact that estimating the tMRCA for entire
virus families or genera requires a broad sampling scheme
in order to cover as much of the variation as possible from
the group in question. However, the wider the sampling, the
higher the variation in substitution rates among lineages.
This will consequently lead to an increasing departure from
a constant molecular clock (Bromham & Penny, 2003; Duffy
et al., 2008; Holmes, 2003a). A high variation in substitution
rates will lead to uncertainty in the reconstructed divergence
times unless calibration points restrict the node ages.
Therefore, using tip dates alone results in a high level of
uncertainty for the deeper nodes and thus internal
calibration becomes crucial (Ho & Phillips, 2009). As the
Beringian calibration constraint (15 000–11 000 years) is
narrower than the TBF calibration constraint (16 100–42 300
years; Heinze et al., 2012), the resulting 95 % HPD intervals
from the Beringian analysis are also narrower.
Our study shows how incorporation of internal calibration
in large-scale virus phylogenies can help reconstruct more
precise divergences times, independent of the substitution
model, given a robust and reliable calibration by narrowing
down the 95 % HPD intervals. Furthermore, as the mean
tMRCA estimates are generally congruent between the
calibration schemes applied (Table S1), we will hereafter
only report and discuss the results from the combined tip
dates and the internal node calibration based on the BeringianPOWV biogeographical event, i.e. the Beringian calibration,
with the most narrow 95 % HPD intervals, unless otherwise
stated. This is because the incorporation of biogeographical
information is essential to date virus origins, especially for
ancient events (Katzourakis et al., 2009; Wertheim &
Kosakovsky Pond, 2011), as is the case with Flavivirus.
Divergence times of the genus Flavivirus:
congruences and incongruences
The genus Flavivirus is defined as sensu lato (node B) or
sensu stricto (node C). Our analysis, inferred from complete
coding nucleotide genomes, indicated that Flavivirus sensu
lato originated ~119 800 (87 100–158 900) years ago if
TABV is to be considered a part of the genus, or ~84 700
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Journal of General Virology 95
Origin of the genus Flavivirus
(63 700–109 600) years ago if TABV is excluded, i.e.
Flavivirus sensu stricto. Either way, this first molecular
dating for the whole genus indicates a significantly older
age than the 10 000 years that was previously suggested for
Flavivirus sensu stricto (Gould et al., 2001).
Our results also contrast the 3 000 years age of the MBFdom
group (node F), including MBFs, ISFs and NKVFs,
estimated in Zanotto et al. (1996). In the present analysis,
the MBFdom group (node F) was instead suggested to have
emerged .41 500 (32 600–51 600) years ago, with a
subsequent diversification of the MBFAedes clade (node
H) and the MBFCulex clade (node K) clade ~25 900 (19 800–
32 000) and ~26 900 (21 000–33 400) years ago, respectively. Previously, the ISFa clade (node E) was indicated to
have emerged between 3500 and 350 000 years ago (Crochu
et al., 2004). The combination of tip date calibration and
internal node calibration allows us to more precisely
pinpoint the origin of the ISFa clade to have occurred
~40 700 (30 800–52 300) years ago, i.e. ~44 000 years after
the split from the last common ancestor of Flavivirus sensu
stricto (node C) that emerged ~84 700 (63 700–110 000)
years ago. We also showed the first proposed dating of the
NKVFa clade (node N), here estimated to have emerged
~24 100 (18 000–30 900) years ago, ~15 000 years after the
split from the last common ancestor of TBFs and NKVFs
(node L).
The sharp contrast between the estimates in the present
study and previous studies can possibly be explained
because evolutionary rates in our study are inferred from a
genus-wide sampled dataset using a codon-based substitution model as compared with a nucleotide-based
substitution model used in many other studies. It is likely
that nucleotide-based substitution models will not be able
to account for variation in selective pressure throughout
evolutionary history, where the effect of purifying selection
is likely to cause underestimation of the actual ages
(Wertheim & Kosakovsky Pond, 2011). Using the SLAC, FEL,
IFEL, FUBAR and MEME methods available within the HyPhy
package (Pond et al., 2005) and at the Datamonkey
webserver (Delport et al., 2010), there were none-to-weak
signs of positive selection, but strong signs of purifying
selection in the alignment used in the present study (results
available upon request). Signs of strong purifying selection
within Flavivirus are in concordance with what has been
found previously for members of the genus Flavivirus and
for vector-borne RNA viruses in general (Holmes, 2003b;
Pybus et al., 2007; Woelk & Holmes, 2002). A codon-based
substitution model can to some extent compensate for
purifying selection, producing older tMRCA estimates
than nucleotide-based substitution models (Wertheim &
Kosakovsky Pond, 2011). However, and more importantly,
to accurately date deep RNA virus origins, the use of other
sources of evidence (such as biogeography) will eventually
become necessary as even codon-based models cannot
account for the complex interactions of events and factors
that have occurred throughout evolution (Katzourakis &
Gifford, 2010; Katzourakis et al., 2009).
