Capripoxvirus G-protein-coupled chemokine receptor: A host-range gene suitable for virus animal origin discrimination

Abstract

The genus Capripoxvirus within the family Poxviridae comprises three closely related viruses, namely goat pox, sheep pox and lumpy skin disease viruses. This nomenclature is based on the animal species from which the virus was first isolated, respectively, goat, sheep and cattle. Since capripoxviruses are serologically identical, their specific identification relies exclusively on the use of molecular tools. We describe here the suitability of the G-protein-coupled chemokine receptor (GPCR) gene for use in host-range grouping of capripoxviruses. The analysis of 58 capripoxviruses showed three tight genetic clusters consisting of goat pox, sheep pox and lumpy skin disease viruses. However, a few discrepancies exist with the classical virus-host origin nomenclature: a virus isolated from sheep is grouped in the goat poxvirus clade and vice versa. Intra-group diversity was further observed for the goat pox and lumpy skin disease virus isolates. Despite the presence of nine vaccine strains, no genetic determinants of virulence were identified on the GPCR gene. For sheep poxviruses, the addition or deletion of 21 nucleic acids (7 aa) was consistently observed in the 5' terminal part of the gene. Specific signatures for each cluster were also identified. Prediction of the capripoxvirus GPCR topology, and its comparison with other known mammalian GPCRs and viral homologues, revealed not only a classical GPCR profile in the last three-quarters of the protein but also unique features such as a longer N-terminal end with a proximal hydrophobic alpha-helix and a shorter serine-rich C-tail.

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Capripoxvirus G-protein-coupled chemokine
receptor: a host-range gene suitable for virus
animal origin discrimination
Christian Le Goff,
1
3Charles Euloge Lamien,
2
3Emna Fakhfakh,
3
Ame
´lie Chadeyras,
1
Elexpeter Aba-Adulugba,
4
Genevie
`ve Libeau,
1
Eeva Tuppurainen,
5
David B. Wallace,
6,7
Tajelser Adam,
8
Roland Silber,
9
Vely´ Gulyaz,
10
Hafsa Madani,
11
Philippe Caufour,
1
Salah Hammami,
3
Adama Diallo
2
and Emmanuel Albina
1
Correspondence
Emmanuel Albina
emmanuel.albina@cirad.fr
1
CIRAD, UMR Contro
ˆle des Maladies, F-34398 Montpellier, France
2
Animal Production Unit, FAO/IAEA Agriculture and Biotechnology Laboratory, IAEA Laboratories
Seibersdorf, International Atomic Energy Agency, Wagramer Strasse 5, PO Box 100, A-1400
Vienna, Austria
3
IRVT, La Rabta, 1006 Tunis, Tunisia
4
National Veterinary Research Institute, Vom, Plateau State, Nigeria
5
Institute of Animal Health, Pirbright Laboratory, Woking, Surrey GU24 ONF, UK
6
Biotechnology Division, ARC-Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort
0110, South Africa
7
Department of Veterinary Tropical Diseases, University of Pretoria, Faculty of Veterinary Science,
Private Bag X4, Onderstepoort 0110, South Africa
8
Department of Viral Vaccines Production, Central Veterinary Research Laboratories Centre,
Animal Resources Research Corporation, Ministry of Science and Technology, Khartoum, Sudan
9
Institute for Veterinary Disease Control, Austrian Agency for Health and Food Security, Robert
Koch Gasse 17, A-2340 Mo¨ dling, Austria
10
Pendik Veterinary Control and Research Institute, Pendik, Istanbul, Turkey
11
Institut National de la Me
´decine Ve
´te
´rinaire, Laboratoire Central Ve
´te
´rinaire d’Alger, BP 205
Hacen Badi, El Harrach, Alger, Algeria
Received 30 January 2009
Accepted 29 March 2009
The genus Capripoxvirus within the family Poxviridae comprises three closely related viruses, namely
goat pox, sheep pox and lumpy skin disease viruses. This nomenclature is based on the animal species
from which the virus was first isolated, respectively, goat, sheep and cattle. Since capripoxviruses are
serologically identical, their specific identification relies exclusively on the use of molecular tools. We
describe here the suitability of the G-protein-coupled chemokine receptor (GPCR) gene for use in
host-range grouping of capripoxviruses. The analysis of 58 capripoxviruses showed three tight genetic
clusters consisting of goat pox, sheep pox and lumpy skin disease viruses. However, a few
discrepancies exist with the classical virus–host origin nomenclature: a virus isolated from sheep is
grouped in the goat poxvirus clade and vice versa. Intra-group diversity was further observed for the
goat pox and lumpy skin disease virus isolates. Despite the presence of nine vaccine strains, no
genetic determinants of virulence were identified on the GPCR gene. For sheep poxviruses, the
addition or deletion of 21 nucleic acids (7 aa) was consistently observed in the 59terminal part of the
gene. Specific signatures for each cluster were also identified. Prediction of the capripoxvirus GPCR
topology, and its comparison with other known mammalian GPCRs and viral homologues, revealed
not only a classical GPCR profile in the last three-quarters of the protein but also unique features such
as a longer N-terminal end with a proximal hydrophobic a-helix and a shorter serine-rich C-tail.
