Download Full genomic analysis of new variant rabbit hemorrhagic disease

Journal of General Virology (2015), 96, 1309–1319
DOI 10.1099/vir.0.000070
Full genomic analysis of new variant rabbit
hemorrhagic disease virus revealed multiple
recombination events
Ana M. Lopes,1,2,3 Kevin P. Dalton,4 Maria J. Magalhães,1
Francisco Parra,4 Pedro J. Esteves,1,2,5 Edward C. Holmes6
and Joana Abrantes1
Correspondence
Joana Abrantes
[email protected]
1
CIBIO, InBIO - Research Network in Biodiversity and Evolutionary Biology, Universidade do Porto,
Campus de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal
2
Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Porto, Portugal
3
INSERM, UMR892, Université de Nantes, Nantes, France
4
Instituto Universitario de Biotecnologı́a de Asturias, Departamento de Bioquı́mica y Biologı́a
Molecular, Universidad de Oviedo, Oviedo, Spain
5
CITS, Centro de Investigação em Tecnologias da Saúde, IPSN, CESPU, Gandra, Portugal
6
Marie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, School of
Biological Sciences and Sydney Medical School, The University of Sydney, Sydney, NSW, Australia
Received 13 November 2014
Accepted 22 January 2015
Rabbit hemorrhagic disease virus (RHDV), a Lagovirus of the family Caliciviridae, causes rabbit
hemorrhagic disease (RHD) in the European rabbit (Oryctolagus cuniculus). The disease was first
documented in 1984 in China and rapidly spread worldwide. In 2010, a new RHDV variant
emerged, tentatively classified as ‘RHDVb’. RHDVb is characterized by affecting vaccinated
rabbits and those ,2 months old, and is genetically distinct (~20 %) from older strains. To
determine the evolution of RHDV, including the new variant, we generated 28 full-genome
sequences from samples collected between 1994 and 2014. Phylogenetic analysis of the gene
encoding the major capsid protein, VP60, indicated that all viruses sampled from 2012 to 2014
were RHDVb. Multiple recombination events were detected in the more recent RHDVb genomes,
with a single major breakpoint located in the 59 region of VP60. This breakpoint divides the
genome into two regions: one that encodes the non-structural proteins and another that encodes
the major and minor structural proteins, VP60 and VP10, respectively. Additional phylogenetic
analysis of each region revealed two types of recombinants with distinct genomic backgrounds.
Recombinants always include the structural proteins of RHDVb, with non-structural proteins from
non-pathogenic lagoviruses or from pathogenic genogroup 1 strains. Our results show that in
contrast to the evolutionary history of older RHDV strains, recombination plays an important role in
generating diversity in the newly emerged RHDVb.
INTRODUCTION
Rabbit hemorrhagic disease (RHD) is an acute fatal necrotizing hepatitis that affects wild and domestic European
rabbits of both the Oryctolagus cuniculus cuniculus and
Oryctolagus cuniculus algirus subspecies (Abrantes et al.,
The GenBank/EMBL/DDBJ accession numbers for the RHDV strains
determined in this study are KF442964, KF442963, KP090974,
KP090975, JF438967, KM115714, KM115715, KM115716, KF442961,
KF442962, KP090976, KM115697, KM115698, KM115680, KM115681,
KM115682, KM115711, KM115712, KM115713, KM115689,
KM115683, KM979445, KM878681, KM979445, KP129395,
KP129396, KP129397, KP129398, KP129399 and KP129400.
000070 G 2015 The Authors
2012b). The disease was first detected in 1984 in China
following the importation of commercially bred Angora
rabbits from Germany (Liu et al., 1984). RHD rapidly
spread worldwide and is currently endemic in several
countries including Portugal, Spain and France. The economic burden of RHD on rabbit farming and hunting
industries and the negative ecological impact on the wild
rabbit populations and in their dependent predators, particularly in the Mediterranean ecosystem, are well known
and of major concern (Delibes-Mateos et al., 2009). In
contrast, in Australia, RHD has been successfully exploited
as a form of biocontrol to reduce rabbit numbers, although
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Printed in Great Britain
1309
A. M. Lopes and others
there is mounting evidence for host resistance (Elsworth
et al., 2012; Kovaliski, 1998; Kovaliski et al., 2014).
RHD is caused by rabbit hemorrhagic disease virus (RHDV),
a Lagovirus of the family Caliciviridae. RHDV has a nonenveloped single-stranded (ss) RNA genome organized into
two narrowly overlapping open reading frames (ORFs).
ORF1 encompasses nucleotide residues 10–7044 and
encodes a large polyprotein that is cleaved by a virusencoded protease to generate several non-structural (NS)
proteins and the major capsid protein, VP60 or VP1 (for
convenience, we use VP60). ORF2 comprises nucleotide
residues 7025–7378 and produces VP10 (or VP2), a minor
structural protein (Wirblich et al., 1996). At the 59 region,
the RHDV genome presents a covalently linked protein,
VPg, and is polyadenylated at the 39 region (Gregg et al.,
1991; Morales et al., 2004). Viral particles also package an
abundant subgenomic RNA (sgRNA) that contains the
coding sequences of VP60 and VP10. As is the case for the
RHDV genomic RNA, the sgRNA is VPg-linked and
polyadenylated (Meyers et al., 1991; Morales et al., 2004;
Ohlinger et al., 1990).
