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Journal of Virology, December 2001, p. 11803-11810, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11803-11810.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular Evolution of Puumala Hantavirus
Tarja
Sironen,
Antti
Vaheri,* and
Alexander
Plyusnin
Department of Virology, Haartman Institute,
University of Helsinki, Helsinki, Finland
Received 21 June 2001/Accepted 29 August 2001
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ABSTRACT |
Puumala virus (PUUV) is a negative-stranded RNA virus in the genus
Hantavirus, family Bunyaviridae. In this
study, detailed phylogenetic analysis was performed on 42 complete S
segment sequences of PUUV originated from several European countries,
Russia, and Japan, the largest set available thus far for hantaviruses.
The results show that PUUV sequences form seven distinct and
well-supported genetic lineages; within these lineages, geographical
clustering of genetic variants is observed. The overall phylogeny of
PUUV is star-like, suggesting an early split of genetic lineages. The individual PUUV lineages appear to be independent, with the only exception to this being the Finnish and the Russian lineages that are
closely connected to each other. Two strains of PUUV-like virus from
Japan form the most ancestral lineage diverging from PUUV.
Recombination points within the S segment were searched for and
evidence for intralineage recombination events was seen in the Finnish,
Russian, Danish, and Belgian lineages of PUUV. Molecular clock analysis
showed that PUUV is a stable virus, evolving slowly at a rate of
0.7 × 10
7 to 2.2 × 106 nt
substitutions per site per year.
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INTRODUCTION |
Puumala virus (PUUV) belongs to the genus
Hantavirus of the family Bunyaviridae
(12). Like other members of this family, PUUV is an
enveloped virus with a segmented, single-stranded RNA genome of
negative polarity. The large (L) segment of 6.5 kb encodes the viral
RNA polymerase, the 3.7-kb medium (M) segment encodes the two surface
glycoproteins, and the 1.8-kb small (S) segment encodes the
nucleocapsid protein (N).
The natural host of PUUV is the bank vole, Clethrionomys
glareolus, which belongs to the Arvicolinae subfamily of the
Muridae family. The bank vole is found in most of Europe, excluding the Mediterranean coast and the northernmost areas (Fig.
1) (40). The virus causes a
life-long persistent and asymptomatic infection in rodents
(47). In contrast, in humans PUUV is pathogenic, causing
nephropathia epidemica, a mild form of hemorrhagic fever with renal
syndrome (HFRS) (8).
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FIG. 1.
Map of Europe showing the distribution of the bank vole
(40). PUUV sequences analyzed in this paper belong to the
following seven distinct lineages: FIN (1), NSCA (2), SSCA (3)
(sequences are from Sweden [A] and Norway [B]), DAN (4), BEL (5)
(sequences are from Belgium [A] and Germany [B]), BAL (6), and RUS
(7) (sequences are from Russia [A] and the Baltics [B]).
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PUUV from the following countries has been genetically characterized
(Fig. 1): Finland (45, 46, 48, 62), Sweden (24, 25,
38), Norway (38), Denmark (3), Russia
(3, 16, 48, 49, 66), Belgium (6, 13), Austria
(1, 5), and Germany (19, 43). So far, over
100 partial or complete PUUV sequences have been deposited in GenBank.
These include 38 complete S segment sequences, 9 complete M segment
sequences, and 2 complete L segment sequences. In general, the
phylogenetic relationships between different hantaviruses and their
rodent hosts mirror each other, supporting the idea of coevolution of the virus and its host (44). Furthermore, different
strains of a given hantavirus type, including PUUV, show geographical clustering (48, 49) reflecting the sometime complicated
history of host range movements. The most recent bank vole range
changes are due to the recolonization of Europe and especially
Fennoscandia after the last ice age 10,000 years ago (29).
Earlier it has been shown that these migrations have had an impact on
the evolution of PUUV (24, 38).