3These authors contributed equally to this work.
Two supplementary tables are available with the online version of this paper.
Journal of General Virology (2009), 90, 1967–1977 DOI 10.1099/vir.0.010686-0
010686 G2009 SGM Printed in Great Britain 1967

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INTRODUCTION
The family Poxviridae consists of large double-stranded
DNA enveloped viruses that replicate in the cell cytoplasm.
Poxviruses affect a wide range of host species, from insects
(subfamily Entomopoxvirinae) to poultry and mammals
(subfamily Chordopoxvirinae). The latter subfamily is
further divided into eight genera that includes the genus
Capripoxvirus. The natural hosts for capripoxviruses
(CaPVs) are ruminants, including cattle, sheep and goats.
CaPVs are subdivided into three virus species according to
their host origins: sheep poxvirus (SPPV), goat poxvirus
(GTPV) and lumpy skin disease virus (LSDV) of cattle. The
diseases caused by these viruses have a significant economic
impact on the livestock industry in Africa and Asia. Both
sheep pox and goat pox are endemic in Africa, the Middle
East and many countries in Asia (Carn, 1993), while lumpy
skin disease (LSD) is widespread throughout most of Africa
and some areas in the Middle-East, but is absent in Asia
(Davies, 1981, 1982; Diallo & Viljoen, 2007). CaPV
infections lead to similar clinical signs in sheep and goats,
mainly characterized by fever, excess salivation, conjunc-
tivitis and rhinitis with ocular and nasal discharges. These
symptoms are followed by eruption of pox lesions
throughout the skin. LSD is a subacute to acute cattle
disease with appearance of skin nodules. During epizootics
of CaPVs in domestic animals, disease in wild ungulates
has never been reported (Davies, 1991), although serology
and isolated cases suggests that wildlife species have a
possible role in virus maintenance.
CaPVs are generally considered to be host-specific, leading
to outbreaks in one preferred host. This is partially true
since some SPPV and GTPV isolates are capable of causing
severe diseases in both sheep and goats (Davies, 1976;
Kitching et al., 1989). An in-depth epidemiological
investigation to specifically identify the viruses involved
in acute small ruminant pox diseases cannot be achieved by
serological testing alone due to the very close antigenic
relationship between CaPVs, with the existence of only one
serotype (Kitching et al., 1986, 1989). The common
immunogenic properties of these viruses have been used
for the preparation of live attenuated vaccines that protect
all ruminants against CaPV infection (Kitching et al.,
1987). Recombinant CaPVs have also been developed for
multivalent vaccination purposes (Berhe et al., 2003; Perrin
et al., 2007; Romero et al., 1993; Wade-Evans et al., 1996;
Wallace et al., 2006). However, although they are
antigenically closely related, restriction enzyme pattern
analysis, cross-hybridization studies and, more recently,
nucleic acid sequencing have shown that nearly all CaPVs
can be grouped according to their host origins (Black et al.,
1986; Cao et al., 1995; Gershon & Black, 1988; Kitching
et al., 1989; Tulman et al., 2002). In this paper, we describe
the suitability of one of the CaPV genes, the G-protein-
coupled chemokine receptor (GPCR) gene described by
Cao et al. (1995), for host range phylogenetic grouping of
CaPVs. Compared with previous studies, we have analysed
a larger number of CaPV isolates (almost 60) from
different geographical regions that support the host-range
discrimination and also provide evidence of specific
signatures for each of the three CaPVs. The CaPV GPCR
was finally compared with other mammalian GPCRs and
herpes- and poxvirus homologues; the differences are
reported here.
METHODS
Viruses. Infectious material isolated directly from lesions or first in
vitro passages of virulent isolates and vaccine strains were used for the
study. Fifty-eight CaPVs from different African and Asian countries
were compared. These included 22 strains isolated from sheep, 12
from goats, 13 from cattle and 2 from springbok antelopes
(Antidorcas marsupialis) (Supplementary Table S1, available in JGV
Online). Eight of the isolates were vaccine strains: a goat pox vaccine
from Kazakhstan [GTPV1 in Supplementary Table S1 (Kitching et al.,
1987)], a sheep and goat vaccine strain (SPPV14 and GTPV4,
respectively) used for routine vaccination in Nigeria (sent by Dr
Majiyagbe, National Veterinary Research Institute, Vom, Plateau
State, Nigeria), the Onderstepoort LSD Neethling vaccine strains
[LSDV1, partial sequence (Fick & Viljoen, 1994) and LSDV3 full
genome sequence (Kara et al., 2003)], an LSD vaccine used in Nigeria
(LSDV8), the Romanian sheep pox vaccine strain produced by
Biopharma, in Morocco (SPPV11), a sheep pox vaccine from
Kazakhstan [SPPV3 (Tulman et al., 2002)], the sheep pox vaccine
strain Nihkhi from Kazakhstan [SPPV3 (Tulman et al., 2002)]and the
Kenya sheep vaccine isolate of LSDV [KS-1, SPPV15 (Kitching et al.,
1987)]. They were compared to the corresponding sequences of three
SPPV, two GTPV and four LSDV isolates retrieved from GenBank in
October 2008. Three additional poxviruses were selected as outgroups
for the phylogenetic study, these were: two deer poxviruses (Afonso
et al., 2005) and one swine poxvirus (Massung et al., 1993).
