Journal of General Virology (2014), 95, 2390–2401
DOI 10.1099/vir.0.069443-0
Evolutionary dynamics and genetic diversity from
three genes of Anguillid rhabdovirus
Laure Bellec,1,2 Joelle Cabon,1,2 Sven Bergmann,3 Claire de Boisséson,4
Marc Engelsma,5 Olga Haenen,5 Thierry Morin,1,2 Niels Jørgen Olesen,6
Heike Schuetze,3 Anna Toffan,7 Keith Way8 and Laurent Bigarré1,2
Correspondence
Laure Bellec
[email protected]
1
French Agency for Food, Environmental and Occupational Health & Safety,
Ploufragan–Plouzané Laboratory, Unit Viral Disease of Fish, Plouzané, France
2
European University of Brittany, France
3
Friedrich-Loeffler Institut, Insel Reims, Germany
4
French Agency for Food, Environmental and Occupational Health & Safety,
Ploufragan-Plouzané Laboratory, Unit Biosecurity and Viral Genetics, Ploufragan, France
5
Central Veterinary Institute of Wageningen, Lelystad, The Netherlands
6
National Veterinary Institute, Technical University of Denmark, Åarhus, Denmark
7
Research & Innovation Department, Division of Biomedical Science, Legnaro, Italy
8
Centre for Environment, Fisheries and Aquaculture Science, Weymouth, UK
Received 23 June 2014
Accepted 30 July 2014
Wild freshwater eel populations have dramatically declined in recent past decades in Europe and
America, partially through the impact of several factors including the wide spread of infectious
diseases. The anguillid rhabdoviruses eel virus European X (EVEX) and eel virus American (EVA)
potentially play a role in this decline, even if their real contribution is still unclear. In this study, we
investigate the evolutionary dynamics and genetic diversity of anguiillid rhabdoviruses by analysing
sequences from the glycoprotein, nucleoprotein and phosphoprotein (P) genes of 57 viral strains
collected from seven countries over 40 years using maximum-likelihood and Bayesian
approaches. Phylogenetic trees from the three genes are congruent and allow two monophyletic
groups, European and American, to be clearly distinguished. Results of nucleotide substitution
rates per site per year indicate that the P gene is expected to evolve most rapidly. The nucleotide
diversity observed is low (2–3 %) for the three genes, with a significantly higher variability within
the P gene, which encodes multiple proteins from a single genomic RNA sequence, particularly a
small C protein. This putative C protein is a potential molecular marker suitable for characterization
of distinct genotypes within anguillid rhabdoviruses. This study provides, to our knowledge, the
first molecular characterization of EVA, brings new insights to the evolutionary dynamics of two
genotypes of Anguillid rhabdovirus, and is a baseline for further investigations on the tracking of
its spread.
INTRODUCTION
Since the 1980s, wild populations of European (Anguilla
anguilla) and American (Anguilla rostrata) eels have shown a
strong decline throughout the world (Casselman, 2003;
Dekker, 2003; Haro et al., 2000; Richkus & Whalen, 2000;
Stone, 2003). The combination of several factors has been
investigated to explain this dramatic decline (Dekker, 2004;
Haenen et al., 2009, 2012); these factors include overfishing,
The GenBank/EMBL/DDBJ accession numbers for the sequences of
the glycoprotein, nucleoprotein and phosphoprotein genes (KJ600650–
KJ600771) of eel virus European X (EVEX) and eel virus American
(EVA) are shown in Table 4.
2390
habitat loss or degradation, migration barriers and infectious diseases (parasitic, bacterial and viral). Viral diseases
are caused by three main viruses isolated from either wild or
farmed eel: the aquabirnavirus eel virus European (EVE), the
alloherpesvirus anguillid herpes virus type 1 (AngHV1) and
the rhabdovirus eel virus European X (EVEX) (Haenen et al.,
2012). The Rhabdoviridae family is composed of over 200
viral pathogens of animals, plants, insects and fish, and is
divided into 11 genera by the International Committee
on Taxonomy of Viruses. Fish rhabdoviruses are presently
assigned to the genera Sprivivirus, Novirhabdovirus and
recently described Perhabdovirus (Hoffmann et al., 2005;
Stone et al., 2013).
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069443
Printed in Great Britain
Diversity and evolution of Anguillid rhabdovirus
In the 1970s, two eel rhabdoviruses were isolated in Japan.
The first one, designated eel virus American (EVA), was
imported from Cuba within a shipment of A. rostrata elvers,
while the second one originated from France through
European eel culture-ponds and was named EVEX (Sano,
1976; Sano et al., 1976, 1977). Their physico-chemical,
morphological, serological and infectivity properties are
strongly similar, suggesting they are two strains of a single
virus (Hill et al., 1980; Nishimura et al., 1981). Since then,
EVEX isolates have been detected in wild and farmed eel
from various geographical regions: France (Castric & Chastel,
1980; Castric et al., 1984), The Netherlands (van Beurden
et al., 2011; van Ginneken et al., 2005), Italy (van Ginneken
et al., 2004), UK, Denmark and Sweden (Jørgensen et al.,
1994), and Germany and Russia (Ahne et al., 1987;
Shchelkunov et al., 1989). In 40 years many isolations have
been made, but no molecular studies were carried out before
2012, when the first complete genome of an EVEX isolate
from The Netherlands (CVI153311) was sequenced (Galinier
et al., 2012). A few months later a second almost-complete
genome of the original EVEX isolate was published (Sano,
1976; Sano et al., 1976; Stone et al., 2013). Stone and coauthors have suggested that EVEX and EVA strains belong
to a new species, Anguillid rhabdovirus, within the genus
Perhabdovirus.