Despite the importance of RHDV for animal health and
ecosystem well-being, its origin and emergence as a pathogenic virus in the European rabbit is not well understood.
On current data the most likely explanation is direct
evolution from a non-pathogenic form of the virus (Kerr
et al., 2009). Indeed, a number of non-pathogenic and a
weakly pathogenic rabbit caliciviruses have been described
in Europe, America and Australia (Bergin et al., 2009;
Capucci et al., 1996; Le Gall-Reculé et al., 2011b; Strive
et al., 2009), and there is evidence for the existence of
RHDV as a non-pathogenic form before the first documented outbreak (reviewed by Abrantes et al., 2012b). To
date, however, the mutations (or recombination events)
responsible for the profound change in virulence are
unknown. It is also theoretically possible that a crossspecies transmission event was central to the emergence of
RHDV as a pathogenic form. Indeed, in the 1990s, RHDV
affected a species other than the European rabbit (Lopes
et al., 2014).
More recently, a new variant of RHDV was detected in
France (Le Gall-Reculé et al., 2011a). As a definitive RHDV
nomenclature has not yet been agreed (various terms such
as ‘new variant’, ‘RHDVb’ and ‘RHDV29 have been used),
for simplicity we use the term ‘RHDVb’. RHDVb resulted
in atypical RHD outbreaks in that it led to mortality in
both vaccinated adult rabbits (Le Gall-Reculé et al., 2011a)
and young rabbits (Dalton et al., 2014, 2012) that are
typically resistant to RHDV. Phylogenetic relationships
inferred from the VP60 gene showed that RHDVb formed
a novel phylogenetic group that fell between the RCV-like
viruses, i.e. European non-pathogenic viruses and the
weakly pathogenic Michigan rabbit calicivirus (MRCV)
and the Australian non-pathogenic rabbit caliciviruses
(RCV-A1) (Le Gall-Reculé et al., 2011a). Further work
demonstrated that RHDVb is antigenically different from
1310
other RHDVs (Dalton et al., 2012; Le Gall-Reculé et al.,
2013), although its genesis is uncertain. Importantly, the
recent detection of RHDVb in leporid species other than
the European rabbit (Camarda et al., 2014; Puggioni et al.,
2013) indicates that it might infect a wider range of host
species.
Both host-jumping and changes in virulence have been
associated with the ability of RNA viruses to rapidly
produce genetic diversity through mutation (Domingo &
Holland, 1997; Holmes, 2010). However, it is also possible
that the genotypes associated with successful emergence are
generated by recombination (Simon-Loriere & Holmes,
2011). Because it requires template switching, (homologous) RNA recombination generally involves regions of
high sequence similarity. Although homologous recombination has been detected in RHDV (Abrantes et al., 2008;
Forrester et al., 2008), its impact on fitness and hence longterm importance remains uncertain. Notably, the RHDV
strain isolated in 1984 in China had a recombinant origin
(Forrester et al., 2008), which may have been in part
responsible for the marked change in virulence observed in
this virus.
RHDV has been divided into six genogroups, denoted G1–
G6 (Le Gall-Reculé et al., 2003). In the Iberian Peninsula,
RHDV is characterized by a high degree of virus isolation
with only G1 detected in wild rabbit populations (Abrantes
et al., 2012a; Alda et al., 2010; Muller et al., 2009), which
differs from the epidemiological pattern seen in other
European countries (Le Gall-Reculé et al., 2003). However,
in 2011 and 2012, RHDVb was detected in both Spain and
Portugal (Abrantes et al., 2013; Dalton et al., 2012). To
better understand the evolution of RHDV and particularly
the occurrence and impact of recombination, we determined the complete coding sequences of Iberian RHDV
strains collected between 1994 and 2014.
RESULTS AND DISCUSSION
We determined the complete coding sequence (excluding
the 59 and 39 UTRs) of 28 RHDV strains from rabbits
found dead in the Iberian Peninsula. Based on their position in the maximum-likelihood (ML) tree of the capsid
gene (VP60) (Fig. 1), 24 strains were identified as RHDVb
and four as genogroup 1 (G1). Considering the distribution of the European rabbit subspecies in the Iberian
Peninsula (Carneiro et al., 2011; Ferrand, 2008), these
results further confirm that both O. cuniculus algirus and
O. cuniculus cuniculus are susceptible to this new virus
(Abrantes et al., 2013). Interestingly, the co-circulation of
RHDVb and other RHDV genogroups, particularly G1, was
not detected. This supports the notion that G1 has been
replaced by RHDVb (Lopes et al., 2015) and is compatible
with the rapid spread of this new variant through the
Iberian Peninsula (Dalton et al., 2014).