Viral DNA isolation, cloning and sequencing. CaPV genomic
DNA was extracted and purified from infected lamb testis cells
(Qiagen DNeasy Tissue System) and amplified using primers derived
from the KS-1 vaccine strain sequence (Cao et al., 1995). Two primers
(59-TTAAGTAAAGCATAACTCCAACAAAAATG-39and 59-
TTTTTTTATTTTTTATCCAATGCTAATACT-39) were designed to
amplify the genome at position 6961–8119 (Tulman et al., 2001).
These primers were used for the amplification of the entire GPCR
gene. Two additional primers (59-GATGAGTATTGATAGATACC-
TAGCTGTAGTT-39and 59-TGAGACAATCCAAACCACCAT-39)
were positioned internally for primer walking sequencing. PCR
products were purified, inserted into a DNA vector using a Blunt End
Cloning kit (Roche) or the pGEM-T DNA plasmid (Promega). GPCR
sequences were amplified using dideoxy sequencing chemistry and
run on an automated DNA sequencer (Applied Biosystem, Prism
377), except for SPPV16–25, GTPV8–15 and LSDV9–18, which were
sent to Agowa (Germany) for sequencing.
Sequence alignment and phylogenetic analysis. Multiple align-
ments of nucleotide and amino acid sequences were generated using
the CLUSTAL W program (Invitrogen). Phylogenetic analysis was
carried out by means of the neighbour-joining method (Saitou & Nei,
1987). Dissimilarities and edge length of dissimilarities between the
sequences were first determined with Darwin software (Perrier et al.,
2003), selecting the correction of Kimura (1980) and considering gaps
as missing data. Tree construction was based on the unweighted
neighbour-joining method proposed by Gascuel (1997). Trees were
generated with the TREECON MATRIXW program of Darwin (Van de
Peer & De Wachter, 1993). Bootstrap confidence values were
calculated on 1000 replicates according to the maximum-likelihood
approach (Felsenstein, 1981).
C. Le Goff and others
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Sequence analysis and protein modelling. In order to assess the
effect of mutations, additions or deletions on the protein secondary
structure and to identify putative functional motifs, the amino acid
sequence of the GPCR (model GTPV1) was submitted to a series of
online bioinformatics tools (Supplementary Table S2, available in
JGV Online). The consensus secondary structure and predicted trans-
membrane helices from all structures generated by these software
programmes were used for further representation and analysis.
Amino acid blocks, conserved motifs and signatures were also
replaced.
RESULTS
For sequence comparison, we selected a non-essential gene
that encodes a homologue of a GPCR. The gene sequence
was obtained from the published sequence for the Kenya
sheep (KS-1) CaPV isolate (Cao et al., 1995). PCR primers
were designed to enable amplification and sequencing of
the full GPCR gene of all CaPV strains. Nucleotide
sequences were aligned and two major variations were
observed: single nucleotide mutations spread over the full
sequence and two addition or deletion regions of 21 and
12 nt (data not shown). To study the relationship between
the 58 CaPVs, a representative phylogenetic analysis using
the neighbour-joining method was carried out on nucleic
acid sequences. The consensus tree showed three tight
genetic clusters consisting of LSDV, GTPV and SPPV
lineages (Fig. 1). The different lineages were supported by
the bootstrap values, suggesting a strong co-adaptation of
the strains and their respective hosts. Considering the edge
length of the branches in the rooted consensus tree and
the nodes placed between the three CaPV clusters, LSDV
and GTPV appeared more closely related to each other
than to SPPV. The LSDV and GTPV clusters showed more
intra-group diversity than the SPPV cluster. In the LSDV
group, the isolates were segregated into two subgroups,
one of these (strains isolated pre-1960) consisting of an
early South African field isolate (LSDV17) and the
Onderstepoort vaccine strain (LSDV1 and 3), the other
comprising an early Kenyan isolate (LSDV2) and other,
more recent, isolates. Similarly, the GTPV consisted of
two subgroups: the first group was Middle Eastern and
Asian isolates (GTPV1–2 and GTPV10–14), whereas the
second group comprised Middle Eastern and African
isolates (GTPV3, GTPV5–7 and GTPV9). Within the first
subgroup, a subdivision was also observed. Subgroup 1.1
contained three GTPV (10–12) and one ‘GTPV-like’ SPPV
(SPPV24) isolate. They were all from the Middle East and
southern Asia. In subgroup 1.2, four GTPV strains (1, 2,
13 and 14) are from the Middle East and western Asia.
Unexpectedly, the SPPV isolate SPPV24 fell within the
GTPV strain cluster, whereas three GTPVs were segre-
gated within the SPPV cluster. The phylogenetic tree
generated using the deduced amino acid sequences was
consistent with that for the nucleic acids (data not
shown).