http://vir.sgmjournals.org
Our study is supported by the fact that both Beringian
calibration and only tip date calibration give similar mean
tMRCA estimates, although only tip date calibration gives
broader 95 % HPD intervals. In contrast to several studies
(Sharp & Simmonds, 2011), we have not found any conflict
between internal node and tip date calibrations. Our results
are also congruent with several other studies (Table S3).
The estimated tMRCA for the TBFs clade [node M, at
27 500 (21 700–34 200) years ago] is supported by previous
estimates for the whole clade (Heinze et al., 2012) (28 600
years ago) and for the lineages within (Bertrand et al., 2012;
Uzcátegui et al., 2012) (Table S3). For the split of the
POWV clade and its sister (node O), our tMRCA estimates
of 14 800 years ago from tip date analysis also match
broadly with previous molecular clock studies (12 300 years
ago; Heinze et al., 2012) and support the use of the
Beringian biogeographical calibration.
Evolutionary rates of the genus Flavivirus and
molecular clocks in RNA viruses
Rates of nucleotide substitution were estimated from the
Beringian calibrated BEAST analyses for the entire ORF of
the genome (Table 3). Rates for the Beringian, TBF and tip
date calibration are summarized in Table S2.
The genomic substitution rate of positive-sense ssRNA
viruses varies between 1022 and 1025 substitutions site–1
year21 (Hanada et al., 2004; Jenkins et al., 2002; Sanjuán,
2012). Even though the estimated mean rates in the present
study for Flavivirus sensu lato (including TABV; node B),
461025 (261025 to 661025) substitutions site–1 year21,
are within range of those estimated for positive-sense
ssRNA viruses, they are at least an order of magnitude
slower than previous estimates for different lineages within
Flavivirus, i.e. 161023 and 261024 substitutions site–1
year21 as found for DENV-4 and St. Louis encephalitis
virus, respectively (Sanjuán, 2012 and references therein).
Our results are not very surprising given that rate
differences of up to 125 times have been shown for simian
immunodeficiency virus as compared with its sister, HIV
(Worobey et al., 2010).
Furthermore, we did not find that the MBFdom group
(node F) evolved at a significantly faster rate than the TBF
clade (node M), as reported previously (Zanotto et al.,
1995, 1996). Instead, our study showed that the MBFdom
group (node F) evolved at a mean rate of 3.261025
(1.761025 to 5.061025) substitutions site–1 year–1 and
that the TBF clade evolved at a mean rate of 3.961025
(2.361025 to 5.761025) substitutions site–1 year–1 (Table
3). Therefore, the implications for Flavivirus evolution
derived from Zanotto et al. (1995, 1996) need to be revised
and reconsidered. The fastest rate of nucleotide substitution was found for the ISFa clade (node E), with a mean
rate of 6.261025 (4.061025 to 8.661025) substitutions
site–1 year–1. Their fast rate of evolution could perhaps be
explained by their vertical mode of cycling transmission in
