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Vol. 11, Issue 9, 1511-1519, September 2001
LETTER
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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Old World monkeys and, recently, African great apes have been shown,
by serology and polymerase chain reaction (PCR), to harbor different
2-herpesviruses closely related to Kaposi's sarcoma-associated Herpesvirus (KSHV). Although the presence of two distinct lineages of
KSHV-like rhadinoviruses, RV1 and RV2, has been revealed in Old World
primates (including African green monkeys, macaques, and, recently,
mandrills), viruses belonging to the RV2 genogroup have not yet been
identified from great apes. Indeed, the three yet known
2-herpesviruses in chimpanzees (PanRHV1a/PtRV1, PanRHV1b) and
gorillas (GorRHV1) belong to the RV1 group. To investigate the putative
existence of a new RV2 Rhadinovirus in chimpanzees and
gorillas we have used the degenerate consensus primer PCR strategy for
the Herpesviral DNA polymerase gene on 40 wild-caught animals. This
study led to the discovery, in common chimpanzees, of a novel
2-herpesvirus belonging to the RV2 genogroup, termed Pan
Rhadino-herpesvirus 2 (PanRHV2). Use of specific primers and internal
oligonucleotide probes demonstrated the presence of this novel
2-herpesvirus in three wild-caught animals. Comparison of a 1092-bp
fragment of the DNA polymerase obtained from these three animals of the
Pan troglodytes troglodytes subspecies, one from Gabon and the
two others from Cameroon, revealed <1% of nucleotide divergence. The
geographic colocalization as well as the phylogenetic "relationship" of the human and simian 2-herpesviruses support the model according to which herpesviruses have diversified from a
common ancestor in a manner mediating cospeciation of herpesviruses with their host species. By demonstrating the existence of two distinct
Rhadinovirus lineages in common chimpanzees, our finding indicates the possible existence of a novel human 2-herpesvirus belonging to the RV2 genogroup.
[The Herpesviral DNA polymerase sequence data determined herein have been deposited at the GenBank database under accession nos. AF290601, AF346488, AF346489, and AF346490.]
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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The members of the family Herpesviridae have been
grouped into three subfamilies, designated Alphaherpesvirinae,
Betaherpesvirinae, and Gammaherpesvirinae (Roizmann et al. 1992
).
Herpesviruses are widespread in vertebrate species, sharing several
moderately to well conserved genes, as determined from amino acid
identity comparisons (e.g., DNA polymerase and glycoprotein B). Among
Gammaherpesvirinae, Epstein-Barr virus (EBV) and Kaposi's
sarcoma-associated Herpesvirus (KSHV), also named Human herpesvirus 8 (HHV8), are the human prototypes of the Lymphocryptovirus
genus and the Rhadinovirus genus, respectively. Both of these
viruses play a critical role in human multistep carcinogenesis,
especially in immunodeficiency patients, leading to Burkitt's lymphoma
(Magrath and Judde 1996) and Kaposi's sarcoma (KS) (Chang et al. 1994;
Schulz 1998), respectively. Rhadinoviruses, or 2-herpesviruses, have
also been found in several animal species including New World monkeys
(Herpesvirus ateles and Herpesvirus saïmiri)
(Albrecht and Fleckenstein 1990; Albrecht 2000) and Old World monkeys
(macaques, African green monkeys, and recently mandrills) (Desrosiers
et al. 1997; Rose et al. 1997; Auerbach et al. 2000; Greensill et al.
2000b; Lacoste et al. 2000c; Strand et al. 2000). Comparison and
phylogenetic analyses of available sequences support the existence of
two distinct genogroups among the Old World monkey rhadinoviruses,
called RV1 and RV2 for Rhadinovirus genogroups 1 and 2 (Bosch
et al. 1998; Greensill et al. 2000b; Lacoste et al. 2000c; Schultz et
al. 2000). KSHV belongs to the RV1 genogroup, whereas no human virus
has yet been discovered in the RV2 group.
Considering that KSHV and Kaposi's sarcoma are highly endemic in
Central Africa (Schulz 1998; Gessain et al. 1999) and no 2-herpesvirus sequence has been described in great apes (Sinkovics and Horvath 1999), the closest primate species to human in the animal
kingdom, we decided to investigate the potential presence of
KSHV-related viruses in chimpanzees and gorillas from Central Africa.