Besides the phylogenetic analysis discriminating the three
lineages on the basis of sequence mutations, amino acid
mutations and two additions/deletions were found in the
GPCR protein in some isolates compared with the others
(Fig. 2). Some of the unique mutations (circled on Fig. 2)
have to be considered cautiously, since only a single clone
for each virus was sequenced and therefore PCR errors
cannot be ruled out. The two additions/deletions can be
differentially ascribed to each of the three groups of viruses.
Amino acids in position 10–16 were missing in all sheep
isolates and in five goat isolates (GTPV8, 10–12 and 15).
However, in the 10 goat isolates that did not have the
‘SPPV-like’ gap, the sequence at positions 10–16 was
GYAMYNS or SYAMYNS compared with SATMYNS that
is found in all LSDV isolates. The absence of amino acid
residues at positions 30–33 was observed exclusively, but
not systematically, in cattle isolates. Due to these additions/
deletions, the length of the GPCR protein was 374 aa for
SPPV, but varied from 374 to 381 aa for GTPV and 377 to
381 aa for LSDV. The GTPV1 sequence was used as the
representative CaPV GPCR sequence and after alignment
with other protein sequences available in public databases,
the maximum amino acid identity with GTPV1 GPCR was
32.4 % for the rhesus monkey C-C chemokine receptor 8
(CCR-8, GenBank accession number O97665) and 30 % for
the GPCR homologues K2 of swinepox (strain Kasza,
GenBank accession no. Q08520).
Table 1 summarizes the differences in amino acid motifs
between the three CaPV species that define eight unique
profiles. Of the 17 positions indicated, LSDV and SPPV
had distinct motifs represented in eight of the positions
which constitute signature motifs for cattle and sheep
CaPVs, respectively, these were: S/R
6
, S/–
10
, A/–
11
, T/–
12
,T/
R
34
, S/L
99
, P/T
199
and M/I
328
. At all positions, GTPV
isolates could contain either LSDV, SPPV or specific
motifs. GTPVs did not have a systematic and exclusive
motif and, therefore, could not be distinguished from the
other CaPVs on the basis of any single amino acid position
changes. However, 87 % of GTPVs had a specific signature
consisting of either (N
6
,G
10
,Y
11
and A
12
)or(V
34
,K
49
,F
99
and S
199
). For example, GTPV10, 11 and 12 contained the
SPPV-like amino acid gap but had a GTPV signature at
positions 34, 49, 99 and 199, and GTPV4 had the GTPV
signature at positions 6, 10, 11 and 12 but a SPPV profile at
the other positions. Taken together, these discriminatory
features placed the GTPV group in an intermediate
position between the LSDV and SPPV clusters. The only
exceptions to the previous rules were GTPV4, 8 and 15,
and SPPV15 and 24. GTPV8 and 15 are field isolates from
Saudi Arabia and Sudan, respectively. They had not only
seven missing amino acid residues, like SPPV strains, but
also the SPPV profile on the other amino acid positions,
making them ‘true’ SPPVs. GTPV4 is a live attenuated
vaccine isolate from Nigeria. It did not exhibit R
6
and the
sheep-like gap, but featured a real SPPV signature for all
other amino acid motifs. SPPV15 (strain KS-1) was
originally isolated from a sheep and later shown by
restriction enzyme digestion analysis to have an LSDV
profile (Kitching et al., 1987). This is confirmed in this
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study, as KS-1 did not possess the SPPV-like gap and had a
clear LSDV signature. SPPV24 is an SPPV isolated in
Oman. It had the SPPV-like gap, but a GTPV signature for
all other positions, grouping this isolate closely to the Asian
and Middle-Eastern strains [GTPV10 (Bangladesh), 11
(India) and 12 (Oman)]. The vaccine strains GTPV1
(Kazakhstan isolate), LSDV3 (South African
Onderstepoort vaccine strain) and LSDV8 (Nigeria isolate
vaccine) were 100 % identical to the virulent strains
GTPV2 (Kazakhstan) and 13 (Turkey isolate) and the
virulent South African LSDV17 and 4 isolates, respectively.
These sequence identities indicate that the attenuation of
these vaccine strains have not required modifications in the
GPCR gene. The two Onderstepoort LSDV Neethling
vaccine strain sequences, LSDV1 and 3, taken from
GenBank, showed one amino acid mutation, D
355
Y,
resulting from a unique base mutation (G
1063
T) between
the two corresponding genes. That the two sequences are
from the same strain was confirmed by the alignment of
12 518 of their nucleotides showing only 19 variations
(0.15 %; data not shown). However, it is not known
whether these variations resulted from sequencing errors or
genetic drift acquired during virus passages in cells in vitro.
Interestingly, half of the amino acid point mutations and
the two additions/deletions between the different CaPV
GPCR homologues were found in the first quarter of the
protein sequence (positions 1–95). This region is probably
Fig. 1. Phylogenetic tree of CaPVs based on the alignment of the nucleotide sequences (6976–8118) of the GPCR gene.
Bootstrap values of 1000 replicates are shown when higher than 70 %. The consensus tree was rooted in reference to the three
poxvirus outgroups and was broken up to show the distant relationship between them and CaPVs. Underlined labels indicate
the vaccine strains. SPPV, GTPV and LSDV strains are shown in green, blue and red text, respectively.