The complete coding sequences of RHDVb exhibited some
length variation due to deletions in the non-structural
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Journal of General Virology 96
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100
99
98
100
GQ166866/MRCV/USA/2001
AM268419/06-11/France/06-11-2006
EF558587/Ashington/UK/1998
G6/RHDVa
CB154/Gaia/Portugal/1997
CB107/Coruche/Portugal/1994
CB110/Santarem/Portugal/1994
EF558578/Eisenhuttenstadt/1989
JF438967/CB156/Fornos/Portugal/1997
100
99*
G2-G5
1311
Fig. 1. ML phylogenetic tree for 211 VP60 sequences of RHDV. Major genetic groups (genogroups) are indicated. For clarity of presentation, the sequences in genogroups
G2–G5 and G6 (RHDVa) have been collapsed and have the following GenBank accession numbers: AB300693, AF231353, AF258618, AJ006019, AJ302016, AJ303106,
AJ495856, AJ535092, AJ535094, AJ969628, AM085133, AY925210, AY926883, AY928268, AY928269, DQ069280–DQ069282, DQ189077, DQ189078,
DQ205345, DQ841708, EF363035, EF558572, EF558574, EF558575, EF558577, EF558581–EF558584, EU003578–EU003582, EU250330, EU650679,
EU650680, FJ212322, FJ212323, FJ794179, FJ794180, FN552800, FR823354, FR823355, GU339228, GU373617, GU373618, GU564448, HE963222,
HM623309, HQ917923, JF412629, JN165233–JN165236, JN851729–JN851735, JQ815391, JQ995154, JX393309–JX393312, KC345614, KC595270,
KF270630, KF494906–KF494952, KF537692, KF537693, KF594474–KF594476, KF677011, KJ579156–KJ579160, KJ606958, KJ606959, KJ683893, KJ683900,
KJ683902, KJ683905–KJ683908, KJ814617–KJ814622, M67473, RHU49726, RHU54983, X87607, Y15424 and Y15427. The accession numbers are indicated for the
remaining sequences. Viruses sequenced here (in bold) fall into two distinct clusters, G1 and RHDVb. All horizontal branch lengths are drawn to a scale of nucleotide
substitutions per site and the tree is mid-point rooted. Bootstrap values greater than 75 % are shown for key nodes. *The main group of sequences in genogroups G2–G5
clustered together with 99 % bootstrap support.
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Recombination in rabbit hemorrhagic disease virus
Z29514/SD/France/1989
L48547/MC/Spain/1989
Z24757/AST89/Spain/1989
Z49271/AST89/Spain/1989
0.05 subs/site
Bar01-11/Spain/01-2011
CB194/Chaves/Portugal/2007
C147/Castro_Marim/Portugal/2004
KJ683894/14266/PT06
KJ683903/23212/08
KJ683904/14393/PT06
G1
100
100
RHDVb
100
94
EU871528/RCV-A1/Australia/2007
KC345611/Ss11
KC345612/Vs11-1
KC345613/Vs11-2
JX106023/Vs11
HE819400/10-08/France/2010
HE800531/10-28/France/2010
FR819781/10-05/France/2010
JQ929052/Ud11/Italy/2011
JX106022/Tn12
KC907712/Tn12-1
HE800532/10-32/France/2010
HE800530/10-07/France/2010
HE800529/10-01/France/2010
Tar06-12/Spain/06-2012
Zar06-12/Spain/06-2012
N11/Spain/10-2011
CBAnd1/Spain/04-2012
Zar11-11/Spain/11-2011
97
Rij06-12/Spain/06-2012
CBVal16/Portugal/11-2012
Seg08-12/Spain/08-2012
91
CBCoruche14-2/Portugal/02-2014
CBCoruche14-1/Portugal
Barrancos7-13/Portugal/01-2013
Barrancos10A-13/Portugal/01-2013
CBLavra10-13-1/Portugal/10-2013
CBEstoi13-7/Portugal/12-2013
CBAlgarve3/Portugal/01-2013
CBAlgarve1/Portugal/01-2013
CBMontemor14-1/Portugal/02-2014
CBEstremoz14-3/Portugal
CBMora14-1/Portugal
CBEstremoz14-1/Portugal/01-2014
CBMertola14-2/Portugal/01-2014
CBAlgarve14-3/Portugal
100 CBMertola14-1/Portugal/01-2014
CBAlgarve14-1/Portugal
CBAlgarve14-4/Portugal
X96868/RCV/Italy/1996
A. M. Lopes and others
proteins p16/NS1 and p37/NS3: a deletion of codon 68
(p16) in CBAnd1/Spain/04-2012 and deletions of codons
134–135 (p16/NS1) and of codon 714 (p37/NS3) in several
strains. While the biological role of p16/NS1 is unknown, a
recent study of RHDV in Australia revealed a relatively
high number of mutations in this protein, which were
tentatively associated with increased virulence (Elsworth
et al., 2014). Protein p37 has an ATPase activity and may
be a member of the helicase superfamily III (Marı́n et al.,
2000). Although helicase activity is crucial for replication
(Kadaré & Haenni, 1997), this deletion is located some
distance from the Walker motifs A (amino acid positions
522–529 in RHDV) and B (amino acid positions 566–567
in RHDV) which are typical of NTP-binding proteins, and
from an additional conserved C motif (amino acid positions 600–614 in RHDV), which is also involved in ATP
hydrolysis (Marı́n et al., 2000). This indicates that the main
biological functions of p37 are not compromised by the
deletion. Additionally, the deletion is present in MRCV,
RCV-A1 and in European brown hare syndrome virus (the
other member of the genus Lagovirus), further suggesting
that the replication activity of p37 is maintained. A
mutation in the initiation codon (T to C in codon ATG,
nucleotide positions 7025–7027) of ORF2 was observed in
one strain, but expression of VP10 is not expected to be
affected as translation of ORF2 relies on the presence of a
sequence element at the 39 end of ORF1 and not on a
particular initiation codon (Meyers, 2003). Additionally,
another ATG in this region (nucleotide positions 7037–
7039) may act as the start site for translation of ORF2.