Perhabdovirus genomes are composed of a single molecule,
linear, negative sense ssRNA encoding five structural genes
in the order 39-nucleoprotein (N), phosphoprotein (P),
matrixprotein (M), glycoprotein (G) and RNA polymerase
(L)-59 (Galinier et al., 2012). These five structural genes are
well described for other rhabdoviruses but their functions
and evolutionary relationships are not fully understood at
this time, especially for eel viruses. The N protein is a major
component for RNA encapsidation and has the highest
conservation rate among all fish rhabdovirus proteins
(Galinier et al., 2012). The G protein is involved in receptor
recognition and allows the viral particle to attach and enter
the host cell. The P protein can be associated with L and N
proteins to form a complex involved in virus transcription
and replication. The P amino acid sequence is the least
conserved, showing a low identity (,20 %) with other
vesiculoviruses. Within the P gene an overlapping ORF
(+1) encodes a putative C protein. Most rhabdoviruses
possess this additional ORF, including vesicular stomatitis
virus (Kretzschmar et al., 1996; Spiropoulou & Nichol,
1993), Cocal virus (Pauszek et al., 2008), Chandipura virus
and Isfahan virus (Marriott, 2005), spring viraemia of carp
virus (Teng et al., 2007), Pike fry rhabdovirus (Chen et al.,
2009) and EVEX (Galinier et al., 2012). These C proteins
have a size varying from 41 to 93 amino acids and share a
very limited identity (,20 %) with putative C proteins
from other rhabdoviruses or other known proteins.
A common characteristic of RNA viruses is their rapid
evolution induced by the multiple errors made by the RNA
polymerase during the replication process. These multiple
errors generate rates per site per replication that are three
to four orders of magnitude greater than those of DNA
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viruses (Domingo & Holland, 1997; Drake et al., 1998).
Mutation, substitution and replication rates are essential to
understand viral evolution (Duffy et al., 2008; Kuzmin
et al., 2009). Within the Rhabdoviridae, mutations appear
to be the main source of genetic variation, although rare
cases suggesting the possibility of recombination have been
observed (Badrane & Tordo, 2001).
In this study, our aim is to describe the genetic diversity and
phylogenetic relationships of anguillid rhabdoviruses from
analysis of more than 50 viruses isolated from European and
American eels, collected from seven countries over a 40 year
period, comparing the complete N and P, and partial
G genes. The characterization of the viral evolution also
requires investigation of the frequencies, nature and localization of mutations as well as the selection mechanisms
constraining this viral population.
RESULTS
Analysis of virus genetic stability during passage
in cell culture
One virus isolate (FR-2013.1) was sequenced for two genes
(N and P) at multiple laboratory passages (2nd, 5th, 11th
and 13th). For neither gene were any mutations observed
in the viral sequences at the four different passages.
Phylogenetic analysis and evolutionary dynamics
of Anguillid rhabdovirus
Three genes of a significant sampling of viruses from two
eel species (A. anguilla and A. rostrata) in seven countries
over a 40 year period (1974–2013) were sequenced. For
the G gene, a partial dataset of 1164 bp was created using
33 viral strains. For the N and P genes, complete ORF
sequences were produced from 44 and 57 strains, respectively. Phylogenetic reconstructions produced the same
topologies for each gene using either maximum likelihood
(ML) or Bayesian inference (BI) (Fig. 1). All eel viral
sequences clustered within the species Anguillid rhabdovirus, far from other species within the genus Perhabdovirus, such as Perch rhabdovirus and Sea trout rhabdovirus
(STRV). Within the anguillid rhabdoviruses, two clades
were clearly distinguished and strongly supported (posterior probabilities of unity and ML bootstrap support of 100).
These two genogroups, A. anguilla viruses and A. rostrata
viruses, have around 10 % divergence (either nucleotides or
amino acids). Nevertheless, the A. rostrata genogroup comprises only two viral strains, from Cuba and Japan. Within
the A. anguilla virus clade some specific groups were present
within the topologies of the three genes. For example, the
DK-1986.2 strain is always in basal position within this
clade with strong support (1/100), the longest branch in
the three trees is the viral strain NL-2010.1, and a group of
seven viral strains from three different countries (IT-1989,
NL-2010.2 and FR-2003; 2006; 2008.1; 2008.2; 2012.3) is
always present and supported [1/70 (gene P), 89 (gene N),
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L. Bellec and others
NL-2011.1
DE-2003
FR-2011.1
1/87
FR-2011.7
FR-2011.9
0.88/FR-2002
FR-2004
1/74
DK-1986.1
DK-1990
0.89/61 NL-2006
NL-1990
0.81/NL-2010.1
NL-1992
FR-1987.1
0.97/63
FR-1985
FR-1978
1/99 NL-2010.2
0.76/74
FR-2012.3
FR-2003
1/90
FR-2008.1
FR-2008.2
1/83
FR-2006
IT-1989
1/94
IT-2001
NL-1994
0.56/NL-1997
1/100
IT-2000
DE-2004
FR-1976
IT-1997
DK-1986.2
1/100 JP-1998
CU-1974
Gene G
PRV
FR-2011.2
FR-2011.1
FR-2011.6
0.97/60 FR-2011.7