The main source of genetic variation of hantaviruses seems to be
genetic drift, i.e., accumulation of base substitutions and deletions
or insertions (3, 38, 48). Additionally, reassortment has
been shown for some hantaviruses (20) and it has been
proposed also for PUUV (45). It has been recently shown
that recombination was involved in the evolution of Tula hantavirus
(TULV) (55). There are indications that this mechanism is
operating in PUUV as well (3, 13).
PUUV has been shown to form quasispecies populations in individual bank
voles (48). In general, this feature is connected to a
high potential for rapid evolution (11). Nevertheless, if
the master genotype and phenotype have high fitness to the environment, the quasispecies populations might be stable for a long
period of time. This is most probably the case for PUUV and other
hantaviruses, which have been well adapted to their natural hosts since
long ago.
Previous publications on PUUV evolution (1, 3, 13, 19, 24, 43,
45, 46, 48, 49) have focused mainly on specific geographical
areas, including only a limited number of sequences, and all the
phylogenetic analyses have been performed with distant matrix methods.
Recently, a substantial set of new complete PUUV S segment sequences
became available (references 3 and 13 and our
unpublished results). In this study, for the first time we have
attempted to analyze all the known complete S segment sequences of PUUV
using different phylogenetic methods in order to gain more insight into
the evolution of this virus. Special attention was paid to studying
whether the concept of a molecular clock fits PUUV evolution or not and
to the role of recombination in the evolution of this virus.
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MATERIALS AND METHODS |
The following PUUV S segment and N protein hantavirus sequences
were analyzed. Sequences from strains of Finnish lineage (FIN) were
strain Sotkamo, GenBank accession number X61035, Evo/12Cg/93, Z30702;
Evo/13Cg/93, Z30703; Evo/14Cg/93, Z30704; Evo/15Cg/93, Z30705;
Virrat/25Cg/95, Z69985; Karhumäki/Cg117/95, AJ238788; Kolodozero/Cg53/95, AJ238789; Gomselga/Cg4/95, AJ238790; Puumala/1324Cg/79, Z46942; and Pallasjarvi/63Cg/98, AJ314597. Sequences
from strains from southern Scandinavia (SSCA) were Eidsvoll/1124v, AJ223368; Eidsvoll/Cg1138/87, AJ223369; Solleftea/Cg3/95, AJ223376; and
Solleftea/Cg6/95, AJ223377. Sequences from strains from northern
Scandinavia (NSCA) were Hundberget/Cg36/95, AJ223371;
Mellansel/Cg47/94, AJ223374; Mellansel/Cg49/95, AJ223375;
Tavelsjo/Cg81/94, AJ223380; Vindeln/L20Cg/83, Z48586; Vindeln/Cg4/94,
AJ223381; and "Vranica"/Hällnäs, U14137. Sequences from
Danish (DAN) strains were Fyn19, AJ238791; Fyn47, AJ238792; and Fyn131,
AJ238793. Sequences from Russian (RUS) strains were Udmurtia/338Cg/92,
Z30708; Udmurtia/444Cg/88, Z30706; Udmurtia/458Cg/88, Z30707;
Udmurtia/894Cg/91, Z21497; Kazan, Z84204; Cg1820, M32750; P360, L11347;
Baltic/49Cg/00, AJ314598; and Baltic/205Cg/00, AJ314599. Sequences from Belgian (BEL) strains were Cg13891, U22423; Cg-Erft, AJ238779; Thuin/33Cg/96, AJ277030; Montbliart/23Cg/96, AJ277031;
Momignies/47Cg/96, AJ277032; Momignies/55Cg/96, AJ277033; and
Couvin/59Cg/97, AJ277034. Sequences from Balkan (BAL) strains were
Balkan/65Cg/00, AJ314600; and Balkan/78Cg/00, AJ314601. Sequences from
Japanese (JPN) strains were Tobetsu-60Cr-93, AB010731; and
Kamiiso-8Cr-95, AB010730. Sequences from other hantaviruses were TULV,
strain Moravia02v (Z69991); Hantaan virus (HTNV), 76-118 (M14626); Dobrava, Dobrava (L41916); Saaremaa, Saaremaa/160V (AJ009773); Seoul,
SR-11 (M34881); El Moro Canyon, RM-97 (U11427); New York, RI-1
(U11427); Sin Nombre, NM H10 (L25784); Bayou, Louisiana (L36929); Black
Creek Canal (L39949); Laguna Negra, 510B (AF005727); Topografov, Ls136V
(AJ011646); Khabarovsk, MF-43 (U35255); Prospect Hill, PH-1 (Z49098);
and Andes, AH-1 (AF324902).