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important for ligand–receptor interaction, but is not
engaged in the important motifs for the chemokine
receptor functionality (Fig. 2). Indeed, using multiple
alignments and sequence homology searches, a profile for
GPCRs was identified between nt 92 and 357, including the
seven helical transmembrane (TM) domains and a
conserved disulfide bond (C
162
–C
239
). This disulfide bond
links the second and fourth loops (two extracellular
domains) of the GPCR receptor. Another putative disulfide
bond was located between the conserved positions C
83
and
C
327
on the GTPV1 sequence. The prediction did not
identify any signal or anchor peptide motifs, which
suggests that this is a non-secretory protein. Sequence
comparisons with the rhesus monkey C-C chemokine
receptor 8 and the bovine interleukin (IL) 8 receptor
identified seven putative TM domains on the GTPV1
sequence (Fig. 2). The SPPV-like gap removed three amino
acid residues, Y
11
,Y
14
and S
16
, that are potentially involved
in protein–protein interactions and also an N-glycosylation
site (NSSS) at nt 15–18. This gap may indicate that the
potential functions of this area, i.e. protein–protein
interaction and N-protein glycosylation, are not used by
CaPVs. Using block and sequence homology searches,
human and monkey chemokine receptor signatures were
located in positions 168–179 and 333–347, respectively.
The first signature is highly conserved amongst the
different CaPVs, whereas the second signature endorses
amino acid variations. A highly conserved intracytoplasmic
block of amino acid residues [DRYLA(V/I)V(H/Y)]that are
also found as a consensus sequence in multiple alignments of
different mammalian chemokine receptors and viral homo-
logues are found close to the first signature (Fig. 3). In this
alignment, we also observed that the N-terminal regions of the
CaPV GPCR are longer and their C-tails are shorter than the
ones from other GPCRs. However, the C-tail of CaPV GPCR
is still rich in serine, the amino acid involved in receptor
internalization. In addition, conserved proline residues are
found in the second, fourth, fifth, sixth and seventh TM
domains, according to the numbering of mammalian GPCRs,
and the conserved
344
NPxxY
348
motif joined the last TM
domain and the C-tail of the protein (Fig. 3).
Secondary protein structure predictions identified an
additional a-helix at positions 17–36 of GTPV1 (Fig. 2).
Three viruses (GTPV1, SPPV1 and LSDV4) were further
selected as representatives of the different CaPV lineages
for TM topology predictions. Out of the nine software
packages used for these predictions (Supplementary Table
S2), five, four and two of them predicted the supplement-
ary a-helix as an eighth TM domain for GTPV1, SPPV1
and LDV4, respectively. Software packages failing to
predict this eighth TM domain, however, did identify an
additional a-helix, but with TM prediction certainties
marginally below the threshold values (data not shown). In
contrast, all packages correctly predicted the 7-TM model
of mammalian GPCRs and other herpes- and poxvirus
homologues. The predicted eighth TM domain in
CaPV GPCRs included the conserved sequence
SNITTIATTIISTILS(V/R)IS at positions 18–36 and 11–29
for GTPV1 and SPPV1, respectively. The absence of the
sequence
32
LSVI
35
, including three hydrophobic leucine,
valine and isoleucine residues, might account for the
absence of the 8-TM prediction in LSDV4. This is
confirmed by the non-deleted LSDV strains that have a
normal 8-TM prediction (data not shown). Due to this TM
topology prediction, the CaPV GPCR conformation in the
cell membrane may differ from classical descriptions.
Table 1. Amino acid specificity of cattle (LSDV), goat (GTPV) and sheep (SPPV) CaPVs
For 17 amino acid positions over the GPCR sequence (GTPV1 numbering), eight unique profiles were distinguished: two for LSDV, five for
GTPV and one for SPPV. The distribution of the different profiles within each CaPV group is indicated as a percentage. Amino acid variations
are shown in each column for each specific position. Dashes and dots indicate missing amino acids and conserved residues at the corresponding
positions, respectively. Black and grey shading denote the LSDV and SPPV amino acid profiles, respectively. GTPV residues often originate from
the two other CaPVs, as shown by the interpenetrations of the LSDV and SPPV profiles; however, GTPV-specific residues (white boxes) could be
identified.
CaPV
group
Proportion in
group (%)
Position
6 10 11–12 34 36 49 60 80 99 117 199 249 268 328 335 370
LSDV 83 S S AT T S E A I S R P I T M L R
17 .. .. ...... . . . IT..
GTPV 27 .. YA V .KSTF C S F .M..
20* .––– .
..... . . . . ..H
33 N G YA .L.AI .R.I..IR
7.. . RSE ..L.T..IL.
13 R – –– ...... . . . . . . .
SPPV 96 .––– ...... . . . . . . .
*This GTPV profile is also found for SPPV24.