Accordingly, we recently reported the detection and molecular characterization of the DNA polymerase gene fragment of three novel
2-herpesviruses in these great apes (Lacoste et al. 2000b). These
three new and different rhadinoviruses, two present in Pan troglodytes (PanRHV1a and PanRHV1b) and the latest in Gorilla gorilla (GorRHV1), were more closely related to KSHV (70%-85% identity at the nucleotide level) than any other previously described virus of this genus. These three novel 2-herpesviruses belong to the
RV1 genogroup as determined by phylogenetic analyses (Lacoste et al.
2000b). Moreover, an independent confirmation of the presence of
PanRHV1a (named PtRV1) in a colony of captive common chimpanzees (Pan troglodytes troglodytes) was published recently
(Greensill and Schulz 2000; Greensill et al. 2000a). The goals of
the present study were therefore to search for other 2-herpesviruses
belonging especially to the RV2 genogroup in African great apes,
chimpanzees, and gorillas, and to study the prevalence and the species
specificity of such identified novel herpesviruses in wild-caught great
apes from Central Africa.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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To look for the presence of KSHV-related viruses in great apes, we
first performed a serological analysis followed by a PCR-based study on
the peripheral blood mononuclear cells (PBMCs) DNA. The plasma of 40 animals, 28 chimpanzees and 12 gorillas, mostly wild-caught and
originating from the Western part of Central Africa, were tested by an
immunofluorescence assay (IFA) that detects both latent and lytic KSHV
antigens (Chatlynne et al. 1998). The results (Table
1) demonstrated a clear fluorescent
reactivity to the KSHV antigens-producing cells (KS-1) in the plasma of
22/28 chimpanzees and 6/12 gorillas, with antibody titers ranging from
1/40 (initial dilution) to 1/640.
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We then attempted to amplify a fragment of the very conserved
Herpesvirus DNA polymerase gene from the PBMCs DNA of 31 out of the 40 great apes, by nested PCR with degenerate primers (Rose et al. 1997).
Twenty DNA samples scored positive on the EtBr gel after the nested
PCR. By using Southern blot analysis and specific probes for the
recently described ape rhadinoviruses (PanRHV1a, PanRHV1b, and
GorRHV1; Tables 1 and 2), we observed that
7 of the 31 studied animals were infected by a PanRHV1a, 6 by a
PanRHV1b, and 1 by GorRHV1. Cloning and sequencing of two nested PCR
products that did not hybridize with such specific viral probes
revealed the presence of a novel Gammaherpesviral sequence. The 172-bp sequences (excluding primers) were identical to each other and exhibited 55%, 56%, 54%, and 59% nucleotide identity with the corresponding KSHV, PanRHV1a, PanRHV1b, and GorRHV1 fragments, respectively. Using the same nested PCR approach with, however, a
specific reverse primer for the second PCR (Pan2as, designed from this
novel sequence), we then amplified a second overlapping fragment of
~400 bp (DFASA-Pan2as) from 3 of the 31 animals and obtained finally
a 476-bp fragment of the DNA polymerase gene for two chimpanzees and a
400-bp fragment for one other (Table 2; Fig.
1). Another heminested PCR, using P2s (a
new degenerate forward primer based on conserved amino acid motifs
within the DNA polymerase of 2-herpesviruses), P2eas, and P2ias (new
virus-specific reverse primers designed from the DFASA-GDTD1B sequence;
Table 2) yielded a further 870 bp of viral sequence. The resulting sequences were assembled to give a total of 1168 bp (excluding primers)
of PanRHV2 for two chimpanzees and 1092 bp for one other.
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Database searches using the BLAST Web server demonstrated
that these novel sequences were most similar to the DNA polymerases of
the 2-Herpesvirus subfamily. Comprehensive comparative analysis of
these novel sequences with all the other available related Herpesviral
sequences (Table 3; Figs. 2 and
3)
revealed the existence of a novel and distinct chimpanzee KSHV-like
viral strain that we propose to name PanRHV2 for Pan
Rhadino-herpesvirus 2. This viral strain exhibited 63% identity at the
nucleotide level and 73% identity at the amino acid level with KSHV in
comparing a 454-bp DNA pol fragment (Table 3). Comparison of
the 364 encoded amino acid sequences showed that the two Cameroonese
PanRHV2 strains were 99.7% identical to each other, and the Gabonese
viral strain compared to the two Cameroonese strains presents 99.2%
and 99.5% amino acid identity, respectively.