Multiple sequence alignments were prepared with ClustalX
(60) using the following parameters: gap opening, 10; gap
extension, 6.66; delay divergent sequences by 40%; DNA transition
weight, 0.50; and no negative matrix.
Phylogenetic analysis.
The Wisconsin Package, version 10.2 (Genetics Computer Group, Madison, Wis.), was used for sequence entry
and analysis. Nucleotide sequences were translated into amino acid
sequences with SeqApp. PHYLIP was used to create phylogenetic trees
using distance matrix (DM) methods (Fitch-Margoliash or neighbor
joining) and maximum parsimony (MP) methods (15). These
methods were applied for both the nucleotide and deduced amino acid
sequences with 500 bootstrap replicates.
The TreePuzzle program was used to reconstruct phylogenetic trees using
the maximum likelihood (ML) approach (
57). Ten thousand
puzzling steps were applied using the Hasegawa-Kishino-Yano
(HKY)
model of substitution (
18). The
transition/transversion ratio
and nucleotide frequencies were estimated
from the data set. Rate
heterogeneity was applied using discrete gamma
distribution with
eight rate categories, and the shape parameter alpha
was estimated
from the data
set.
Split decomposition analysis (
4,
28) was performed with
the program SplitsTree using the LogDet method for computing
distances.
This method presents conflicting evolutionary signals
in the data set
as a network instead of a dichotomously branching
phylogenetic
tree.
Similarity plots were created using Stuart Ray's SimPlot 2.5 (
34). The window size was 200 to 300 nucleotides (nt) and
the
step size was 20 nt. Jukes-Cantor corrections were applied.
Consensus
sequences of PUUV lineages were used as query or reference
sequences.
Rate of evolution.
To calculate the evolution rate of PUUV,
the number of synonymous and nonsynonymous substitutions per 100 sites
(dS and dN) was estimated using the program Diverge from the Genetics Computer Group Wisconsin package. Values were calculated comparing PUUV to two
different hantavirus species, HTNV and TULV. Alternatively, the ML
branch lengths derived from trees with contemporary tips (calculated
using TreePuzzle) were used. Both the mean number of
dS and the ML branch lengths were then
divided by the time of divergence of rodents carrying these viruses to
gain an estimate of the substitution rate.
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RESULTS |
Phylogeny of PUUV.
On a phylogenetic tree hantaviruses form
three clades carried by Murinae, Arvicolinae, and Sigmodontinae rodents
(Fig. 2). PUUV is placed within the
second clade, which also includes TULV, Bloodland Lake, Prospect Hill,
Isla Vista, and Khabarovsk viruses, all carried by voles, and
Topografov virus, whose natural hosts are lemmings. TULV is used as an
outgroup sequence in the phylogenetic analyses of PUUV in this paper.
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FIG. 2.
A phylogenetic tree of hantavirus N protein sequences
calculated using TreePuzzle (55). An enlarged phylogenetic
tree of PUUV S-segment coding sequences created using FITCH of the
PHYLIP package (15) is shown. The bootstrap support values
for the PUUV lineages are given in Table 2.