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DISCUSSION
Ruminant CaPVs are antigenically and genetically closely
related to each other. SPPV protects cattle against LSD;
sheep immunized with GTPV have been reported to be
protected against sheep pox and vice versa in goats
(Bhanuprakash et al., 2006). However, the geographical
distribution of LSD differs markedly from that of sheep
and goat pox, which tend to coexist over most of their
distribution range. Though mixed flocks often comprise
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sheep and goats, outbreaks of sheep or goat pox can be
confined to only sheep or goats, respectively, or can involve
both species. The host preference for goat poxviruses was
recently demonstrated experimentally (Babiuk et al., 2009);
however, the breed of the sheep and goat used might
account for this difference. There is at least one confirmed
isolation of an LSDV isolate from a sheep, the Kenyan O-
240 isolate (KS-1) (Davies, 1976, 1982; Kitching et al.,
1987). In southern Africa, there have never been reports of
CaPV-like diseases in sheep or goats, whereas there are
outbreaks of LSD in cattle every few years (Hunter &
Wallace, 2001; Weiss, 1968). The exact pattern of
circulation of CaPVs between cattle, sheep and goats
remains to be established but the approach has long been
hampered by the lack of differential identification tools.
Recent studies have shown that the three CaPVs can be
distinguished genetically (Le Goff et al., 2005; Tulman et
al., 2002). The Q2/3L gene, which encodes a homologue of
a GPCR (Tulman et al., 2001), known to be a single copy
gene located in the left terminus of the genome, is likely to
affect the virus virulence (Kara et al., 2003; Tulman et al.,
2002). In mammals, the GPCR superfamily represents the
largest family of transmembrane proteins responsible for
the transmission of extracellular signals to intracellular
responses by stimuli as diverse as light, odorants, nucle-
otides and proteins (Kostenis, 2004). GPCRs are located in
five major families based on their relation to either
rhodopsin (family A, type I, rhodopsin-like), calcitonin
(family B, type II, secretin-like), glutamate (family C, type
III, metabotropic glutamate receptor-like), adhesion or
odorant/taste/frizzled receptors (Fredriksson et al., 2003).
Chemokine receptors are members of subgroups A1 and
Fig. 2. Multiple alignment of the deduced
amino acid sequence of the GPCR of 58
CaPVs. The strains were isolated from goats
(GTPV), cattle and springbok antelope (LSD)
and sheep (SPPV), and are presented in
numerical order. LSDV4 is representative of
isolates LSDV5, 7, 9, 10, 13, 14, 16 and 18 as
they share 100 % identity with each other.
Similarly, GTPV3 represents GTPV5, 6 and 7,
GTPV2 represents GTPV13, GTPV10 repre-
sents GTPV11, SPPV1 represents SPPV4, 5,
7, 9, 10, 12, 13, 22 and 23, and SPPV18
represents SPPV19. Underlined labels indic-
ate the vaccine strains. Dots represent ident-
ical residues and dashes indicate missing
amino acids. Unique amino acid mutations
among the 58 viruses are circled. A probable
N-glycosylation site, NSSS, in position 15–18
(in bold and boxed) is deleted in the ‘sheep-
like’ genotype and in GTPV8, 10–12 and 15.
The secondary structure of the proteins is
shown above the alignment and homology with
the profile of the GPCR family is indicated by
the ovoid shading. Potential protein–protein
interaction sites are indicated by a bold capital
letter ‘P’ above the sequence of GTPV1. The
seven homologues of the TM helices of the
bovine IL-8 receptor B (GenBank accession
no. Q28003) and of the rhesus monkey C-C
chemokine receptor 8 (GenBank accession
no. O97665) are aligned to the sequence of
the GTPV1 and GTPV2 sequences, respect-
ively (indicated by grey shading). Human and
rhesus monkey C-C chemokine receptor 8
signatures retrieved from databases using
sequence homology searches are indicated
by bold underlined letters (positions 168–179
and 334–347, respectively; accession no.s
P51685 and O97665). Two putative disulfide
bonds are indicated.
Analysis of capripoxvirus GPCR gene
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A2 of the rhodopsin-like family A (Joost & Methner, 2002;
Kawasawa et al., 2003). Like the other GPCRs, they are
integral membrane proteins that transduce extracellular
signals to the intracellular environment through hetero-
trimeric guanine nucleotide-binding (G) domains
(Cabrera-Vera et al., 2003; Oppermann, 2004; Schoneberg
et al., 1999). They are composed of seven helical TM
domains with an extracellular N-terminal segment and a
cytoplasmic C-terminal tail. During the course of their
evolution, herpes- and poxviruses have probably acquired
their chemokine receptor genes from their hosts and
adapted them for their own benefit, either as viral ligand-
to-cell receptors or for cell activation, increased propaga-
tion or control of the host antiviral responses (Rosenkilde,
2005). Although still largely unknown, the pathogenic
effects of such virally encoded GPCRs may include
increased cell trafficking and proliferation (angiogenesis
and tumorigenesis), cell lysis, chemokine removing and
sequestering, and cytokine downregulation (Randolph-
Habecker et al., 2002; Rosenkilde et al., 2008). It is
tempting to surmise that the CaPV homologue of GPCR
may play a role in the cell proliferative lesions and
immunosuppression induced by CaPV infections. Because
it was previously shown to be one of the most variable
genes within the CaPVs (Tulman et al., 2002), we
hypothesized that this gene would be a suitable target for
genetic discrimination between ruminant poxviruses.