FR-2011.9
FR-2013.2
0.94/63 FR-2011.8
FR-2011.4
FR-2011.3
NL-2011.1
1/66
FR-2011.11
FR-2004
FR-2002
FR-2011.10
0.94/85
FR-2011.5
FR-2012.1
DE-2003
Gene N
0.95/85
0.9/100
NL-2010.3
NL-1990
NL-1992
1/97 FR-1987.1
FR-1985
0.7/70
DK-1999
DK-1986.1
FR-1978
0.98/65 FR-2008.1
0.94/62 FR-2008.2
FR-2006
1/63
NL-2006
NL-2010.2
1/89
FR-2003
FR-2012.3
IT-1989
DE-2004
IT-1997
IT-20001
IT-2001
1/66
NL-1994
NL-1997
FR-1976
DK-1986.2
1/100
JP-1998
CU-1974
Gene P
FR-2011.3
FR-2011.4
FR-2011.8
FR-2011.7
0.97/63
NL-2001.2
NL-2011.1
0.96/64
FR-2011.11
FR-2013.2
0.99/63
FR-2013.3
FR-2011.1
FR-2011.2
FR-2011.5
0.68/58
FR-2011.6
FR-2011.9
FR-2011.10
FR-2004
FR-2012.2
FR-2012.1
FR-2013.1
NL-2011.2
NL-1999.1
0.64/62
NL-1992
0.64/0.85/62
E
A
NL-2010.1
DE-2003
FR-2002
NL-1997
FR-1999
1/88
NL-2010.2
FR-2012.3
FR-2003
0.98/76
FR-2008.1
1/98
FR-2008.2
1/70
FR-2006
IT-1989
1/82
NL-2010.3
NL-1999.2
1/85
NL-2006
1/70
NL-2009
NL-2001.1
NL-1990
NL-2010.1
E
FR-1987.2
FR-1987.3
FR-1987.4
FR-1987.1
FR-1985
0.76/62
0.83/-1/70
A
PRV
0.96/-
1/73
1/100
1/100
E
DE-2004
IT-1997
DK-1986.1
DK-1990
FR-1978
FR-1976
NL-1994
0.84/- IT-2000
IT-2001
DK-1986.2
JP-1998
CU-1974
A
PRV
Fig. 1. Phylogenetic tree of Anguillid rhabdovirus strains computed from the partial G gene (33 sequences), the complete N
gene (44 sequences) and the complete P gene (57 sequences) by BI and ML. For simplicity, only the BI trees are shown; the
ML trees have the same topologies. The numbers are posterior probabilities (BI) and bootstrap proportions (ML) reflecting clade
support (values below 50 are indicated by dashes). The two genogroups E (European type) and A (American type) are
indicated. Sequence names include countries of origin (international code) and dates of isolation. Perch rhabdovirus (PRV/
JX679246) was used as the outgroup. For clarity, the branches of the outgroup are cut.
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Journal of General Virology 95
Diversity and evolution of Anguillid rhabdovirus
83 (gene G)]. Based on these phylogenetic relationships, we
propose to define two genogroups of eel viruses: anguillid
rhabdovirus type European (E) and anguillid rhabdovirus
type American (A). These names and initial letters were
chosen to take into account the hosts (the European and
American eel) and the historical acronyms of the virus (EVEX
and EVA).
The nucleotide substitution rates (per site and per year)
and the time for the most recent common ancestor
(TMRCA) were estimated for each gene (Table 1). For a
more accurate analysis, three separate datasets were
defined: strains isolated from Europe (named European);
a group of 33 viral strains amplified for the three genes
(named ‘common’); and a set containing all sequences
available (named ‘all’). For all analyses, the hypothesis of a
strict molecular clock was rejected and a relaxed molecular
clock was used because each coefficient of variation (CoV)
value (and 95 % highest posterior density, HPD) was .0.
For each gene, an increase in the rate of nucleotide
substitution was observed for the European group. For the
three datasets, significant differences (P,0.0001) between
the three genes were found: substitution rates in the P gene
were higher than for the G gene, and both were higher than
for the N gene. Based on these rates, the mean TMRCA
calculated for the three genes is around the second half of
the nineteenth century (1847–1883). For the European
viruses only, the mean TMRCA is around the middle of the
twentieth century (1942–1972).
Maximum clade credibility (MCC) trees and the demographic history (using the Bayesian skyline plot) of
different anguillid rhabdovirus datasets were also constructed (data not shown). We illustrate only the results
from the 31 combined sequences of the three genes from
the European genogroup (Fig. 2). The MCC tree shows the
same topology as those obtained by phylogenetic analyses
with the European viruses. The results suggest that the
population history increased until the mid-1980s, followed
by a decline. The period of increase corresponds to the rise
in diversity also detected within the MCC tree.
Genetic diversity
Viral sequences displayed a low genetic diversity for each
gene, with mean nucleotide identity ranging from 97.41 to
98.36 %, with a similar trend for amino acids (97.58 to
99.22 %) (Table 2). The number of variable nucleotide
mutations was highest for the P gene (24.12 %) and lowest
for the N gene (14.33 %). Only 11.57 % of the mutations of
the N gene were non-synonymous compared with 32.46 %
for the P gene. The ratio dN/dS (non-synonymous to synonymous mutations) and Tajima’s D (test based on polymorphism frequencies) were calculated for each gene. All
three genes displayed significantly more synonymous than
non-synonymous changes (ratio ,1), suggesting a purifying selection on the viral population. Tajima tests were
significantly negative for each gene, assuming a negative
selection on the viral population. Significant differences
were observed among the three genes for the 31 common
sequences from the European virus type (P,0.0001) (Fig.
3). The N gene displayed the lowest value (dN/dS50.07),
followed by the G gene (dN/dS50.13), and the P gene
showed the highest (dN/dS50.22).