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The best collection of sequences is available for the S segment, which
also seems to be a good representative of the whole
PUUV genome
(
3). These sequences vary in length from 1,784
nt in
strain CG1820 to 1,882 nt in strain Sollefteå-6 and contain
an open
reading frame coding for the N protein of 433 amino acids
(aa). The 5'
noncoding region (NCR) in the positive strand is
42 nt in length and
the 3' NCR varies from 442 to 540 nt. Except
for the last
100 nt,
the S segment 3' NCR of different strains
could be aligned only within
given genetic lineages of PUUV (
3,
13,
38) and was
therefore excluded from our
analysis.
Phylogeny of PUUV S segment nucleotide sequences shows eight distinct
genetic lineages, FIN, RUS, NSCA, SSCA, DAN, BEL, BAL,
and JPN, which
share a common ancient ancestor (Fig.
2). PUUV
strains in each lineage
are given in Table
1. The first seven
lineages share a common more recent ancestor, while the JPN lineage
occupies the most ancestral node. This lineage includes two wild-type
strains recovered from tissue samples of
Clethrionomys
rufocanus trapped in Hokkaido (
32). Being associated
with a distinct host
species, these strains cannot be strictly referred
to as PUUV
but rather are considered PUUV-like.
All genetic lineages of PUUV possess specific amino acid
"signatures" (Table
1). In addition to signatures characteristic
of
the FIN and the RUS lineages, there are 2 aa residues
(Val
34 and Tyr
61) shared by
these lineages, indicating a closer relationship
between them. The JPN
lineage has the longest amino acid
signature.
Comparison of the S segment sequence identities (reference
3 and our unpublished data) shows that the variation
between
the lineages ranges at the nucleotide level from 15 to 27%,
with
the smallest difference being observed between the RUS and the
FIN
lineages. The intralineage nucleotide diversity is 0.3 to
9.0% for all
the lineages except SSCA, which shows diversity up
to 13.4%, and RUS
(15.6%). The SSCA lineage is actually formed
by two sublineages
constituted by strains from central Sweden
and Norway, respectively,
with the intrasublineage diversity ranging
from 0.3 to 5.7%
(
38). The RUS lineage seems to be formed also
by two
sublineages formed by strains from the European part of
Russia and the
Baltics.
At the amino acid level, the interlineage variation translates to
substantially lower values, of 0 to 7.8%, indicating that
a strong
purifying selection occurred at the N protein level.
Notably, the PUUV
N protein sequence diversity is higher than
in other hantaviruses
(
23,
35) and in several cases even exceeds
the cutoff
level of 7% arbitrarily selected to define distinct
hantavirus species
(
12).
The overall topology of the PUUV phylogenetic tree is star-like,
suggesting an early split of the genetic lineages. In general,
all
these lineages are well supported (Table
2). Although the
topology of all trees
calculated with DM, MP, and ML is the same,
the bootstrap support
values vary. Both the MP and DM methods
show different bootstrap
support values depending on whether the
calculations are based on
nucleotide or amino acid sequences.
MP gives more consistent results
than the distance methods, but
there are still values lower than 70%
(Table
2), the widely accepted
confidence limit (
22). The
support values calculated using the
ML method are the most consistent.
The early split of PUUV genetic lineages manifested as the star-like
topology of the trees infers that the relationships between
distinct
lineages remain obscure. The FIN and RUS lineages represent
the only
exception to this, being more closely connected to each
other.
As the bootstrap support values sometimes varied depending on whether
the calculations were based on nucleotide or amino acid
sequences, this
was further evaluated using the "likelihood mapping"
option of the
TreePuzzle program. This method can be used to visualize
the
phylogenetic content of a sequence alignment as follows. Different
topologies of quartet trees are plotted in a triangle so that
the
corners represent the completely resolved, tree-like quartets;
the
sides represent quartets that are partially resolved and the
center
represents the unresolved quartets (
56). It appeared
that
only a very low number (1.6%) of the quartets based on PUUV
nucleotide
sequences was partially or completely unresolved (Fig.