The phylogenetic study on this gene that is reported here
confirms that the CaPVs can be divided into three distinct
clusters, as previously shown using the comparison of the
full genome sequences (Tulman et al., 2002). Although
GTPV and SPPV are distinct viruses, isolates of both are
able to affect sheep and goats in the field with or without
differences in the degree of pathogenicity induced. This
study shows that GTPV and LSDV are more closely related
to each other than to SPPV. This would support the
hypothesis that they both emerged from a common
ancestor close to the SPPVs, as proposed by others who
carried out phylogenetic studies on different genome
segments (Hosamani et al., 2004; Stram et al., 2008).
This assumption is, however, in contradiction with another
study, which concluded that small ruminant poxviruses
may have emerged from a common LSDV-like ancestor,
Fig. 3. Multiple alignments of GPCRs of the three lineages of CaPVs [GTPV1 (AY077836), LSDV4 (AF409137) and SPPV1
(AY077832)], four mammalian chemokine receptors (O97665, P51680, Q2HJ17 and Q28003) and three viral homologues
(Q08520, Q9J5I0 and P69332). White letters on black shading and letters on grey shading show identical and conserved amino
acids, respectively, compared with the GTPV1 sequence. Framed blocks represent the position of the helical TM domains.
C. Le Goff and others
1974 Journal of General Virology 90

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based on the observation in GTPV and SPPV of disrupted
LSDV genes with putative virulence and host range
functions (Tulman et al., 2002). The GTPVs, and to a
lesser extent the LSDV, lineages show more diversity than
in the SPPV group. This observation was also observed
previously for the P32 gene, but using a very limited
number of isolates (Hosamani et al., 2004). Our GTPVs fall
into three subgroups. For LSDV, the subdivision into
subgroups 1 and 2 was also supported by the work on P32
for our strains LSDV2, 3 and 4 (Hosamani et al., 2004).
The reasons for such diversity within the GTPV and LSDV
isolates are unclear, especially in light of the higher degree
of conservation of the SPPV isolates. The answer may lie in
the split from a common ancestor and differences in
selection pressures experienced in the different host species.
Although Stram et al. (2008) claimed the use of one of the
most variable regions of the CaPV genome, located at the
left terminus, for distinguishing LSDV isolates, they did
not show substantially more genetic variability than we
have done by using the GPCR gene. We even observed
more divergence within each lineage than shown previously
(Hosamani et al., 2004; Stram et al., 2008), thus identifying
clearly separated sublineages with high bootstrap support.
The multiple sequence alignments of the GPCR gene and
the corresponding phylogenetic tree did not reveal specific
differences between the vaccine and virulent strains of
GTPV, LSDV and SPPV. Furthermore, we noticed that one
vaccine strain (LSDV8) and two virulent isolates (LSDV5
and 17) demonstrated the virulent and vaccine-like amino
acid profiles, respectively, of LSDV described by Kara et al.
(2003). This observation was partly supported by restric-
tion enzyme digestion studies on DNA isolated from
LSDV1/3 and 17 (data not shown), which suggested that
the LSDV Onderstepoort vaccine strain (LSDV1/3)
developed from the virulent Haden field isolate (LSDV17).
In this study, an amino acid addition or deletion in positions
10–16 allowed the separation of LSDV isolates from other
CaPVs, since they always had the complete sequence.
Instead, LSDV isolates have the characteristic AT signature
at positions 11–12. The gap at positions 10–16 cannot
discriminate between SPPVs and GTPVs, since some of the
GTPV isolates may display this gap, while others may not.
However, GTPV isolates, which do not possess the gap, have
a specific YA amino acid signature in positions 11–12
instead. These differences might prove useful for diagnostic
applications. Indeed, the rational design of CaPV subspecies
primers in this region may permit the development of
conventional and real-time PCR allowing the direct
differential detection of the three CaPVs. Such a tool would
be more robust and less sensitive to genetic drift than the
two conventional PCRs described before, which both rely on
subsequent analysis of the restriction profiles of the PCR
products (Heine et al., 1999; Hosamani et al., 2004). This
tool would also supersede a recently developed conventional
PCR for LSDV, which does not allow the detection of SPPV
(Stram et al., 2008). Unexpectedly, three GTPV isolates were
found to have an SPPV-like gap, but with a GTPV amino
acid signature in other positions; one other isolate did not
have the gap, but had an SPPV profile in other positions.
Since the host specificity of CaPVs is less stringent than
initially thought, the viruses may cross between sheep and
goats living in close contact, resulting in genetic adaptations
to their new hosts. Under these conditions, it can be foreseen
that some CaPVs may evolve with an intermediate status.
This may be the case with the Oman sheep isolate, SPPV24,
which has the sheep 10–16 gap but the GTPV signature and
groups in the GTPV cluster. This observation confirms a
previous study, carried out by analysing DNA fragments
resulting from DNA digestion with restriction enzymes,
which highlighted the oddity of this Oman sheep strain
(Black et al., 1986). Atypical CaPV strains with intermediate
profiles, such as SPPV24, GTPV4, 8 and 15, may account for
some of the conflicting reports describing severe disease
inducement in both small ruminant species, while others
only indicate that one species is affected (Davies, 1976;
Kitching et al., 1989; Kitching & Taylor, 1985).