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Phylogenetic analyses using different methods (Neighbor Joining, DNA
Maximum Parsimony) clearly placed this novel chimpanzee virus (PanRHV2)
within the Rhadinovirus genus (Fig. 2). Significantly, even
though only partial fragments of the DNA polymerases were studied, the
phylogenetic analysis presented in Figure 2 is in close agreement with
the known clustering of Herpesviruses into 
, 
, and subfamilies. Moreover, phylogenetic analyses performed on the 1168-bp
DNA polymerase gene fragment provide an identical tree topology (Table
3; data not shown). PanRHV2 clusters with the macaque (RRV, MneRV2,
MGVMn, MGVMm, and MGVMf), the African green monkey (ChRV2), and the
recently reported mandrill (MndRHV2) viral strains in the RV2 genogroup.
A second analysis restricted only to all the available primate
Rhadinovirus polymerase genes (Fig. 3) demonstrated the
existence of three major distinct separate lineages, supported by high
bootstrap values among 2-herpesviruses. The first corresponds to the
RV1 genogroup that comprises the human (KSHV), the chimpanzee (PanRHV1a and PanRHV1b), the gorilla (GorRHV1), the mandrill (MndRHV1) as well as
the macaque (RFHVMm and RFHVMn), and the African green monkey (ChRV1)
strains. This main lineage could be separated into three sublineages,
each one being well supported (bootstrap values >75%). Among them, we
can distinguish the lineage that contains only the Herpesviral strains
from Old World monkeys (macaques, mandrills, and African green monkeys)
from the two others constituted by Herpesviral strains of apes
(gorilla, pan, and homo). The second main lineage, that is, the RV2
genogroup, comprises Old World monkeys strains (ChRV2, RRV, MneRV2,
MGVMn, MGVMm, MGVMf, and MndRHV2) and the only new Herpesviral strain
of apes, PanRHV2, present in common chimpanzees. At least, this RV2
lineage is more closely connected to the third lineage of New World
monkey (Spider and Squirrel monkeys) rhadinoviruses than is the RV1 genogroup.
To study the prevalence of PanRHV2 infection, we hybridized the products of the heminested PCR (VYGA-GDTD1B) with a specific PanRHV2 oligonucleotide-labeled probe (PanRHV2-1). Three DNAs scored positive. We also developed a heminested PCR system with specific primers for the new PanRHV2 polymerase gene (Table 2). Using these primers in a nested PCR assay followed by hybridization of the PCR products with another internal specific oligonucleotide probe (PanRHV2-2), we detect the same three positive samples among the 31 DNAs.
Significantly, in our series only one case of multiple 2-herpesviral
infection was observed in a SIVcpz-infected chimpanzee among the 31 great apes we tested. This common chimpanzee, described as a natural
host of HIV-1-related viruses (Corbet et al. 2000), is therefore also a
natural host for distinct 2-herpesviruses belonging to the two
Rhadinovirus genogroups, PanRHV1a and PanRHV2.
To explore whether host-dependent evolution of chimpanzee
rhadinoviruses exists, we determined the subspecies identity of the
animals from which these novel viruses were derived. Four chimpanzee
subspecies with nonoverlapping geographic ranges have been proposed on
the basis of genetic differences in mitochondrial DNA sequences (Morin
et al. 1994; Gonder et al. 1997; Gagneux et al. 1999). We amplified and
sequenced a 498-bp fragment of mitochondrial DNA displacement loop
(mtDNA D-loop) for the 15 chimpanzees infected by PanRHV1a, PanRHV1b,
or PanRHV2. Comparison of these newly derived mtDNA sequences to
representative sequences from the four different chimpanzee subspecies
revealed that 9 out of the 15 infected chimpanzees belonged to the
Pan troglodytes troglodytes subspecies, and the 6 others
belonged to Pan troglodytes vellerosus (Table 1).
Classification of the chimpanzees was unambiguous as their mtDNA
sequences fell within well-defined subspecies clusters and was further
corroborated by the known geographic origins of these animals (Cameroon
and Gabon).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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The novel data presented in this paper as well as recently published
data (Greensill et al. 2000a; Lacoste et al. 2000b) indicate that
chimpanzees and gorillas are natural hosts for at least four novel and
distinct gammaherpesviruses belonging to the two known 2-herpesvirus
lineages. The new 2-herpesvirus PanRHV2 described in this report
corresponds to the first strain of the RV2 genogroup identified in
great apes. In contrast, the three other DNA sequences previously
detected in chimpanzees and gorillas (PanRHV1a/PtRV1, PanRHV1b, and GorRHV1) belonging to the RV1 genogroup are the closest known homologs to KSHV.