P and C gene variation
Among all viruses characterized above, a complete ORF C
of 195 bp (65 amino acids) located at the 59-end of the P
gene, precisely between P protein amino acids 137 and 203,
was identified for 47 viral strains. Ten partial sequences of
the C gene, composed of two American viruses (CU-1974
and JP-1998) and eight European viruses (DK-1986.2; IT2001; NL-2009; NL-2010.1; FR-1987.1–4), were deleted
from our dataset because of a stop codon appearing too
early or a start codon not being present. Sequence variations from the 47 remaining viral strains were analysed for
the P-total (full-length of the gene P), the C (full-length),
and a P–C region defined as the position within P determined by the same nucleotides encoding the C gene (Table
3). The amino acid identity of the C protein was lower than
both P–C region and P-total identities. Distributions of the
Table 1. Estimates of evolutionary rates (nucleotide substitutions per site per year), TMRCA (year) and CoV inferred from N, P and G
genes using the relaxed molecular clock model
Gene
N
P
G
Dataset
n
Date range
All
Common
European
All
Common
European
All–Common
European
44
33
42
57
33
55
33
31
1974–2013
1974–2012
1976–2012
1974–2013
1974–2012
1976–2012
1974–2012
1976–2012
Mean rate, ¾10”4 site”1 year”1
(95 % HPD)
3.37
3.26
4.14
5.71
5.05
7
4.23
4.75
(1.86–4.99)
(1.6–4.94)
(2.37–5.75)
(2.88–8.67)
(2.27–7.92)
(4.61–9.65)
(2.81–5.77)
(3.43–6.15)
TMRCA, years
(95 % HPD)
1882
1880
1957
1883
1859
1972
1847
1942
(1790–1955)
(1879–1957)
(1928–1977)
(1749–1969)
(1697–1969)
(1954–1977)
(1749–1933)
(1908–1977)
CoV (95 % HPD)
0.71
0.75
0.68
1.42
1.2
1.6
0.55
0.53
(0.38–1.09)
(0.38–1.17)
(0.31–1.1)
(0.79–2.16)
(0.55–2.01)
(0.9–2.41)
(0.25–0.88)
(0.1–1.23)
Three separate datasets were defined: All (all sequences available); Common (strains amplified for the three genes); European (strains isolated from
Europe). n, Number of sequences.
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variable nucleotides were almost the same for the three
regions, but there was an important increase (more than
20 %) of non-synonymous substitutions within both P–C
and C regions. The slight difference (3.5 %) between the P–
C and C regions was established by the distribution of the
third codon positions within their sequences. Anguillid
rhabdovirus C proteins have a very limited identity (,20 %)
with other C proteins observed within Rhabdoviridae (data
not shown).
Some variations were observed within full-length sequences
of the P gene. The 39-start and 59-end of the gene seemed
to correspond to highly conserved domains with almost no
substitutions. Other regions such as the P–C region possess
more mutations (data not shown). Moreover, two European
viruses (FR-2012.3 and NL-2010.2) with 100 % similarity
had a deletion of four amino acids between positions 173
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98.36 (93–100)
97.98 (89.6–100)
97.41 (88.9–100)
1284 (428)
825 (275)D
1164 (388)
Fig. 2. MCC tree and Bayesian skyline plot inferred from the
European genogroup with the 31 combined sequences of the
three genes. The y-axis of the Bayesian plot represents the relative
genetic diversity (Net), which is the product of the effective
population size and the generation length in years. The thick solid
line is the median estimate, and the grey area shows the 95 %
HDP limits.
44
57
33
2014
Gene N
Gene P
Gene G
% AA ID,
(min–max)
2004
% Nt ID,
(min–max)
1994
Year
N nucleotides
(amino acids)
1984
Table 2. Molecular data for N, P and G sequences from Anguillid rhabdovirus
1974
99.22 (97.6–100)
97.58 (90.9–100)
97.76 (91.7–100)
10
1
2394
14.33
24.12
18.47
% Variable
nucleotides
100
N sequences
Relative genetic diversity
1000
N, Number; Nt ID, mean nucleotide identity; AA ID, mean amino acid identity; dN, number of non-synonymous mutations; dS, number of synonymous mutations.
*Ratio dN/dS and Tajima’s D differ at the P,0.0001 level by the Kruskal–Wallis test.
DTwo sequences (FR-2012.3 and NL-2010.2) had a deletion of four amino acids.
22.05*
22.39*
21.93*
0.06*
0.19*
0.13*
11.57
32.46
27.48
% Non-synonymous
mutations
dN/dS
Tajima’s D
L. Bellec and others
Journal of General Virology 95
Diversity and evolution of Anguillid rhabdovirus
P (0.22)
0.035
0.025
G (0.13)
dN
0.020
0.015
N (0.07)
0.010
0.005
0
0
0.05
0.10
0.15
0.20
0.25
dS
Fig. 3. Numbers of non-synonymous (dN) versus synonymous (dS)
substitutions per nucleotide site from the 31 common European
viruses for the three genes. Triangles, gene P; squares, gene N;
circles, gene G. Linear trends were added with the name of the
gene (N, small-dashed line; G, large-dashed line; P, solid line) with
the mean ratio dN/dS in parentheses.
and 176, whereas all other European viruses include the
motif QDPK, and both American viruses display the motif
QDKR.
DISCUSSION
The present study on genetic diversity and evolutionary
dynamics in 57 collected viral strains of A. anguilla and A.
rostrata reveals three main results: (1) these eel viruses
belong to the Rhabdoviridae and more precisely to the
species Anguillid rhabdovirus, where two genotypes corresponding to European type and American type viruses can
be delimited; (2) the global genetic diversity is low (less
than 3 %); (3) a putative protein C is present within the
majority of the European viruses.
Classically, first molecular studies and genogroup definition are based on partial sequences of genes such as the polymerase or the glycoprotein. For example, viral hemorrhagic
septicemia virus (VHSV) strain characterization was initially
carried out on two short regions of the G gene (Benmansour
et al., 1997) and has led to the identification of three
genogroups. A fourth was defined using the N gene (Snow
et al., 1999) and was confirmed by a study on the entire G
gene (Einer-Jensen et al., 2004). To avoid this common issue
in viral studies, an alternative solution consists of conducting
analysis of multiple genes at the same time. For VHSV, a
phylogenetic analysis using N, G and Nv genes has been
performed and led to a good resolution of the four
genogroups previously described (Einer-Jensen et al., 2005).
Here we applied this approach to make analyses on three
genes representing 35 % of the complete genome of EVEX
(Galinier et al., 2012).