combination with their specificity to insects with relatively
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1975
J. H.-O Pettersson and O. Fiz-Palacios
Beringian calibration
(11 000–15 000 years)
TBF
O
TBF calibration
(16 100–44 929 years)
M
L
N
D
TBEV-Eu_1971_TEU27495
TBEV-Eu_2006_FJ572210.1
SSEV_1987_DQ235152.1
LIV_1963_NC_001809.1
GGEV_1969_DQ235153.1
TSEV_1969_DQ235151.1
TBEV-FE_1995_AB062063.2
TBEV-FE_1937_AB062064.1
TBEV_1979_EF469661.1
TBEV_1984_EF469662.1
TBEV-Sib_1969_AF069066.1
TBEV-Sib_2000_GU183384.1
OHFV_1947_AY438626.1
OHFV_1947_AB507800.1
LGTV_1956_AF253419.1
AHFV_1995_AF331718.1
KFDV_1957_JF416959.1
POWV_1958_L06436.1
DTV_1995_NC_003218.1
GGYV_1975_DQ235145.1
KSIV_1972_NC_006947.1
RFV_1968_DQ235149.1
KADV_1967_DQ235146.1
SREV_1974_DQ235150.1
MEAV_1981_DQ235144.1
TYUV_1971_DQ235148.1
MMLV_1958_AJ299445.1
RBV_1954_AF144692.1
MODV_1958_AJ242984.1
APOIV_1954_AF160193.1
KUNV_1960_D00246.1
WNV_1999_AF196835.2
WNV_1937_NC_001563.2
WNV_1997_AY765264.1
KOUV_1968_EU082200.1
YAOV_1968_EU082199.1
MVEV_1956_AF161266.1
ALFV_1966_AY898809.1
JEV_1982_M18370.1
USUV_1958_AY453412.1
SLEV_1975_DQ359217.1
ROCV_1975_AY632542.4
ILHV_1944_AY632539.4
TMUV_2010_JQ314465.1
BYDV_2010_JF312912.1
STWV_2000_JX477686.1
BAGV_1966_AY632545.2
BAGV_2010_HQ644143.1
NTAV_1966_JX236040.1
IGUV_1979_AY632538.4
BSQV_1956_NC_009026.2
NMV_1998_KC788512.1
KOKV_1960_NC_009029.2
DENV-1_1974_U88536.1
DENV-3_1956_M93130.1
DENV-2_1944_AF038403.1
DENV-4_1956_M14931.2
ZIKV_1947_DQ859059.1
SPOV_1955_DQ859064.1
KEDV_1972_AY632540.2
NOUV_2004_FJ711167.1
ISF
CHAOV_2003_JQ068102.1
LAMV_2004_FJ606789.1
DGV_2009_NC_016997.1
SABV_1968_DQ859062.1
POTV_1989_DQ859067.1
JUGV_1968_DQ859066.1
BANV_1956_DQ859056.1
UGSV_1947_DQ859065.1
BOUV_1967_DQ859057.1
EHV_1969_DQ859060.1
WESSV_1955_DQ859058.1
SEPV_1966_DQ859063.1
YFV_1979_AF094612.1
YOKV_1971_AB114858.1
ENTV_1957_DQ837641.1
CxFV_2003_AB262759.2
QBV_2002_NC_012671.1
NAKV_2008_GQ165809.2
PCV_2010_KC505248.1
KRV_1999_NC_005064.1
AEFV_2011_KC181923.1
CFAV_1975_NC_001564.1
HANKV_2005_JQ268258.1
TABV_1973_NC_003996.1 NKVF
BVDV-1_1963_NC_001461.1
NKVF
MBF
K
J
I
C
ISF
F
H
G
B
MBF
NKVF
A
322
E
200
150
land bridge
glacier
16 000–14 000 years
0 Time (×103 years)
50
land bridge
land bridge
glacier
25 000–18 000 years
100
ISF
migration path
POWV
13 000–11 000 years
glacier
glacier
POWV
mig
rati
on
pa
th
<10 500 years
Fig. 1. Chronogram based on tip date calibrated terminals and internal nodes using Beringian calibration (see text for details).
All named nodes have maximum posterior probability support (1.0 pp). Terminal tips are named by virus name (abbreviations,
1976
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Journal of General Virology 95
Origin of the genus Flavivirus
see Table 1), year of isolation and GenBank accession number. Development of the Beringia region between the last glacial
maximum until the Bering Strait was formed (25 000–10 500 years ago). Beringia development after Elias et al. (1996) and
Dixon (2013).
short generation times (Bolling et al., 2012; Lutomiah et al.,
2007).