The most striking result of our study was the occurrence of
recombination in a number of recent RHDVb viruses with
strong statistical support (P values ,0.001, Table 1). In
these recombinant viruses, there was consistent evidence
for a single recombination breakpoint located in a region
close to the VP60 initiation codon (although two
recombination breakpoints were detected they fall in very
similar and overlapping locations, which is strongly
suggestive of a single recombination event; Table 1). This
was further confirmed with the SimPlot analysis (Fig. 2).
Notably, this recombination breakpoint is located within a
region of high sequence similarity (data not shown) and
divides the genome into two regions that include different
protein subsets: one that includes the non-structural
proteins and another that comprises the structural proteins
VP60 and VP10. To confirm this recombinant history, ML
phylogenetic trees were inferred for the different genome
fragments on either side of the recombination breakpoint
identified above. For simplicity, we based this phylogenetic
analysis on the recombinant breakpoint at nucleotide position 5305. For the phylogenetic tree of the genes encoding
the structural proteins (VP60 and VP10 genes; Fig. 3a), all
viruses previously identified as RHDVb (Fig. 1) clustered
together in a single strongly supported monophyletic
group (identified as RHDVb and shaded light grey). This
cluster fell deep in the VP60 and VP10 phylogeny, close to
the position of the mid-point root of the tree and between
the non-pathogenic Australian strain (RCV-A1) and the
1312
weakly pathogenic MRCV. Hence, RHDVb is phylogenetically
distinct from the pathogenic RHDV sequences that form
a separate highly supported cluster. In contrast, in the
phylogeny inferred for the non-structural proteins (Fig. 3b),
the RHDVb sequences fall into three distinct clusters: (i) one
comprising likely non-recombinant RHDVb sequences
(shaded light grey) that share a common ancestor with
pathogenic RHDV strains, (ii) another composed of likely
recombinant RHDVb sequences that cluster with MRCV
and RCV-A1 (medium grey) and (iii) a group of RHDVb
recombinant sequences that cluster closely with recently
sampled pathogenic G1 viruses (dark grey).
The occurrence of multiple recombination events, i.e. RHDVb
clusters (i) and (ii) mentioned above, makes it difficult to
clearly distinguish recombinants from parents, but it is clearly
necessary to invoke at least two independent recombination
events involving RHDVb to explain these phylogenetic data.
Moreover, the divergent position of the CBAnd1/Spain/
04-2012 sequence within the RHDVb cluster (ii) may be
indicative of an additional recombination event. It is also
evident that one of the recombination events involved
the combination of the RHDVb VP60 and VP10 coding
sequences with a backbone composed of the coding sequences
of the non-structural proteins of a G1-like strain, while in the
other the RHDVb VP60 and VP10 coding sequences became
linked with the coding sequences of the non-structural
proteins of non-pathogenic lagoviruses (Fig. 3b).
Since some of the recombinant viruses possess nonstructural genes that are closely related to the Australian
non-pathogenic virus (as supported by the clustering of
such recombinant viruses with RCV-A1 with 98 % bootstrap support), this evolutionary history implies that
lineages related to the Australian viruses must circulate in
the Iberian Peninsula but have not yet been detected [indeed,
it is clear that RCV-A1 was imported into Australia (Jahnke
et al., 2010)]. In addition, recombinant sequences were
retrieved from animals found dead in the field with lesions
compatible with RHD, and thus these strains are likely
pathogenic despite their genome deriving mostly from nonpathogenic viruses.
Although circulation of ‘true’ G1 viruses has not been
detected in this study from 2011 onwards, the cocirculation of G1-like and RHDVb strains within the same
geographical region and time-frame must have occurred
for recombination to take place. Interestingly, the two
different types of recombinants were detected from the same
geographical area only one year apart (e.g. CBAlgarve14-1/
Portugal, detected in January 2014, and CBAlgarve1/
Portugal/01-2013, detected in January 2013). These observations indicate that there is a relatively high diversity of
RHDV strains circulating in this region, which clearly merits
additional surveillance. In addition, the same type of
recombinant was observed in several and distantly located
populations and in different years, confirming its viability
and capacity to spread at the epidemiological scale.
Two mechanisms of RNA recombination are possible for
RNA viruses: replicative and non-replicative. The (copy-choice)
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Table 1. Results of the recombination analysis
Recombinant strains
Most likely ‘parental’ strain
NS*
Genogroup
VP60 and
VP10
NS*
VP60 and
VP10
BreakpointD
Average P value
RDP
GENECONV
BootScan
EU871528/RCV-A1/
Australia/2007
Seg08-12/Spain/
08-2012
NPd
RHDVb
5305
4.2726102125 2.8986102132 2.9716102137
(5267–5326)
CBMertola14-2/Portugal/01-2014 CBMertola14-1/
Portugal/01-2014 CBAlgarve14-1/Portugal
CBAlgarve14-3/Portugal CBAlgarve14-4/Portugal
JX886001/CB194/Chaves/
Portugal/2006
Seg08-12/Spain/
08-2012
G1
RHDVb
5336
6.2836102109 3.6106102106 6.3166102109
(5264–5343)
*Non-structural proteins.