Relatively simple rules were used to delimit clades within
our phylogenies and defined them as: (1) groups
distributed similarly in all single-gene phylogenetic reconstructions, (2) branches strongly supported within all
topologies (ML bootstrap support .70 and posterior
probabilities .0.9). On this basis, two virus clades were
defined according to their specific host species, American
type for A. rostrata and European type for A. anguilla, but
only two of the 57 isolates were from one of the hosts, A.
rostrata. In our study, only two host species were examined
of the 18 species/subspecies described in the genus
Anguilla. The limited numbers of hosts used may have
artificially led to these host-specific clades. Monophyletic
groups from several fish species had already been observed
(Stone et al., 2003) and only further improvement of our
phylogenetic trees with viral strains from other eel species
would allow confirmation of this host-specific repartition.
In this work, European viruses were isolated from five
countries across a 40 year period, and their evolutionary
relationships were not correlated with their geographical
isolation, date or host life stage. This is unusual for fish
rhabdvoviruses, which are more frequently defined according to their major geographical area of isolation. For
example, phylogenetic analyses of infectious haematopoietic necrosis virus (IHNV) revealed three major genogroups,
designated U, M and L for upper, middle and lower
geographical distribution in North America (Kurath et al.,
2003). A. anguilla is the only eel species present in Europe
and is exploited at all freshwater stages. Indeed, elvers landed
on the European coast lead to important commercial
Table 3. Anguillid rhabdovirus P and C protein variations among 47 isolates
Taxa
P-total
P–C region
C
% Nt ID
(min–max)
% AA ID
(min–max)
% Variable
nucleotides
% Non-synonymous
mutations
98.81 (95.3–100)
97.97 (89.2–100)
97.97 (89.2–100)
98.21 (93.4–100)
96.31 (84.6–100)
95.9 (84.6–100)
12.4
14.9
14.9
43.13
62
65.51
Codon position
1
2
3
9
9
9
11
11
9
Nt ID, Mean nucleotide identity; AA ID, mean amino acid identity.
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L. Bellec and others
Table 4. Virus isolates used in this study
Virus isolate
DK-1986.1
DK-1986.2
DK-1990
FR-1976
FR-1978
FR-1985
FR-1987.1
FR-1987.2
FR-1987.3
FR-1987.4
FR-1999
FR-2002
FR-2003
FR-2004
FR-2006
FR-2008.1
FR-2008.2
FR-2011.1
FR-2011.2
FR-2011.3
FR-2011.4
FR-2011.5
FR-2011.6
FR-2011.7
FR-2011.8
FR-2011.9
FR-2011.10
FR-2011.11
FR-2012.1
FR-2012.2
FR-2012.3
FR-2013.1
FR-2013.2
FR-2013.3
DE-2003
DE-2004
IT-1989
IT-1997
IT-2000
IT-2001
NL-1990
NL-1992
NL-1994
NL-1997
NL-1999.1
NL-1999.2
NL-2001.1
NL-2001.2
NL-2006
NL-2009
NL-2010.1
NL-2010.2
NL-2010.3
NL-2011.1
2396
Lab. name
DK-3545
DK-3631
DK-5743
EVEX
C30
J14
L40
L42
L61
L72
X23
M5366
BB13
P2086
06/03565
GG129
GG184
JJ16
JJ17
JJ20
JJ23
JJ25
JJ26
JJ27
JJ28
JJ29
JJ30
JJ33
KK16
KK107
KK110
LL13
331.2
332.3
DF03/03
DF25/04
690/89
25-1/97
393/I00
260/I01
CVI108778
CVI153311
CVI313006
CVI459083
CVI524012-1
CVI531138
CVI574597
CVI574856
CVI6010366-244135
CVI9009703
CVI10008555
CVI10012078
CVI10015091
CVI11003032
Date of isolation
24.1.1986
4.6.1986
14.2.1990
24.3.1976
4.1978
7.3.1985
24.4.1987
29.4.1987
29.4.1987
29.4.1987
24.2.1999
10.4.2002
8.1.2003
12.2.2004
8.12.2006
11.2008
15.12.2008
25.3.2011
25.3.2011
25.3.2011
25.3.2011
25.3.2011
25.3.2011
3.25.2011
25.3.2011
25.3.2011
25.3.2011
25.3.2011
27.1.2012
22.3.2012
28.3.2012
28.1.2013
2.2013
2.2013
2003
2004
1989
20.1.1997
29.9.2000
14.6.2001
31.5.1990
20.10.1992
8.12.1994
2.9.1997
8.3.1999
31.5.1999
11.1.2001
16.1.2001
27.3.2006
3.6.2009
21.5.2010
20.7.2010
13.9.2010
23.2.2011
No. in vitro
passages
ND
ND
ND
ND
ND
2
6
6
5
6
1
1
2
1
1
ND
1
2
2
2
2
2
2
2
2
2
2
2
1
1
1
2
2
2
6
4
ND
ND
ND
ND
4
3
2
4
GenBank accession number
Gene N
Gene P
Gene G
KJ600732
KJ600733
KJ600734
JX827265*
JN639009*
KJ600735
KJ600736
KJ600650
KJ600651
KJ600652
JX827265*
JN639009*
KJ600653
KJ600654
KJ600655
KJ600656
KJ600657
KJ600658
KJ600659
KJ600660
KJ600661
KJ600662
JN639010*
KJ600663
KJ600664
KJ600665
KJ600666
KJ600667
KJ600668
KJ600669
KJ600670
KJ600671
KJ600672
KJ600673
KJ600674
KJ600675
KJ600676
KJ600677
KJ600678
KJ600679
KJ600680
KJ600681
KJ600682
KJ600683
KJ600684
KJ600685
KJ600686
KJ600687
FN557213*
KJ600688
KJ600689
KJ600690
KJ600691
KJ600692
KJ600693
KJ600694
KJ600695
KJ600696
KJ600697
KJ600698
KJ600699
KJ600703
KJ600704
KJ600705
JX827265*
JN639009*
KJ600706
KJ600707
KJ600737
KJ600738
KJ600739
KJ600740
JN639010*
KJ600741
KJ600742
KJ600743
KJ600744
KJ600745
KJ600746
KJ600747
KJ600748
KJ600749
KJ600750
KJ600751
KJ600752
KJ600753
KJ600754
KJ600755
KJ600756
KJ600757
KJ600758
KJ600759
KJ600760
KJ600761
KJ600762
FN557213*
KJ600763
KJ600764
ND
ND
2
ND
1
2
2
1
2
1
KJ600765
KJ600766
KJ600767
KJ600768
KJ600769
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KJ600708
KJ600709
KJ600710
KJ600711
JN639010*
KJ600712
KJ600713
KJ600714
KJ600715
KJ600716
KJ600717
KJ600718
KJ600719
KJ600720
KJ600721
KJ600722
KJ600723
FN557213*
KJ600724
KJ600725
KJ600726
KJ600727
KJ600728
KJ600729
Journal of General Virology 95
Diversity and evolution of Anguillid rhabdovirus
Table 4. cont.