In instances in which particular animal species (macaques, Mandrills,
and African green monkeys) have been thoroughly analyzed for the
presence of 2-herpesviruses, at least two distinct viruses, each one
belonging to a particular genogroup RV1 or RV2, have been identified
(Desrosiers et al. 1997; Rose et al. 1997; Auerbach et al. 2000;
Greensill et al. 2000b; Lacoste et al. 2000c; Schultz et al. 2000). To
date, therefore, five distinct macaque 2-herpesviruses have been
characterized, two in Macaca mulatta (RFHVMm and RRV/MGVMm), two in Macaca nemestrina (RFHVMn and MneRV2/MGVMn), one in
Macaca fascicularis (MGVMf), and two in African green monkeys
(ChRV1 and ChRV2) as well as in mandrills (MndRHV1 and MndRHV2). Among these viruses, RFHVMm, RFHVMn, ChRV1, and MndRHV1 belong to the RV1
genogroup, and RRV, MGVMm, MneRV2, MGVMn, MGVMf, ChRV2, and MndRHV2
belong to RV2. Data obtained here from great apes extend the existence
of the two phylogenetically distinct groups of 2-herpesviruses to
the chimpanzees and demonstrate the existence of sublineages, within
RV1 and RV2, of both Old World monkey and ape rhadinoviruses.
Regarding the comparison of the serological results for KSHV with the PCR detection of the new viruses, it is difficult to provide convincing epidemiological findings. This is because of the limited series of animal tested (40 by serology and 31 by nPCR), but also the fact that there is no serological assay specific for any of these new viruses. However, there is an overall good concordance, as seen in Table 1, between the serological results and the presence of PanRHV1a or PanRHV1b because 12 of 13 PCR positive samples were found in KSHV seropositive animals, but only one PCR positive DNA was detected among the 8 seronegative individuals.
We found that among the PanRHV1a-infected chimpanzees, there were 3 P. t. vellerosus and 4 P. t. troglodytes, and among the PanRHV1b-infected animals, there were 3 P. t. vellerosus and 3 P. t. troglodytes (Table 1). This indicates that for PanRHV1 viruses there is no specific association between a peculiar virus strain (1a or 1b) and a chimpanzee subspecies. Nevertheless, among the PanRHV2-infected chimpanzees, there were only 3 Pan t. troglodytes individuals. These data indicate a possible species specificity for this new virus, but such preliminary findings need to be confirmed on a larger series of animals.
It is worth noting that several of these animals, despite living in
close contact in the same enclosure for several months or years, harbor
different viruses. For example, PanCamDja and PanCamJac were housed in
the same enclosure for 5 yr before being tested and they harbor
different gammaherpesviruses. This suggests that viral infection took
place before their arrival in the rescue center. Mother-to-offspring
transmission is a possibility that has been suggested for KSHV in
highly endemic areas of Central and East Africa (Plancoulaine et al. 2000).
Such data, taken as a whole, indicate that Central African great apes
constitute an important reservoir of novel 2-herpesviruses. Although
there are no available data supporting this hypothesis, the close
identity of these viruses with their human pathogenic counterpart KSHV,
their presence in peripheral blood mononuclear cells, and the high
genetic relationship between apes and humans indicate that they are
potentially transmissible to humans.
Regarding the diseases associated with 2-herpesviruses, RFHVMm and
RFHVMn have been identified in retroperitoneal fibromatosis, a vascular
fibroproliferative neoplasm with many morphological and histological
similarities to Kaposi's sarcoma (Rose et al. 1997). RRV has also been
isolated from simian immunodeficiency-virus-infected macaques with
lymphoproliferative disorder reminiscent of multicentric Castleman's
disease (Searles et al. 1999). Although the four 2-herpesviruses found in chimpanzees and gorillas are closely related to their human
pathogenic counterpart KSHV, no clinical pathology has yet been
identified in association with infection. The question therefore remains as to whether there is any disease associated with these novel
herpesviruses in their natural hosts and especially in the case of
multiple infection by SIVcpz, PanRHV1a, and PanRHV2 as observed in one
wild-caught animal of our series. Followup of both experimentally
HIV-infected chimpanzees (Greensill et al. 2000a) and of naturally
SIV-infected animals may therefore provide some important clues
regarding the physiopathology and natural history of infection
by these novel 2-herpesviruses. Efforts are ongoing to establish a
cell culture system for the propagation and extensive characterization
of these viruses, which may allow the comparison with KSHV strains.