3); the corresponding number for the
amino acid sequences is higher
(9.4%) but is still low enough to
consider the tree reconstruction
to be accurate (
56).
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FIG. 3.
Likelihood mapping of PUUV nucleotide (A) and amino acid
(B) sequences. Dots in the corners of triangles represent fully
resolved tree topologies and dots in the center represent unresolved
ones.
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Molecular clock and the rate of PUUV evolution.
TreePuzzle was
also used to perform a likelihood ratio test in order to check a
molecular clock hypothesis. In our case, a clock-like tree did not pass
the test when either all or only a few PUUV strains were considered
assuming a uniform rate of nucleotide substitutions. Only when applying
gamma distribution of rate heterogeneity was the clock no longer
rejected (Fig. 4). The shape parameter
alpha estimated from the data set is 0.23 ± 0.01, indicating that
most of the sites evolve slowly but that a few sites have a
moderate-to-high rate of evolution.
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FIG. 4.
A phylogenetic tree of PUUV with contemporary tips and
estimates of dates of divergence of the host rodents (9, 10,
30).
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Passing the likelihood ratio test of the molecular clock showed that
the data set can be used to estimate the evolution rate
of PUUV. The
mean values of
dS and
dN (the number of synonymous
and
nonsynonymous substitutions per 100 sites) calculated for
all PUUV
sequences were 88 ± 29 and 2.37 ± 0.83, respectively.
Within a genetic lineage
dS varied from 6 to 45, and between lineages
it varied from 85 to 122.
dS was highest within the SSCA and RUS
lineages, probably reflecting the fact that these lineages actually
contain well-separated sublineages; the corresponding values for
dN were 0.1 to 1.2 and 0.1 to 3.8. In all
cases, the
dN/
dS
ratio
was extremely low, indicating that positive selection is
not the
primary mechanism driving the evolution of this
virus.
In order to estimate the rate of nucleotide substitutions in PUUV
evolution, the
dS values between PUUV,
Japanese PUUV-like
viruses, TULV, and HTNV were calculated (Table
3). Assuming that
the viruses coevolved
with their hosts, the evolutionary rate
of PUUV will be 1.9 × 10
7 to 2.2 × 10
6
synonymous nucleotide substitutions per site per year. The evolutionary
rate of PUUV was also estimated using the ML branch lengths of
the
clock-like trees (Fig.
4). The values ranged from 0.7 × 10
7 to 1.1 × 10
6
nt per site per year depending on whether HTNV was included in
the
calculations in addition to PUUV and TULV (Table
3). The
rate
estimations obtained by these two methods gave encouragingly
similar
results, with all values being between 0.7 × 10
7 and 2.2 × 10
6
nt per site per year.
Recombination in PUUV evolution.
Recombination of hantaviruses
has been shown for TULV (55) and suggested for PUUV as
well (3, 13). To study this issue further, similarity
plots were created to visualize the pattern of sequence similarity
between the distinct PUUV lineages. These plots (Fig.
5A) revealed within the S-segment
sequence some regions with higher-than-average similarity between the
FIN and RUS lineages. This was particularly pronounced on the PUUV
sequences from the Baltics within the RUS lineage (data not shown),
suggesting a recombinant origin for these strains. Such a suggestion
was further strengthened by our observation that inclusion of either
one of these Baltic strains in a clock-like tree led to rejection of the molecular clock, just as would be expected from a recombinant sequence (54).
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FIG. 5.
Similarity analysis based on consensus sequences of
distinct PUUV lineages. The query sequence is the Balkan consensus
sequence (A) or the Baltic consensus sequence (B).
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A closer analysis has shown that two regions in the Baltic sequences,
nt 440 to 630 and 940 to 1130, are in fact more similar
to the FIN
sequences (Fig.