Estimates of substitution rates are often found to be much
higher for tips than for deep nodes (Wertheim &
Kosakovsky Pond, 2011). From our perspective, a highly
variable non-constant molecular clock with pulses of very
high substitution rates for short periods of time may reflect
the difference of orders of magnitude seen when comparing
evolutionary rates in short and recent times (e.g. 161023
for DENV-4 in Klungthong et al., 2004) with long and deep
times (e.g. 461025 for Flaviviruses sensu lato in this study).
Beringia and the emergence of POWV in North
America
The Beringian land bridge was open for land migration
between 15 000 and 11 000 years ago until it was flooded
and the Bering Strait was formed (Dixon, 2013; Elias, 2001;
Elias et al., 1996; Kelly, 2003), effectively blocking
migration between Asia and North America for animals
other than humans (Fig. 1). Our molecular reconstructions
using only tip dates are congruent with POWV being
introduced into North America when the Beringian land
bridge was accessible and supports the use of tip dates in
combination with an internal Beringian calibration (Fig. 1,
Table 2).
How then did POWV enter North America? Although
humans might have been responsible for carrying POWV,
as humans do get infected with POWV, perhaps it is more
likely that ticks and associated tick hosts jointly brought
POWV into North America. Human infections with
POWV in present times are infrequent (Ebel, 2010),
although numbers appear to be on the increase (Hinten
et al., 2008). Between 15 000 and 11 000 years ago, the
migrating human population who settled the Americas had
an effective population size of perhaps ,80 individuals
(Hey, 2005). This, in combination with the fact that
POWV is considered to be maintained solely in an enzootic
cycle between ixodid ticks and their vertebrate hosts (Ebel,
2010; Ebel et al., 2000), is a strong argument against
humans being the likely vectors by which POWV first
entered North America. Nor is it likely that POWV entered
the Americas by the coastal route. Even though the coastal
areas did support marine mammals and had terrestrial
mammals living in refugia (Heaton & Grady, 2003), the
deglaciated coastal areas had sparse vegetation that did not
support large populations of terrestrial mammals (Dixon,
2013). Also, animals were isolated and movement was
inhibited due to the glaciers, and the coastal route was, in
general, not considered to be a migratory route for
mammals other than humans (Dixon, 2013).
In view of the results from the present study, the most
likely time period of introduction was after the deglaciation
corridor became habitable ~13 500 years ago (Catto, 1996;
Dixon, 2013) and before sea levels rose by 40 m and the
Bering strait reached its present width before 10 500 years
ago (Elias et al., 1996). During this period, populations of
Table 2. Mean estimated tMRCA (¾103 years) and 95 % HPD interval: results for the Beringian, TBF and tip date calibration analyses
Node
A: Root
B: TABV/ISF–MBF–TBF–NKVF
C: ISFa/MBFdom–TBF–NKVFa
D: MBFdom/TBF–NKVFa
E: ISFa
F: MBFdom
G: NKVFb/MBFAedes
H: MBFAedes
I: ISFb/NOUV–MBFCulex
J: NOUV/MBFCulex
K: MBFCulex
L: TBF/NKVFa
M: TBF
N: NKVFa
O: POWV/sister clade of TBF
http://vir.sgmjournals.org
Beringian (11 000–15 000 years)
TBF (16 100–42 300 years)
Tip dates only
tMRCA
95 % HPD
tMRCA
95 % HPD
tMRCA
95 % HPD
230.5
119.8
84.7
47.2
40.7
41.5
33.2
25.9
33.8
31.0
26.9
39.3
27.5
24.1
12.8
156.1–322.7
87.1–158.9
63.7–109.6
37.3–58.7
30.8–52.3
32.6–51.6
25.7–42.0
19.8–33.0
26.6–42.1
24.3–38.6
21.0–3343
30.7–49.2
21.7–34.2
18.0–30.9
11.0–14.8
219.4
114.0
80.6
44.9
38.5
39.4
31.5
24.6
32.1
29.5
25.5
37.5
26.2
22.9
12.2
110.6–387.6