Virus isolate
NL-2011.2
CU-1974
JP-1998
Lab. name
CVI11007014
EVA-J6BF4D
EVA Y.HD
Date of isolation
No. in vitro
passages
19.4.2011
8.1974
1.12.1998
2
5
6
GenBank accession number
Gene N
Gene P
Gene G
KJ600770
KJ600771
KJ600700
KJ600701
KJ600702
KJ600730
KJ600731
Prefixes: FR, French isolates; DK, Danish isolates; DE, German isolates; IT, Italian isolates; NL, Dutch isolates; CU, Cuban isolate; JP, Japanese
isolate. ND, Not determined.
*Sequences not amplified in this study.
DA. rostrata host species; all other isolates were from A. anguilla.
transactions across all of Europe. In fact, high yield
farming in The Netherlands, Denmark and Italy relies on
the importation of elvers from France, Portugal and the
UK. Viral strains are consequently spreading in Europe
through this intense circulation of eels, which can partially
explain this lack of geographical distribution for European
viruses.
inconsistency between molecular dating of the TMRCA and
epidemiological, historical evidence. Without correction of
the molecular dating the purifying pressure seems to lead to
underestimating the age of the viral lineage (Wertheim &
Kosakovsky Pond, 2011). For Anguillid rhabdovirus, with
no epidemiological or historical data available we can not
exclude the same issue for TMRCA estimation date.
Rates of nucleotide substitution are one of the major
components to help in understanding viral evolution. The
method of estimating nucleotide substitution rates 2based
on a Bayesian Markov chain Monte Carlo (MCMC) coalescent framework – was used with anguillid rhabdoviruses,
which allows time-structured phylogeny (i.e. evolution is
over the timescale of human observation and tip times of
each sequence correspond to the date of viral sampling).
RNA viruses are often assumed to evolve quickly with a rate
close to 161023 nucleotide substitutions per site per year
and a range between 1022 and 1025 substitutions per site per
year (Duffy et al., 2008; Jenkins et al., 2002). The results of
our analyses based on three genes are within the range for
RNA viruses, but close to the low boundaries with rates
around 1024. These results are consistent with other fish
viruses, such as infectious salmon anaemia virus (ISAV),
for which the substitution rate is estimated at 7.8361024
substitutions per site per year (Castro-Nallar et al., 2011), or
a betanodavirus with 4.8961024 (Panzarin et al., 2012). We
found significant differences between the three genes for the
common group of viruses, with nucleotide substitution rates
from 4.7561024 (G gene) to 5.0561024 (P gene). Previous
studies were carried out using multiple genes for different
viruses, such as spring viraemia of carp virus (SVCV; rates of
5.4761024 and 4.7161024 for the G and P genes,
respectively; Padhi & Verghese, 2012) and VHSV (rates of
5.9161024 for the G gene and 5.1161024 for the P gene;
He et al., 2014). Thus in both SVCV and VHSV, the P gene
evolves more slowly than the G gene, contrary to Anguillid
rhabdovirus. One hypothesis from this interesting finding is
that purifying selection may be present in the G gene
sequences. This negative pressure seems to be a dominant
evolutionary force of these RNA viruses but could also be
a source of TMRCA bias. Indeed, some rapidly evolving
pathogens such as Ebola virus and avian influenza virus show
Nucleotide substitution rates inferred here were significantly higher for the European group. Such differences
between genogroups have already been observed for other
fish rhabdoviruses. The evolutionary rate of the European
freshwater SVCV strains genogroup Ia is at least 5–7 times
higher than genogroup Id (Padhi & Verghese, 2012), and
the freshwater VHSV strains evolve 2.5 times faster than
marine strains (Einer-Jensen et al., 2004). Nucleotide
substitutions are generated during the viral replication
cycle, which can be modified by aquaculture practices. For
example, high fish densities and water temperature increase
the stress level in fish. In addition, water temperature plays
a crucial role in the virulence of anguillid rhabdoviruses.
Experimental trials have suggested that mortality is lowest
at 10 uC and highest at 20 uC (van Beurden et al., 2012).
Thus, an increase of stress level (due to farming practices)
or a change in virulence may be linked to an increase in
replication rate and could explain the high rate of
nucleotide substitution observed for European viruses.
http://vir.sgmjournals.org
The global nucleotide diversity observed in Anguillid
rhabdovirus is low (2–3 %) for the three genes studied.
These results are supported by the comparison of other
complete genomes available to date (Stone et al., 2013).