In the RV2 genogroup, only one viral sequence PanRHV2 has yet been
identified among great apes. This sequence branches off alone in the
RV2 group, independently of the Old World monkey viral strains,
indicating that this sequence may represent the prototype strain of a
great ape lineage within this group. These comparative data, obtained
for all the nonhuman primate species, raise the possibility of the
existence of another 2-herpesvirus, belonging to the RV2 lineage, in
humans, in which only KSHV, belonging to the RV1 genogroup, has been
identified to date. The identification of novel RV2 2-herpesvirus
sequences in other nonhuman primate species and the generation of new
consensus degenerate primers targeted to the Herpesviral DNA polymerase
may be helpful in the detection and identification of this putative
human RV2 herpesvirus. Furthermore, the use of degenerate and consensus
primers derived from all the primate -herpesvirus polymerase genes,
including the four novel ones recently described, will allow us to
understand the full extent of the natural infection by these viruses
among great apes and the frequency of the eventual zoonotic
transmission to humans. This virus hunt could be initially focused on
captive or free living great apes and on persons at high risk through contact with such animals, including personnel of zoos and animal centers, as well as hunters and their relatives in Central Africa.
The dogma has always been that herpesviruses have diversified from a
common ancestor, in a manner mediating cospeciation of herpesviruses
with their host species through latent infection. Indeed, analyses of
our phylogenetic results strongly support the notion of host-linked
evolution of the 2-herpesviruses, at least for the RV1 genogroup,
because chimpanzees and gorillas, the nonhuman primate species closest
to humans, are infected by the 2-herpesvirus homologs closest to
KSHV, the human 2-herpesvirus prototype.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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Animals
Blood specimens from 40 great apes including 28 chimpanzees and 12 gorillas were studied. The larger series comprises 27 wild-born animals
(23 chimpanzees and 4 gorillas), originating from different parts of
Cameroon, where they were originally kept as pets after their mothers
had been killed by hunters. They were then gathered in a wildlife
rescue center in the South West province of Cameroon, in which some of
them were kept in the same enclosure, often in close contact (Corbet et
al. 2000). The second group (three chimpanzees and two gorillas)
originated from the large animal center of the Centre International de
Recherches Médicales, Franceville (CIRMF) in Gabon (Georges-Courbot
et al. 1996). The other animals came from two different zoos in France,
five gorillas and two chimpanzees from La Palmyre Zoo (kindly provided
by T. Petit) and one gorilla from Saint Martin la Plaine Zoo (kindly
provided by P. Thivillon). For all the animals from France, except one
(Gorph682), only serum was available. All these great apes were
seronegative for simian immunodeficiency viruses/human immunodeficiency
viruses (SIV/HIV) except two animals from Cameroon (Cam 3 and Cam 4, named in our study PanCamDja and PanCamJac, respectively) from which a
SIVcpz has recently been isolated and characterized (Corbet et al.
2000). Great apes from the CIRMF exhibited an HTLV Western blot
seroindeterminate profile (Georges-Courbot et al. 1996), but all the 35 other animals were negative for HTLV-1/STLV-1 infection. Data in Table
1 concern only animals for which we had both serum and DNA.
KSHV Serological Analysis
All the plasma were tested, at a 1/40 dilution, for the
KSHV-specific IgG, using an immunofluorescence assay (KSHV IFA, ABI). This assay, using the KS-1 cell line as the KSHV source of antigens, detects antibodies directed against both latent and lytic KSHV antigens, and is well adapted to conduct epidemiological works (Chatlynne et al. 1998; Plancoulaine et al. 2000). This assay does not
detect any other human herpesviruses than KSHV.
DNA Extraction and Herpesvirus DNA Polymerase Gene Amplification
DNA was extracted from buffy coats with the QIAamp DNA Blood mini kit (QIAGEN) following the manufacturer's instructions.
Herpesvirus DNA polymerase gene sequences were amplified by consensus
heminested PCR based on a previously described method (Rose et al.