5B). However, when trees were calculated
for these
regions, the Baltic sequences were not placed within
the FIN lineage
but formed a cluster of their own. A similar pattern
has been seen
earlier for the DAN lineage, which in one part was
most closely related
to the NSCA lineage while in another part
it was most closely related
to the SSCA lineage and thus was suggested
to contain recombinant
sequences (
3).
Since some evidence for recombination was revealed, we studied if a
phylogenetic network showing the conflicting signals would
illustrate
the evolution of PUUV better than a phylogenetic tree.
This was done
using the program SplitsTree based on the split
decomposition theory
(
4). On the SplitsTree (Fig.
6A), the
RUS, BEL, NSCA, and DAN lineages
are represented as networks,
suggesting that they contain conflicting
phylogenetic signals.
This indicates that these lineages include
sequences that might
have undergone recombination during their
evolution. Since the
Baltic sequences were suspected to be recombinants
between the
FIN and RUS lineages, they were studied in more detail
(Fig.
6B
to D). At the 5' end (nt 1 to 400) and the 3' end (nt 1100 to
1344) of the S segment coding sequence the Baltic strains form
a
cluster of their own. In contrast, the middle section of the
S segment
(nt 400 to 1100) placed the Baltic sequences into a
network which also
includes FIN and RUS sequences. This finding
provides further evidence
that the Baltic sequences are recombinants
of the ancestors of the FIN
and RUS lineages.
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FIG. 6.
(A) Splitstree based on the complete S-segment coding
sequence of all PUUV strains. (B to D) Splitstrees based on partial
sequences. (B) nt 1 to 400. (C) nt 401 to 1100. (D) nt 1101 to 1344. Two strains of each PUUV lineage are included together with two Baltic
PUUV strains.
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As for the networks seen in the BEL, NSCA, and DAN lineages, they are
not contradictory to earlier suggestions on recombination
within those
lineages as well (
3,
13).
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DISCUSSION |
Different phylogenetic methods show the same overall picture of
PUUV evolution.
The earlier inferred PUUV phylogenies (1, 3,
13, 19, 24, 43, 45, 46, 48, 49) were obtained using DM methods.
Since they are robust they are widely used, but they should be applied
with careful validation. One of our goals was to compare different
phylogenetic approaches in order to (i) confirm the earlier results and
(ii) study the differences between these approaches and their effect on
the results. Our analyses have shown that DM, MP, ML, and split
decomposition methods all give the same overall picture of a star-like
phylogeny of PUUV, assigning strains to their correct and
well-supported phylogeographic genetic lineages. On the other hand, the
connections between the lineages, with only one exception, are not
pronounced, indicating their early split. The most ancestral position
of the Japanese PUUV-like virus seems expected, since it originated
from a distinct host species and cannot be strictly referred to as
PUUV. Notably, unlike the bona fide PUUV, the Japanese variant appears
to be nonpathogenic for humans (31).
Further support for the distinct genetic lineages comes from the amino
acid signatures which can be assigned to each of them
as well as from
levels of intra- and interlineage variation. Sequence
analysis also
shows that the ratio of synonymous substitutions
to nonsynonymous
substitutions is extremely high, suggesting a
neutral mode of evolution
(
17). As no immunological pressure
seems to operate in the
rodent host (
37), which is the main
evolutionary scene of
PUUV, mechanisms like sampling, population
bottlenecks, and founder
effects (
63) have more likely contributed
to the formation
of the distinct PUUV lineages. These events could
be linked to the
well-known temporal population fluctuations of
the bank vole
(
42).
The different methods used for inferring phylogenies are based on
different evolutionary assumptions. Although they give the
same overall
picture of PUUV evolution, the bootstrap support
values observed in MP
and DM methods differ depending on whether
the calculations were based
on nucleotide or amino acid sequences.