62.2–194.6
45.0–135.1
26.4–73.9
21.7–64.6
23.1–64.9
18.2–52.3
14.0–41.0
18.7–52.9
17.2–48.7
14.8–42.1
21.9–61.4
16.1–42.3
12.8–38.4
7.0–20.0
265.0
138.7
98.4
54.0
46.8
47.4
37.9
29.5
38.7
35.5
30.7
44.9
31.5
27.5
14.8
25.6–2.768.1
12.9–1.456.1
9.8–1.024.7
5.5–560.2
4.6–486.7
4.9–492.7
4.0–390.7
2.9–304.1
3.8–401.9
3.8–369.4
3.3–320.0
4.2–468.5
3.1–329.4
2.9–286.6
1.5–152.3
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1977
J. H.-O Pettersson and O. Fiz-Palacios
Table 3. Mean estimated evolutionary rates (¾10”5 substitutions site–1 year–1) and 95 % HPD intervals for the Beringian
calibration (11 000–15 000 years)
Node
B: TABV/ISF–MBF–TBF–NKVF
C: ISFa/MBFdom–TBF–NKVF
D: MBFdom/TBF–NKVFa
E: ISFa
F: MBFdom
G: NKVFb/MBFAedes
H: MBFAedes
I: ISFb/NOUV–MBFCulex
J: NOUV/MBFCulex
K: MBFCulex
L: TBF/NKVFa
M: TBF
N: NKVFa
O: POWV/sister clade of TBF
Mean rate
95 % HPD
3.96
3.60
2.56
6.24
3.24
2.92
3.11
4.02
3.95
4.19
4.27
3.89
5.18
4.30
2.04–6.14
1.81–5.57
1.57–3.70
4.00–8.65
1.73–5.00
1.63–4.43
1.70–4.72
2.28–6.03
2.12–6.06
2.30–6.37
2.39–6.38
2.32–5.73
2.32–6.15
3.31–7.23
ixodid ticks and land mammals (including humans) could
have migrated from eastern Beringia through the then
habitable deglaciated corridor to the south of the glaciers
(Dixon, 2013; Goebel & Buvit, 2011; Shapiro et al., 2004).
Ticks and tick hosts maintaining POWV might initially
have been restricted to eastern Beringia, north-west of the
glaciers. As the deglaciated corridor became habitable and
animals could migrate southwards, and after the Bering
Strait was formed preventing land migration back to Asia,
POWV became established in North America. Using an
internal calibration of 15 000–11 000 or 16 000–10 000 years
for the split of POWV from closely related TBFs gives
similar tMRCA dates for POWV crossing into the Americas
(12 800 and 12 500 years ago, respectively), which is in
agreement with dates of the opening and closing of the
Beringian land bridge.
Did flaviviruses and humans spread throughout
the globe together?
Viruses, and RNA viruses in particular, are among the most
successful evolving entities on the planet, having colonized
and adapted to an array of different environments and
modes of transmission within all domains of life (Wasik &
Turner, 2012). The most significant event that might have
helped shape the distribution of flaviviruses is perhaps the
spread of modern humans out of Africa. Modern humans
arose in Africa (Cavalli-Sforza & Feldman, 2003; McDougall
et al., 2005; Stringer & Andrews, 1988), and began migrating
to other continents between 80 000 and 40 000 years ago,
having populated all continents except Antarctica by 10 000
years ago (Blome et al., 2012; Henn et al., 2012; Macaulay
et al., 2005; Rasmussen et al., 2011). The migration of
humans out of Africa has been accompanied by several other
pathogens. The pathogenic bacterium Helicobacter pylori
appears to have accompanied human migration out of Africa
and is estimated to have spread from east Africa ~58 000
1978
years ago (Falush et al., 2003; Linz et al., 2007; Moodley et al.,
2012). Likewise, the Mycobacterium tuberculosis complex is
estimated to have started to diverge ~70 000 years ago,
consistent with an African origin linked to human expansion
and migration (Comas et al., 2013; Wirth et al., 2008).
Similarly, several human pathogenic viruses also show
patterns of co-divergence associated with human diversification (Holmes, 2004). Therefore, it is perhaps not unlikely
that the spread and evolution of several Flavivirus strains,
especially if much of the Flavivirus diversity originated in
Africa (Gould et al., 2001, 2003), was influenced by human
migration and expansion across the globe.