D99 % confidence interval is indicated.
dNon-pathogenic strain.
1313
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Recombination in rabbit hemorrhagic disease virus
CBAnd1/Spain/04-2012
CBMontemor14-1/Portugal/02-2014
CBEstoi13-7/Portugal/12-2013
CBMora14-1/Portugal
CBEstremoz14-1/Portugal/01-2014 CBEstremoz143/Portugal
CBLavra10-13-1/Portugal/10-2013 CBAlgarve1/
Portugal/01-2013 CBAlgarve3/Portugal/01-2013
Barrancos7-13/Portugal/01-2013
Barrancos10A-13/Portugal/01-2013 CBCoruche141/Portugal
CBCoruche14-2/Portugal/02-2014
A. M. Lopes and others
(a)
p16 ▼ p23
▼
p37
▼
p29
▼VPg▼ pro ▼
RdRp
VP60
VP10
1.0
0.9
0.8
0.7
0.5
101
341
581
821
1061
1301
1541
1781
2021
2261
2501
2741
2981
3221
3461
3701
3941
4181
4421
4661
4901
5141
5381
5621
5861
6101
6341
6581
6821
7061
0.6
EU871528/RCV-A1/Australia/2007
(b)
p16 ▼ p23
▼
p37
▼
p29
Seg08-12/Spain/08-2012
▼VPg▼ pro ▼
RdRp
VP60
VP10
1.0
0.9
0.8
0.7
0.5
101
341
581
821
1061
1301
1541
1781
2021
2261
2501
2741
2981
3221
3461
3701
3941
4181
4421
4661
4901
5141
5381
5621
5861
6101
6341
6581
6821
7061
0.6
CB194/Chaves/Portugal/2007
Seg08-12/Spain/08-2012
Fig. 2. Similarity plot for two types of RHDVb recombinants: (a) CBAlgarve1/Portugal/01-2013 (non-pathogenic lagovirus/
RHDVb) and (b) CBAlgarve14-1/Portugal (G1/RHDVb). The vertical axis represents the percentage of sequence identity of
each putative parental strain (grey and black lines) with the recombinant. The horizontal axis indicates the nucleotide positions.
A window size of 200 bp with a step size of 20 bp was used. The site where the parental strains are identical in sequence to the
recombinant is the predicted recombination breakpoint. A scheme of the genomic organization is shown above each plot with
the encoded proteins indicated (pro, protease); arrowheads indicate cleavage sites.
replicative mechanism involves template switching by the
viral polymerase during RNA synthesis. In other Caliciviridae
genera, such as Sapovirus, Vesivirus and Norovirus (NoV), a
recombination hotspot is similarly observed between the
non-structural proteins and the capsid junction (Bull &
White, 2010). In the case of NoV, a template switching
mechanism has been proposed that reflects a combination of
the copy-choice and the internal initiation model (Bull et al.,
2005). Accordingly, recombination occurs in the second
round of replication when the polymerase starts the synthesis
of a positive-sense RNA strand from a full-length negative
strand. Due to the presence of complex secondary structures,
1314
such as stem–loops, at the ORF1/ORF2 overlap that
coincides with the RNA promoter sequence, the polymerase
loses processivity and switches to a negative strand of sgRNA
synthesized by a co-infecting virus, thereby creating a hybrid
virus (Bull et al., 2005). Alternatively, the polymerase switches
from a genomic RNA to another in regions with high
sequence homology, such as the one found where ORF1 and
ORF2 overlap (Bull et al., 2005). Although the genomic
organization of NoV differs from RHDV with the nonstructural proteins and the capsid protein encoded by
separate ORFs, the synthesis of the sgRNA by an internal
initiation mechanism and the presence of a subgenomic
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Journal of General Virology 96
Recombination in rabbit hemorrhagic disease virus
0.05 subs/site
G6/RHDVa
G1
G2-G5
100
EU871528/RCV-A1/Australia/2007
Tar06-12/Spain/06-2012
Zar06-12/Spain/06-2012
100
CBAnd1/Spain/04-2012
Zar11-11/Spain/11-2011
Rij06-12/Spain/06-2012
96
CBVal16/Portugal/11-2012
Seg08-12/Spain/08-2012
100 CBMertola14-1/Portugal/01-2014
CBAlgarve14-3/Portugal
CBMertola14-2/Portugal/01-2014
CBAlgarve14-1/Portugal
CBAlgarve14-4/Portugal
CBCoruche14-2/Portugal/02-2014
CBCoruche14-1/Portugal
Barrancos10A-13/Portugal/01-2013
98 Barrancos7-13/Portugal/01-2013
CBEstremoz14-3/Portugal
CBEstremoz14-1/Portugal/01-2014
CBMora14-1/Portugal
CBMontemor14-1/Portugal/02-2014
CBLavra10-13-1/Portugal/10-2013
CBEstoi13-7/Portugal/12-2013