Within the new genus Perhabdovirus, isolates STRV and
lake trout rhabdovirus (LTRV) shared a high degree of
similarity (over 97 %) and therefore are considered to
belong to the same species, STRV (Johansson et al., 2002;
Stone et al., 2013). However, the third species from this
genus, Perch rhabdovirus, displayed opposite results with a
divergence of 32.9 % between the four genogroups (Talbi
et al., 2011). The low diversity demonstrated in this study
could be related to a purifying selection process, suggesting
complex relationships within this host–virus system. A.
anguilla and A. rostrata are recognized as panmictic (or
quasi-panmictic) populations with a relative genetic
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2397
L. Bellec and others
homogeneity and a constant level of diversity (Dannewitz
et al., 2005; Wirth & Bernatchez, 2003). Molecular
phylogenetic analyses of the genus Anguilla clearly support
the notion that the European and American eels form an
Atlantic group with a speciation time estimated to 5.8
million years (Minegishi et al., 2005). On the other hand,
viruses in this system are specific (they were only found in
eel) and are adapted to their hosts. The population history of
the European genogroup of viruses and that of the European
A. anguilla host populations show the same decline since
the mid-1980s. The evolution of viruses seems constrained
by adaptation mechanisms, suggesting congruence of the
evolutionary relationship of this eel–Anguillid rhabdovirus
system.
To date, the function of the P gene is not still completely
understood within the Rhabdoviridae but different domains
have been characterized. The vesicular stomatitis virus P
protein is structured in an N-terminal, a central and a Cterminal domains (Leyrat et al., 2012), and there is evidence
of the presence of two conserved but also two variable
domains within the P gene of Lyssavirus (Nadin-Davis et al.,
2002). For the Anguillid rhabdovirus, polymorphism analyses suggest that regions located at the 39- and 59-ends
(approx. 150 nt) are conserved while the central region is
more variable. Within this variable domain, a specific region
(P–C, 195 nt) seems to be subjected to important changes,
with more than 60 % non-synonymous substitutions. For
example, within this P–C region a deletion of 12 nt for two
European strains and two amino acid changes for the
American group were observed. Further work on in vitro
adaptation needs to be carried out to evaluate the genetic
stability of these positions.
Many rhabdoviruses encode an additional ORF, such as an
NV protein in novirhabdoviruses or a small highly basic
protein C in most vesiculoviruses (Walker et al., 2011).
Among our dataset of 57 P sequences, we discovered and
analysed 47 full-length C proteins. Sequence alignments
revealed that this ORF was shorter in the American
genogroup; this observation is similar in VHSV where the
genotype II presents a shorter NV protein (Einer-Jensen
et al., 2005). The function of this putative C protein is still
unclear (Kretzschmar et al., 1996) but it may play a role in
RNA synthesis (Peluso et al., 1996).
In conclusion, data and analyses provided in this study
support the idea that Anguillid rhabdovirus is host-specific
and has European and American genogroups despite its
low genetic diversity.
available in GenBank were added to the sequences produced in this
study.
RNA extraction, primers and sequencing. RNA was extracted
from 150 ml cell culture supernatant using the Nucleospin RNA virus
kit (Macherey-Nagel). The nucleoprotein (gene N), the phosphoprotein (gene P) and the glycoprotein (gene G) were respectively
amplified with the primer sets oPVP301 (59-GGCTATTCTTTAACAGACATC-39) and oPVP302 (59-AAATGACTCATTTCTGCTTC39), oPVP303 (59-CTTTAACAGGGATAAACGTAG-39) and oPVP304
(59-AATAGTCAAGAGGTTCAGAC-39), oPVP284 (59-TTGAGACATTTGTCACTGTG-39) and oPVP285 (59-ACCTGAAGTATCACTTGTAC-39) for A. anguilla viruses. For gene G, only a partial sequence
(1164 bp) was amplified for A. rostrata viruses with the group-specific
primers oPVP299 (59-ACTGTATCATCTCACGAGGT-39) and oPVP286
(59-TACACAGAAATGAGAGTTCC-39). One-step reverse transcription
(RT)-PCRs were performed with the SuperScript III One-Step RT-PCR
System with Platinum Taq High Fidelity (Invitrogen) using the
following mix: around 1 mg of extracted RNA was added to 20 mM each
primer, 1 ml Taq and 25 ml of reaction mix in a final volume of 50 ml.
The RT-PCR was conducted in a Mastercycler (Eppendorf) with an
initial step of 55 uC (30 min) followed by an initial denaturation step
at 94 uC for 2 min then 40 cycles at 94 uC (15 s), 51 uC (30 s), 68 uC
(60 s) for both genes N and P. For gene G, 40 cycles were used:
94 uC (15 s), 56 uC (30 s), 68 uC (90 s), and a final extension at 68 uC
(5 min). All PCR products were purified with a NucleoSpin gel and
PCR Clean-up kit (Macherey-Nagel) and cloned using the TOPO TA
Cloning kit (Invitrogen). For each PCR product, three clones were
selected and sequenced in both directions using a BigDye terminator
v3.1 reaction kit (Applied Biosystems) and a 3130 Genetic Analyzer
(Applied Biosystems). All nucleotide differences were checked visually
using the chromatograms.
Phylogenetic reconstructions. All consensus sequences were
assembled using VectorNTI software v11.5. Alignments were performed with Muscle using SeaView 4 (Gouy et al., 2010). Gaps were
removed using Gblocks (Castresana, 2000) carried out on the
phylogeny.fr platform (Dereeper et al., 2008). Phylogenetic reconstructions were performed using both BI and ML with the Phylemon2, webtools suite (Sánchez et al., 2011). Bayesian analyses used MrBayes 3.1.2
(Ronquist & Huelsenbeck, 2003) with four chains of 106 or 26106
generations, trees sampled every 100 generations, and a burn-in value
set to 20 % of the sampled trees. Sequences were considered with an
evolutionary model designed for coding sequences and taking the
genetic code into account (Goldman & Yang, 1994; Muse & Gaut, 1994;
Shapiro et al., 2006). ML analyses were performed using PhyML
(Guindon & Gascuel, 2003; Guindon et al., 2005) with an evolutionary
model selected via the Akaike information criterion with jModelTest 2
(Darriba et al., 2012), and validated with 1000 bootstrap replicates. For
the complete ORF N (44 sequences of 1284 bp), a general time
reversible model with a gamma distribution (C50.95) and a
proportion of invariable sites (I50.54) was chosen. For the complete
ORF P (57 sequences of 825 bp), a transitional model plus a gamma
distribution (C50.86) was selected. For the partial sequence dataset
of 33 ORF G (1164 bp), a three-phase model with unequal base
frequencies (1 uf) with a gamma distribution (C50.24) was preferred.