This suggests that the chosen
models for correcting distances,
Kimura's two-parameter model of
nucleotide substitutions or the
Dayhoff's model for amino acid
substitutions, fail to adequately
describe the evolutionary processes
in PUUV. MP gives more consistent
support values than distance methods,
but still not all the values
reach the confidence limit of 70%
(
22). In general, MP performs
best when the number of
actual sequence changes is small, and
thus this method might be
affected by the fact that PUUV sequences
are highly
divergent.
ML is often considered the best method for inferring phylogenies due to
an explicit evolutionary model implemented in it (
59).
The
practical problem is that ML is very time-consuming, and with
a large
data set, the use of traditional ML programs is usually
not convenient.
We therefore performed ML analysis using the TreePuzzle
program, which
speeds up the calculations by considering only
four sequences at a
time. Our phylogenetic reconstruction of PUUV
evolution confirmed that
the ML approach gives the most consistent
results, with the support
values for distinct genetic lineages
on the trees based on nucleotide
and amino acid sequences being
essentially the
same.
Molecular clock and a slow evolution of hantaviruses.
A
molecular clock has not yet been reported for hantaviruses. Here, for
the first time, the clock assumption was tested using the ML ratio test
(14). It should be emphasized that the explicit evolutionary model implemented in the ML approach allows for more detailed analysis of PUUV S sequences that differ in many aspects from
an average sequence set. There exists an extremely high
dS/dN ratio,
and the transition/transversion ratio (3.5) is unusually high.
Furthermore, different sites seem to evolve at different rates and
there is a bias in the overall nucleotide composition of the viral
genome (e.g., the A content is 33% while the C content is only 19%).
The S-segment coding and noncoding regions are functioning under
different pressures that seem to be unequal for different parts of the
coding region as well. For instance, in the N protein there is a
hypervariable region (aa 233 to 275) carrying epitopes recognized by
both monoclonal antibodies and human patient sera (36,
61). Within this region, the rate of nonsynonymous substitutions is unusually high, suggesting that positive selection may favor amino
acid replacements (26). However, the ratio of
dN/dS does not exceed 1, which would be supportive of a positive selection (41). Instead, the nonsynonymous changes seem to occur at
random (26, 27), leading to the conclusion that this
region is more likely under low functional or structural constraints.
Taking these factors into account, it is not surprising that the
molecular clock test on PUUV S sequences is passed only when
the gamma
distribution of rate heterogeneity among sites is applied.
The
estimated substitution rate ranged from 0.7 × 10
7 to 2.2 × 10
6
nt per site per year. This slow rate of evolution is indirectly
supported by the low value (0.23) of the shape parameter alpha
of the
gamma distribution. Here it should be stressed that our
estimations of
the substitution rates are based on present knowledge
on evolution of
the rodent hosts. Paleontological records and
other molecular data
range widely, and there are also controversial
reports on the
phylogenies of rodents (
9,
10,
30,
51).
This leads to
different estimations of the PUUV evolutionary rate
ranging
approximately 20-fold. Notably, the rate of evolution
of the M segment
estimated for nine complete sequences known so
far (3.7 × 10
7 to 8.7 × 10
7
nt per site per year) is in the range determined for the S segment.
Thus, both genes of PUUV evolve at a similar rate, and the overall
conclusion that PUUV is evolving rather slowly seems to be well
justified.
The nucleotide substitution rate in hantavirus evolution presented in
this paper correlates well with the estimation based
on the separation
of Old World and New World
Microtus voles of
2.41 × 10
7 to 2.68 × 10
7
nt per site per year (
26). It is also comparable with the
rates
suggested for other stable RNA viruses like human T-cell
lymphotropic
virus type 2 in tribes infected in areas of endemicity
(1.71 ×
10
7 to 7.31 × 10
7) (
52) and hepatitis G virus
(9 × 10
6) (
58). These slowly
evolving viruses infect their primary hosts
(humans) persistently and
are well adapted to them. Thus, they
remain most of the time in
equilibrium close to an adaptive peak,
as the vast majority of new
mutations would probably decrease
the fitness of the virus
(
52). In contrast, the substitution
rates estimated for
more rapidly evolving RNA viruses such as
human immunodeficiency virus
(HIV) and hepatitis C virus are much
higher, being on the order
of 10
2 to 10
5 nt per
site per year (
2,
33).