METHODS
Virus genomes, sequence alignments and models of molecular
evolution. Complete coding genomes of the genus Flavivirus,
excluding the untranslated 59 and 39 flanking regions, were retrieved
from GenBank (Table 1). Genomes were selected based on available
background information on the year of virus strain isolation (isolated
between 1937 and 2011). BVDV-1 of the genus Pestivirus within the
family Flaviviridae was included as an outgroup based on a previously
published Flavivirus phylogeny (Cook et al., 2012). To construct a
robust alignment without frameshifts, all sequences were translated to
amino acids in SeaView 4.4.2 (Gouy et al., 2010), aligned with MAFFT
7.037b using the default G-INS-i algorithm and parameters (Katoh &
Standley, 2013), and then back-translated into nucleotides. The
resulting alignments were inspected visually and edited with AliView
0.8 (http://ormbunkar.se/aliview). To estimate the best-fitting model
of evolution for the sequence alignments, model tests were performed
for the nucleotide alignment using jModelTest 2.1.3 (Darriba et al.,
2012) and for the amino acid alignment using ProtTest 3.2.1 (Darriba
et al., 2011). To test the null hypothesis of a strict molecular clock, a
maximum-likelihood clock test was performed using MEGA 5.22
(Tamura et al., 2011). The best-fitting models were then specified in
the respective analyses.
Phylogenetic analysis: maximum-likelihood and Bayesian
analysis. Maximum-likelihood trees were estimated using RAxML
7.6.3 (Stamatakis, 2006) with 1000 rapid bootstraps under the GTR+
I+G model for the nucleotide alignment and the WAG+I+G model
for the amino acid alignment, respectively. Bayesian phylogenetic trees
were inferred using MrBayes 3.2.1 (Ronquist et al., 2012) by executing
two parallel runs with four Metropolis-coupled chains for 20 million and
9 million Markov chain Monte Carlo (MCMC) generations, using
GTR+I+G for nucleotides and WAG+I+G for amino acids as model
of evolution, respectively, sampling every 1000 generations and run with
default priors, discarding the first 25 % as burn-in.
Estimating the tMRCA: Bayesian analysis with BEAST. To explore
the temporal scale of the entire genus Flavivirus, evolutionary rates
and the tMRCA were estimated using a Bayesian MCMC method as
implemented in BEAST 1.7.5 (Drummond et al., 2012). All BEAST runs
were performed with calibrated tip dates where the sequence with the
most recent sampling date (2011) was set to represent the present.
The SRD06 codon-based partition model (Shapiro et al., 2006) and
HKY85+G nucleotide substitution model were used together with a
non-parametric Bayesian skyline population coalescent tree prior
with a piecewise-constant skyline demographic model (Drummond
et al., 2005), along with a log-normal uncorrelated relaxed molecular
clock. The effect of using the Yang96 codon model (Yang, 1996) was
also explored in a separate analysis. All analyses were run for 100
million generations in triplicate, sampling every 1000 generations to
ensure mixing of chains and that a sufficiently effective sample size
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Journal of General Virology 95
Origin of the genus Flavivirus
(.200) was reached. Convergence of chains and effective sample size
statistics were analysed with Tracer 1.5 (http://beast.bio.ed.ac.uk/
Tracer). Log and tree files were combined in LogCombiner (BEAST
package) to produce consensus files of the different runs. Finally,
maximum clade credibility trees of the three different analyses were
produced using TreeAnnotator (BEAST package). Chronograms were
viewed and annotated in FigTree 1.3.1 (http://tree.bio.ed.ac.uk/software/
figtree). Computations were performed at the Bioportal webportal at the
University of Oslo (www.bioportal.uio.no), the CIPRES webportal
(Miller et al., 2010) and at the Uppsala Multidisciplinary Center for
Advanced Computational Science (www.uppmax.uu.se).
complete coding sequences of arthropod-borne viruses and viruses
with no known vector. J Gen Virol 81, 781–790.
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Bolling, B. G., Olea-Popelka, F. J., Eisen, L., Moore, C. G. & Blair,
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ACKNOWLEDGEMENTS
We are grateful to Allison Perrigo, Martin Ryberg and Petra Korall for
their constructive comments and feedback on the manuscript. We
acknowledge Allison Perrigo and Stina Weststrand for their help with
the design of the figures. We are also grateful to James E. Dixon for
valuable information and discussions on Beringian biogeography.
Finally, we wish to acknowledge all the hard work by people all over
the world making the sequences used in the present study available to
everyone. J. H.-O. P. was supported by Stiftelsen Olle Engkvist
Byggmästare.
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