CBAlgarve1/Portugal/01-2013
CBAlgarve3/Portugal/01-2013
GQ166866/MRCV/USA/2001
EF558586/Hartmannsdorf/1989
EU003582/UT-01/2001
99
EF558583/Triptis/1996
EF558584/Rossi/2002
99
DQ205345/JX/CHA/97/1997
AB300693/Hokkaido/JPN/2002
EU003581/NY-01/2001
99
AF258618/Iowa2000/2000
EF558582/Dachswald/2000
JF412629/HYD/China/2005
DQ280493/ChinaWHNRH
HM623309/NJ-2009/China/2009
EF558581/Erfurt/2000
AY523410/CD/China
100
EU003578/IN-05/2005
CB110/Santarem/Portugal/1994
CB154/Gaia/Portugal/1997
Z49271/RHDV-AST89/1989
EF558578/Eisenhuttenstadt/1989
96
Z29514/SD/France/1989
CB156/Fornos/Portugal/1997
JX886002/CB137/Alpiarca/Portugal/1995
Bar01-11/Spain/01-2011
JX886001/CB194/Chaves/Portugal/2006
DQ189078/Saudi Arabia
AF295785/Mexico89/1989
EU003580/Korea90/1990
M67473/FRG/1989
RHU54983/RHDV-V351/1987
EU003579/Italy90/1990
EF558579/NZ54/2003
78
EF558580/NZ61/2003
EF558585/Hagenow/1990
EF558575/Ascot/UK/1992
X87607/BS89/1989
EF558574/Wika/Germany/1996
EF558572/Frankfurt12/1996
EF558573/Frankfurt5
EF558577/Meiningen/Germany/1993
EF363035/clonepJG-RHDV-DD06/1999
DQ189077/Bahrain/2001
EF558576/Jena/Germany
RHDVb
(a)
Fig. 3. ML phylogenetic trees for the genome regions defined by the recombination analysis. Major genetic groups
(genogroups) are indicated and strains sequenced here are shown in bold. Accession numbers of the sequences retrieved from
GenBank are indicated. All horizontal branch lengths are drawn to a scale of nucleotide substitutions per site and the tree is
mid-point rooted. Bootstrap values greater than 75 % are shown for key nodes. (a) ML tree based on the sequence fragment
including VP60 and VP10 (nucleotide positions 5305–7378). RHDVb sequences are shaded light grey. (b) ML tree based on
non-structural proteins sequences (nucleotide positions 10–5304). RHDVb strains fall in to three distinct clusters
corresponding to: (i) non-recombinant RHDVb sequences (shaded light grey), (ii) non-pathogenic lagovirus/RHDVb
recombinants (medium grey) and (iii) G1/RHDVb recombinants (dark grey).
promoter upstream of the sgRNA starting site in RHDV
(Morales et al., 2004) suggests that a similar recombination
mechanism may occur. In addition, the presence of a region
of high sequence homology upstream of the initiation codon
of VP60 in RHDV (Simmonds et al., 2008), which is compatible with the location of the sgRNA promoter (Boga et al.,
1992; Miller & Koev, 2000; Morales et al., 2004; Sibilia et al.,
1995), as well as the presence of a stem–loop (Simmonds
http://vir.sgmjournals.org
et al., 2008), tentatively suggest that the recombination
events observed could have been due to the template
switching mechanism.
However, it is also possible that the recombinant RHDV
genomes observed might have been generated by a nonreplicative recombination mechanism. Such a mechanism,
demonstrated for other positive-sense ssRNA viruses such
as polioviruses and pestiviruses (Gallei et al., 2004; Gmyl
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1315
100
97
0.05 subs/site
et al., 1999), involves the cleavage of the recombining RNAs
that are then ligated to form the recombinant molecule. In
contrast to the template switching mechanism of recombination that is promoted by sequence conservation, the
non-replicative recombination seems to be largely mediated
by the presence of a RNA secondary structure (Chetverin
et al., 1997) such as the stem–loop predicted for RHDV in
the sgRNA promoter region (Simmonds et al., 2008).
While the impact of recombination on the fitness of
RHDVb remains to be assessed, this process has clearly
created substantial changes in the viral genome, although
whether this has led to differences in virulence is unclear.
Indeed, the recombination events described involve the
combination of structural and non-structural protein
subsets with different ancestries: protein coding sequences
are combined as ‘modules’ with non-structural proteins
originating either from non-pathogenic forms or G1
viruses and structural proteins originating from RHDVb.