In conclusion, the molecular characterization of the GPCR
allows grouping the CaPVs since (A
11
,T
12
,T
34
,S
99
and
P
199
) is the unique signature of LSDVs, (N
6
,G
10
,Y
11
and
A
12
)or(V
34
,K
49
,F
99
and S
199
) are unique signatures of
GTPVs and (R
6
,R
34
,S
99
and T
199
) is, mostly, a specific
signature of SPPVs. SPPV15 (strain KS-1) (Davies, 1982;
Kitching et al., 1987; Kitching & Taylor, 1985) has a clear
LSDV signature. This supports previous observations
indicating that KS-1 is actually an LSDV strain (Black et
al., 1986; Kitching et al., 1989; Tulman et al., 2002) that is
still pathogenic for high-production dairy cattle (Kitching
et al., 1989; Kitching & Taylor, 1985; Yeruham et al., 1994).
LSD is, however, rarely associated with sheep and goat pox.
Indeed, while LSD is widespread in southern Africa, goat
and sheep pox are never reported in that region (Diallo &
Viljoen, 2007). Thus, SPPV15, an LSDV isolated from a
sheep (Davies & Otema, 1981), is apparently a unique case.
Of note is the isolation of LSDV on two separate occasions
from the springbok antelope in South Africa (LSDV12 and
15) (D. B. Wallace & T. Gerdes, unpublished data). These
isolations from a wild game animal species again raise the
question as to the original natural host species for this
virus, as it only appeared in cattle in 1929.
The observed variations in the N-terminal region of the
GPCR protein between the three CaPV lineages are
probably linked with the host specificity in view of
optimized interactions with the corresponding immune
system. It is noteworthy that the SPPV-like gap affects a site
with three potential protein–protein interaction motifs and
results in the suppression of an N-glycosylation site. Such
modifications in SPPV isolates may be strongly selected by
the sheep host due to an essential role in ligand–receptor
affinity and subsequent virus–host interactions in this
species. The second gap of 4 aa residues is not constant
within LSDV isolates, suggesting a minor effect on the
protein function. In contrast with the N-terminal region of
the CaPV GPCR, the central and C-terminal regions are
more conserved because they contain the functional domains
Analysis of capripoxvirus GPCR gene
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of the GPCR family, including the seven helical TM domains
(Cabrera-Vera et al., 2003; Cao et al., 1995; Wells et al., 1996),
potential disulfide bonds between the first and second
extracellular loops, and the extracellular N-terminal region
and third extracellular loop (Oppermann, 2004). The IL
receptor motif (C–X*–C) and IL 8 receptor signatures are
also located within these regions. Another structural feature,
which is found in chemokine receptors, is a conserved
DRYLAVVHA sequence within the second intracellular loop
(end of the third TM domain) that is putatively involved in G
protein interactions. Other GPCR motifs are conserved in
CaPVs, such as the conserved proline residues P
266
,P
308
and
P
352
(GTPV1 numbering), which are considered to be
important for the a-helix structure and function in the GPCR
(Rosenkilde et al., 2008). Although not yet reported, the
conserved P
144
and P
223
residues in the second and fourth
TM domains may play exactly the same role. The motif
344
NPxxY
348
is also present in the CaPV GPCR and, through
interactions with F
362
and the proximal TM6 loop or the
conserved D
136
in TM2, it might regulate the conformation
of the receptor and act as an activation switch.
Bioinformatic predictions identified an additional a-helix
in the long N-terminal region of the CaPV GPCR with the
potential for being a TM domain for GTPV and SPPV.
Conversely, none of the other poxviral homologues or
mammalian chemokine receptors were predicted to have
this extra TM domain, and their amino acid sequences did
not align with the new CaPV a-helix. In this 8-TM domain
model for the CaPV GPCR, the preferential prediction for
the short N-terminal region was intracytoplasmic (data not
shown) and the following long loop was extracellular. This
is in clear contrast with the predicted structure of the
prototype monkey CCR8 with its 7-TM domains (Case et
al., 2008; Oppermann, 2004; Schoneberg et al., 1999).
Although shorter than in other known GPCRs, the C-tail of
CaPV GPCR is still rich in serine residues, which, upon
phosphorylation, is involved in the clathrin-mediated
endocytosis of the receptor (Fraile-Ramos et al., 2001). In
the prediction, the four conserved cysteine residues
putatively involved in disulfide bond formation remain
in the extracellular compartment, which is in agreement
with a proper 3D cylindrical folding model of the receptor
(Cabrera-Vera et al., 2003), although the exact conforma-
tion of the CaPV GPCR still remains to be established.
ACKNOWLEDGEMENTS
This work was mainly supported by the French Ministry of Foreign
Affairs (MAE) via the FSP project (LABOVET 2003-24) and partly by
the EU network of excellence project [EPIZONE (016236, 01/06/
2006–31/05/2011)].
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