1997). We slightly modified the reported cycling conditions as : 10 min
at 94°C, 5 cycles of 30 sec at 94°C, 1 min at 60°C, 1 min at
72°C; followed by 30 cycles of 30 sec at 94°C, 30 sec at 46°C, 30 sec at 72°C. An extension of 10 min at 72°C was realized on the
last cycle (Perkin Elmer GeneAmp PCR system 9600 thermal cycler). The
initial round of PCR contained 500 ng of genomic DNA, 30 pmoles of
degenerate primers, 2 mM MgCl2, 0.2 mM each dNTP, 5 µL of
10× PCR buffer, and 0.5 µL of Taq Gold DNA polymerase in a volume of
50 µL; 1 µL of this reaction was used in the heminested reaction.
After two rounds of heminested PCR (GDTD1B and DFASA primers in the
initial round followed by GDTD1B and VYGA), 172-bp fragments (excluding
primer sequences) were obtained. Specific reverse primer (Pan2as) was
designed from these sequences (Table 2; Fig. 1) and used in heminested
PCRs with the primer DFASA to finally obtain a 476-bp fragment of viral DNA polymerase sequence (excluding primers).
Finally, we designed an additional primer (P2s) derived from a conserved amino acid motif within the DNA pol gene of herpesviruses to allow amplification of a longer DNA polymerase fragment (Table 2; Fig. 1). PCR cycling conditions were 10 min at 94°C, 30 cycles of 30 sec at 94°C, 30 sec at 52°C, 1 min at 72°C; followed by a final extension of 10 min at 72°C. After two rounds of heminested PCR (P2s and P2eas in the initial round then P2s and P2ias) a 870-bp fragment of viral sequence was obtained. Primers P2eas and P2ias were derived from the DFASA-GDTD1B sequences previously determined. For all experiments, stringent precautions against PCR contamination were taken. The amplification mixes were made in a special room physically separated from the laboratory, and at least two negative controls (mix, water, or cellular DNA prepared from a KSHV-negative sample) were included.
Specific PanRHV2 DNA Polymerase Gene Amplification
To determine the PanRHV2 prevalence, PanRHV2-specific PCR primers were designed from alignments of PanRHV2 sequences obtained by the degenerate DFASA-GDTD1B PCR procedure. PanRHV2-specific PCR outer primer pair Pp2es and Pp2as generates a 312-bp product, whereas the inner primer set Pp2is and Pp2as (Table 2; Fig. 1) amplifies a 274-bp product. Template volume and reagent concentrations were identical to the DNA pol consensus assay, and PCR conditions were 10 min at 94°C, 30 cycles of 30 sec at 94°C, 30 sec at 53°C, 30 sec at 72°C; and a final extension of 10 min at 72°C. Nested reactions used 2% of primary reaction product as template with the reaction component concentrations and PCR cycling conditions identical to the primary reaction.
Mitochondrial DNA (mtDNA) Amplification
Single-round PCR amplification and sequence analysis, without
interim cloning, of chimpanzee mitochondrial (mt) DNA was performed on
a 498-bp segment of the mitochondrial D-loop control region (corresponding to position 15998-16497 of the human mitochondrial sequence; Anderson et al. 1981) from PBMC DNA as previously described (Gao et al. 1999; Corbet et al. 2000). The sequences of the primers used for chimpanzee mtDNA amplification are given in Table 2.
Southern Blot Analysis
Nested PCR products (VYGA-GDTD1B or Pp2is-Pp2as) were
size-fractionated by 1.5% agarose gel electrophoresis. Following
electrophoresis, gels were incubated for 30 min in 0.5 M NaOH-1.5 M
NaCl and then for 30 min in 3 M sodium acetate (pH 5.0), after which
they were transferred overnight by capillarity onto Biodyne A nylon
membranes (Pall Corporation). DNA was cross-linked to the membranes by
exposure to UV light in a UV Stratalinker (Stratagene Cloning Systems) and incubated for at least 6 h in hybridization buffer containing 6×
Saline Sodium Phosphate EDTA (SSPE), 0.1% SDS, 5× Denhardt's solution, 50% deionized formamide, and 100 µg/mL of fragmented salmon sperm DNA at 42°C (prehybridization). Hybridizations were performed in the same buffer after the addition of the
[32P]dATP end-labeled internal corresponding probes. The
hydridized membranes were washed first for 1 h in 2× SSPE and 0.1%
SDS and then for 15 min in 0.2× SSPE and 0.1% SDS at temperatures
ranging from 45°C to 65°C, depending on the probe used. Washed
membranes were exposed to phosphor screens and analyzed in a
Phosphorimager (Molecular Dynamics, Amersham-France SA). The sequences
of the probes are given in Table 2.