A similar model of modular viral evolution has been
1316
G6/RHDVa
84
G2-G5
98
GQ166866/MRCV/USA/2001
EU871528/RCV-A1/Australia/2007
CBAnd1/Spain/04-2012
CBMontemor14-1/Portugal/02-2014
CBEstoi13-7/Portugal/12-2013
CBMora14-1/Portugal
CBEstremoz14-1/Portugal/01-2014
100
CBEstremoz14-3/Portugal
CBLavra10-13-1/Portugal/10-2013
CBAlgarve1/Portugal/01-2013
CBAlgarve3/Portugal/01-2013
Barrancos10A-13/Portugal/01-2013
Barrancos7-13/Portuga/01-2013
CBCoruche14-1/Portugal
CBCoruche14-2/Portugal/02-2014
Zar06-12/Spain/06-2012
Tar06-12/Spain/06-2012
Zar11-11/Spain/11-2011
100
Seg08-12/Spain/08-2012
CBVal16/Portugal/11-2012
Rij06-12/Spain/06-2012
EF558586/Hartmannsdorf/1989
EU003582/UT-01/2001
EF558584/Rossi/2002
100
EF558583/Triptis/1996
AF258618/Iowa2000/2000
EF558582/Dachswald/2000
DQ205345/JX/CHA/97/1997
AB300693/Hokkaido/JPN/2002
EU003581/NY-01/2001
EF558585/Hagenow/1990
JF412629/HYD/China/2005
DQ280493/ChinaWHNRH
HM623309/NJ-2009/China/2009
EF558581/Erfurt/2000
AY523410/CD/China
EU003578/IN-05/2005
EU003580/Korea90/1990
100
DQ189078/Saudi Arabia
AF295785/Mexico89/1989
EU003579/Italy90/1990
M67473/FRG/1989
RHU54983/RHDV-V351/1987
EF558579/NZ54/2003
EF558580/NZ61/2003
EF558577/Meiningen/Germany/1993
100
100
EF363035/clonepJG-RHDV-DD06/1999
DQ189077/Bahrain/2001
EF558576/Jena/Germany
EF558575/Ascot/UK/1992
X87607/BS89/1989
EF558572/Frankfurt12/1996
100
EF558574/Wika/Germany/1996
EF558573/Frankfurt5
CB110/Santarem/Portugal/1994
CB154/Gaia/Portugal/1997
EF558578/Eisenhuttenstadt/1989
100
Z29514/SD/France/1989
Z49271/RHDV-AST89/1989
CB156/Fornos/Portugal/1997
JX886002/CB137/Alpiarca/Portugal/1995
Bar01-11/Spain/01-2011
100
JX886001/CB194/Chaves/Portugal/2006
CBAlgarve14-1/Portugal
100
CBMertola14-2/Portugal/01-2014
100 CBMertola14-1/Portugal/01-2014
CBAlgarve14-4/Portugal
CBAlgarve14-3/Portugal
G1
(b)
RHDVb
A. M. Lopes and others
described for enteroviruses (family Picornaviridae), in which
the structural and non-structural regions of the genome
evolve semi-independently (Lukashev, 2005; Lukashev et al.,
2005; Oberste et al., 2004), giving rise to new virus variants.
RNA signals and sequence similarity are among the factors
that might impose such a recombination pattern (Runckel
et al., 2013), although other constraints exist at several stages
of viral infection and replication in the host. These constraints most likely reflect functional protein ‘blocks’ that
work together for protein compatibility. Interestingly, previous studies revealed recombination breakpoints throughout the RHDV genome (Abrantes et al., 2008; Forrester et al.,
2008), although most of these recombination events
occurred between closely related RHDV strains. Hence, it
is likely that the sequence similarity between the recombining strains allows the functional integrity to be maintained.
As there is prior evidence that recombination may have been
central to the emergence of RHDV as a pathogenic form
(Forrester et al., 2008), we contend that more effort should
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Journal of General Virology 96
Recombination in rabbit hemorrhagic disease virus
Table 2. List of primers and PCR conditions used in the amplification of the RHDV genome
Forward primer (5§–3§)
GTGAAAGTTATGGCGGCTATGTCG
AGGTGCACCCTGCCATCATACAA
CAACATCTTTGGCGCATGGT
GCAAACCACCTTGTCAACCT
GTTGGCGTTGACATGACATC
GTGTATGCCATGACTCCGAT
TGCTGAGCCAGATGTACGCT
CACCTGTGGGTAAGAACACA
Reverse primer (5§–3§)
TTCCCAAGGACCATGGTGTGT
ATGAGCGTGGTCTGTGAGTGGAA
TACGCCAGCACGTCAATCTT
ATGGCGTCAAGTATGGTCATGG
CTAGTAGTGGCCACAACACC
GCGTCGATGACAACATGAG
AGTCCAATTGTCACTGGCAG
ATAGCTTACTTTAAACTATAAACCCAA
be directed toward revealing the frequency and fitness
consequences of recombination in lagoviruses.
METHODS
Virus samples and genome amplification. Liver samples were
collected from wild rabbits found dead in the field in different regions
of Portugal and from domestic rabbits from Spanish rabbitries, and
were kept frozen at 220 uC. No live animals were shot, trapped or
handled to obtain tissues, as such, no animal ethics permit was
required. Each frozen liver was thawed and up to 30 mg of the
liver was collected. The tissue was homogenized in a rotor-stator
homogenizer (Mixer Mill MM400; Retsch) at 30 Hz for 7 min. Total
RNA was extracted using the RNeasy Mini kit (Qiagen) according to
the protocol provided by the manufacturer. Reverse transcription was
performed using oligo(dT) as primers and SuperScript III Reverse
Transcriptase (Invitrogen) according to the provided protocol. The
complete genome of each RHDV strain was obtained by the amplification of several overlapping fragments ranging from ~700 to 1700 bp
using 1 ml cDNA reaction mix, 2 pmol of each oligonucleotide, 5 ml
Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific) and
water to a final volume of 10 ml. The primers and PCR conditions used
are summarized in Table 2. For recombinant sequences, each PCR was
performed at least twice independently. After purification, PCR
products were sequenced on an automatic sequencer ABI PRISM 310
Genetic Analyzer (Applied Biosystems). Sequences were determined
using the amplification primers and are available in GenBank.