Evolutionary parameters and demographic history.
METHODS
Viral isolates. A total of 53 anguillid rhabdoviruses were collected as
cell culture supernatants from different laboratory collections: Cuba
(n51), Denmark (n53), France (n528), Germany (n52), Italy
(n54), The Netherlands (n514) and Japan (n51). Isolation dates,
host (A. anguilla or A. rostrata), country and number of laboratory
passages are reported in Table 4. Sequences from four isolates already
2398
BEAST
software package v1.7.5 was used to estimate the rates of nucleotide
substitution and the TMRCA with a Bayesian MCMC (Drummond &
Rambaut, 2007). All datasets were analysed with the Yang 96 codon
model (Shapiro et al., 2006), and both a strict (constant) and a
relaxed uncorrelated lognormal (vary along branches) molecular
clock (Drummond et al., 2006). These models were evaluated with the
CoV (Drummond et al., 2006), where CoV values .0 were considered
as evidence of non-clock evolutionary behaviour. The Bayesian skyline
coalescent tree prior was utilized to infer the complex population
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Journal of General Virology 95
Diversity and evolution of Anguillid rhabdovirus
dynamics of Anguillid rhabdovirus (Drummond et al., 2005). For each
dataset, three independent Bayesian MCMC runs were carried out for
20–35 million generations (to obtain effective sample size values of at
least 200 for each parameter), to retain a sample of 10 000 trees.
Convergences of the runs were confirmed using Tracer v1.5 software
(http://tree.bio.ed.ac.uk/software/tracer/). The results of the three
independent runs were then combined using LogCombiner v1.7.5
(with a burn-in value of 10 %), and MCC trees were generated using
TreeAnnotator v1.5 and visualized using FigTree v1.4 (http://tree.bio.
ed.ac.uk/software/figtree/). After that the normality and homogeneity
of variances were tested for all substitution rates. We used a nonparametric test (Kruskal–Wallis) with the XLStat program (Addinsoft)
to give inferred differences in substitutions rates between the three
genes.
Chen, H. L., Liu, H., Liu, Z. X., He, J. Q., Gao, L. Y., Shi, X. J. & Jiang,
Y. L. (2009). Characterization of the complete genome sequence of
pike fry rhabdovirus. Arch Virol 154, 1489–1494.
Dannewitz, J., Maes, G. E., Johansson, L., Wickström, H., Volckaert,
F. A. & Järvi, T. (2005). Panmixia in the European eel: a matter of
time. Proc Biol Sci 272, 1129–1137.
Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. (2012).
jModelTest 2: more models, new heuristics and parallel computing.
Nat Methods 9, 772.
Dekker, W. (2003). Status of the European eel stock and fisheries. In
Eel Biology, part 4, pp. 237–254. Edited by K. Aida, K. Tsukamoto &
K. Yamauchi. Tokyo: Springer.
Dekker, W. (2004). Slipping Through our Hands. Population Dynamics
of the European Eel. Amsterdam: Universiteit van Amsterdam.
Genetic diversity. Basic population statistics and single nucleotide
polymorphisms were calculated using the program DnaSp v5
(Librado & Rozas, 2009; Rozas et al., 2003). We examined possible
selection pressures with the Tajima’s D test (Tajima, 1989). For the
three genes, the relationships between non-synonymous (dN) and
synonymous (dS) substitutions per nucleotide site were investigated
using the SNAP program (http://www.hiv.lanl.gov) (Korber, 2000).
Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet,
F., Dufayard, J.-F., Guindon, S., Lefort, V. & other authors (2008).
Phylogeny.fr: Robust phylogenetic analysis for the non-specialist.
Nucleic Acids Res 36 (Web Server issue ), W465–W469.
Domingo, E. & Holland, J. J. (1997). RNA virus mutations and fitness
for survival. Annu Rev Microbiol 51, 151–178.
Drake, J. W., Charlesworth, B., Charlesworth, D. & Crow, J. F. (1998).
Rates of spontaneous mutation. Genetics 148, 1667–1686.
ACKNOWLEDGEMENTS
Drummond, A. J. & Rambaut, A. (2007). BEAST: Bayesian evolu-
We thank Célie Dupuy for stimulating discussions and invaluable
advice. We also thank Yves Desdevises for insightful comments. We
thank the Laboratory of Fish Pathology at the Tokyo University of
Fisheries for the isolation of the EVA strain. Y. H. Michael Cieslak is
acknowledged. This work was supported by an EMIDA ERA-NET
project ‘MOLTRAQ’.
tionary analysis by sampling trees. BMC Evol Biol 7, 214.
Drummond, A. J., Rambaut, A., Shapiro, B. & Pybus, O. G. (2005).
Bayesian coalescent inference of past population dynamics from
molecular sequences. Mol Biol Evol 22, 1185–1192.
Drummond, A. J., Ho, S. Y., Phillips, M. J. & Rambaut, A. (2006).
Relaxed phylogenetics and dating with confidence. PLoS Biol 4, e88.
Duffy, S., Shackelton, L. A. & Holmes, E. C. (2008). Rates of
evolutionary change in viruses: patterns and determinants. Nat Rev
Genet 9, 267–276.
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