Cloning, DNA Sequencing, and Phylogenetic Analyses
The TA cloning procedure, DNA sequencing, as well as the
phylogenetic tree constructions using the PHYLIP package have been described previously (Lacoste et al. 2000a, 2000c).
Regarding the names of the new primate herpesvirus described in this paper, we have tentatively and provisionally named it PanRHV2 for Pan Rhadino-herpesvirus 2. However, among the specialists in the field, there is discussion and some debate about a new proposal for primate Rhadinovirus nomenclature. When new names are approved by their consensus, we will naturally modify the names of these new herpesviruses.
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ACKNOWLEDGMENTS |
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V.L. is a fellow of the CANAM. This work was partly supported by grants from the Agence Nationale de Recherches sur le SIDA (ANRS), SIDACTION, Association de Recherches sur le Cancer (ARC), and Action Concertée from the network of the Institut Pasteur. We acknowledge Peter Jenkins and Liza Gadsby (the Pandrillus Directors) for their great help in obtaining some of the blood samples studied and their continuous interest in this work. We also thank Thierry Petit from la Palmyre Zoo and Pierre Thivillon from the Saint Martin la Plaine Zoo for providing some of the studied samples.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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FOOTNOTES |
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5 Corresponding author.
E-MAIL agessain{at}pasteur.fr; FAX 33 0 140-61-34-65.
Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.158601.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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Received February 14, 2001; accepted in revised form June 4, 2001.
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  B. Ehlers, G. Dural, N. Yasmum, T. Lembo, B. de Thoisy, M.-P. Ryser-Degiorgis, R. G. Ulrich, and D. J. McGeoch Novel Mammalian Herpesviruses and Lineages within the Gammaherpesvirinae: Cospeciation and Interspecies Transfer J. Virol., April 1, 2008; 82(7): 3509 - 3516. [Abstract] [Full Text] [PDF]
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V. Lacoste, E. J. Verschoor, E. Nerrienet, and A. Gessain A novel homologue of Human herpesvirus 6 in chimpanzees J. Gen. Virol., August 1, 2005; 86(8): 2135 - 2140. [Abstract] [Full Text] [PDF]
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S. Calattini, E. Nerrienet, P. Mauclere, M.-C. Georges-Courbot, A. Saib, and A. Gessain Natural simian foamy virus infection in wild-caught gorillas, mandrills and drills from Cameroon and Gabon J. Gen. Virol., November 1, 2004; 85(11): 3313 - 3317. [Abstract] [Full Text] [PDF]
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B. de Thoisy, J.-F. Pouliquen, V. Lacoste, A. Gessain, and M. Kazanji Novel Gamma-1 Herpesviruses Identified in Free-Ranging New World Monkeys (Golden-Handed Tamarin [Saguinus midas], Squirrel Monkey [Saimiri sciureus], and White-Faced Saki [Pithecia pithecia]) in French Guiana J. Virol., August 15, 2003; 77(16): 9099 - 9105. [Abstract] [Full Text] [PDF]
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D. Whitby, A. Stossel, C. Gamache, J. Papin, M. Bosch, A. Smith, D. H. Kedes, G. White, R. Kennedy, and D. P. Dittmer Novel Kaposi's Sarcoma-Associated Herpesvirus Homolog in Baboons J. Virol., July 15, 2003; 77(14): 8159 - 8165. [Abstract] [Full Text] [PDF]
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T. M. Rose, J. T. Ryan, E. R. Schultz, B. W. Raden, and C.-C. Tsai Analysis of 4.3 Kilobases of Divergent Locus B of Macaque Retroperitoneal Fibromatosis-Associated Herpesvirus Reveals a Close Similarity in Gene Sequence and Genome Organization to Kaposi's Sarcoma-Associated Herpesvirus J. Virol., May 1, 2003; 77(9): 5084 - 5097. [Abstract] [Full Text] [PDF]
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2-Herpesvirus of the Rhadinovirus 2 Lineage in Chimpanzees