No incongruences were detected between the overlapping fragments
that could indicate preferential amplification of templates in each
PCR. While it is highly unlikely that the Phusion DNA Polymerase
generates crossovers at the same position repeatedly, a long range
PCR was also performed to obtain complete genomic sequences and
exclude the possibility of PCR-mediated artefacts in generating the
recombinant sequences. This PCR was conducted with the following
conditions: 0.25 ml of Takara LA Taq (Clontech), 2.5 ml buffer,
2.5 mM MgCl2, 2.5 mM of each dNTP, 0.4 mM of each primer, 1 ml
of template and water to a final volume of 25 ml. Cycle conditions
consisted of 2 min at 94 uC, 35 cycles of 30 s at 94 uC, 30 s at 63 uC
and 8 min 30 s at 68 uC and a final extension of 15 min at 68 uC. The
amplicons were sequenced as described above using amplification
primers listed in Table 2 (primer 59-GTGTATGCCATGACTCCGAT39 covers the regions located both upstream and downstream of the
recombination breakpoint).
Recombination analysis. The complete genome sequences obtained
(excluding the 59 and 39 UTRs) were aligned using the BioEdit
software, version 7.0.9.0 (Hall, 1999). Full-length RHDV genome
sequences available in public databases were retrieved and included in
http://vir.sgmjournals.org
Annealing
temperature (6C)
55
53
51
48
52
48
52
50
Extension
time
1 min 30
1 min 15
1 min
1 min 30
1 min 15
1 min 30
1 min 15
45 s
s
s
s
s
s
s
the alignment. This produced a final dataset of 68 sequences of
7369 nt, including representatives from all major groups of RHDV.
This sequence alignment was screened for recombination using the
RDP, GENECONV and BootScan methods implemented in the RDP
software (version 4.26) (Martin et al., 2010) under the following
parameters: sequences were set to linear, Bonferroni correction,
highest acceptable P value 0.05 and 100 permutations. Only
recombination events detected by all three methods were considered
as viable and hence merited additional analysis (see Phylogenetic
analysis section). Strains detected as recombinants were further
analysed using the SimPlot software (version 3.5.1) (Lole et al., 1999)
with the default parameters (window size, 200 bp; step, 20 bp; Ts/Tv
ratio, 2 : 0).
Phylogenetic analysis. An initial reconstructed phylogenetic tree
was inferred for the VP60 gene to provide a provisional picture of the
evolutionary relationships of RHDVb to the remaining pathogenic
and non-pathogenic lagoviruses. For this analysis, all available VP60
sequences were retrieved from public databases, producing a final
dataset of 211 sequences of 1740 nt. A phylogenetic tree was then
estimated using the ML method available in PhyML program
(Guindon et al., 2010) utilizing the GTR+I+C model of nucleotide
substitution and employing a combination of nearest-neighbour
interchange (NNI) and subtree pruning and regrafting branchswapping. The support for each node was determined from 1000
bootstrap replicate ML trees, employing the same substitution model
and parameter values as above, and with NNI branch-swapping.
For the complete genome sequences, the dataset was partitioned at
the putative recombination breakpoint(s) detected in the recombination analysis described in the Results and Discussion section and
then subjected to phylogenetic analysis. This partitioning was as
follows: (i) ORF1 except VP60 (nucleotide residue positions 10–5304)
and (ii) VP60 and VP10 (nucleotide residue positions 5305–7378).
Phylogenetic trees were then estimated for both partitions using the
same phylogenetic procedure as described above (i.e. ML trees in
PhyML).
ACKNOWLEDGEMENTS
We acknowledge all the collaborators who provided samples: Dr
Paulo Célio Alves (CIBIO/InBIO-UP), Vı́tor Palmilha, José Manuel
Maia and Jacinto Amaro. We thank INTERCUN and Spanish
Ministerio de Economı́a y Competitividad (grant AGL2013-48550C2-1-R co-financed by FEDER) for the logistical and financial
support provided to the laboratory of F. P. This work was funded by
National Funds through the FCT-Foundation for Science and
Technology under the project ref. FCT-ANR/BIA-BIC/0043/2012.
This work was also supported by ‘Genomics Applied to Genetic
Resources’, co-financed by North Portugal Regional Operational
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1317
A. M. Lopes and others
Programme 2007/2013 (ON.2 - O Novo Norte), under the National
Strategic Reference Framework (NSRF), through the European
Regional Development Fund (ERDF). FCT supported the doctoral
fellowship of A. M. L. (ref. SFRH/BD/78738/2011) and the FCT
Investigator grant of J. A. (ref. IF/01396/2013). E. C. H. is supported
by an NHMRC Australia Fellowship (AF30) and by the ARC (grant
no. DP140103362).
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Delibes-Mateos, M., Farfán, M. A., Olivero, J., Márquez, A. L. &
Vargas, J. M. (2009). Long-term changes in game species over a long
period of transformation in the Iberian Mediterranean landscape.
Environ Manage 43, 1256–1268.
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