Some key events in the PUUV history may now be dated, albeit with not
very high precision, based on our estimation of its
substitution rate.
Thus, the Japanese PUUV-like strains seemed
to diverge from the branch
leading to PUUV not later than 100,000
years ago (YA). This may be
related to the last Weichselian glaciation
of the northern hemisphere
starting about 115,000 YA. It looks
like hypothetical founder
populations of the distinct PUUV lineages
were established not later
than 85,000 YA, and the present lineages
were geographically separated
during the last deglaciation, which
started 21,000 to 17,000 YA. The
retreating glacial ice sheet
left behind several immigration routes for
flora and fauna to
colonize the revealed land, greatly affecting the
evolution of
these species (
21), including bank voles
carrying PUUV. This
is reflected in the closer connections of some PUUV
lineages and
the existence of clearly distinct
sublineages.
Recombination (genetic shift) in hantavirus evolution.
For
many RNA viruses, recombination has been shown to be an important
feature of their evolution (for review, see reference 64).
These include, e.g., the well-established case of HIV (39) as well as more recent reports on enteroviruses (53) and
dengue virus (65). The first evidence of recombination in
negative-stranded RNA viruses has been reported for TULV
(55). In our study several indications of recombination in
PUUV were seen. First, the molecular clock analysis indirectly
suggested that out data set includes recombinant sequences, since the
evolutionary clock was rejected when a large set of sequences was used
in the calculations. Recently, it has been shown that even a few
recombination events can lead to rejection of the clock
(54). Indeed, when the Baltic PUUV sequences of a
suspected recombination origin were excluded from the calculations, the
molecular clock was no longer rejected. Second, as some lineages are
represented by networks, they probably include recombinant sequences
(64). Third, similarity analysis of the Baltic strains
revealed that two regions of their S segment have higher-than-average
similarity to the FIN PUUV strains, while other regions are most
closely related to the RUS strains. This pattern may be interpreted as
evidence of recombination between these two lineages of PUUV. A similar
pattern has been seen earlier for the DAN PUUV strains
(3).
Another variety of genetic shift, reassortment, has been demonstrated
for some members of the genus
Bunyavirus (
50).
In
the genus
Hantavirus it has been shown for Sin Nombre
virus (
20)
and suggested for PUUV as well
(
45). Thus, both mechanisms seem
to play a role in the
evolution of PUUV in cooperation with the
classical genetic
drift.
Detailed knowledge of the rate and mode of genetic variation is
essential for understanding how hantaviruses induce disease
in humans
as well as for molecular epidemiology. The pattern of
PUUV evolution
revealed in this study (the low rate of evolution
obeying the molecular
clock and the early split of genetic lineages
and recombination) would
most probably be applicable to other
hantavirus types, such as Hantaan,
Dobrava, Sin Nombre, and the
like, thus advancing the research of these
severe human
pathogens.
| |
ACKNOWLEDGMENTS |
We thank Heikki Henttonen, Olli Vapalahti, and Vincent Moulton
for their helpful comments.
This study was supported by grants from the Academy of Finland, by EC
grant QLK2-CT-1999-01119, and by the Sigrid Jusélius Foundation,
Helsinki, Finland.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Haartman
Institute, Department of Virology, Haartmaninkatu 3, POB 21, FIN-00014
University of Helsinki, Finland. Phone: 358-9-1912 6490. Fax:
358-9-1912 6491. E-mail: Antti.Vaheri{at}helsinki.fi.
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Journal of Virology, December 2001, p. 11803-11810, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11803-11810.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
