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Dispatch
Novel γ-2-Herpesvirus
of the Rhadinovirus 2 Lineage in Gibbons
Renan Duprez,* Emmanuelle Boulanger,* Yannick Roman,† and Antoine
Gessain*
*Institut Pasteur, Paris, France; and †Museum National d’Histoire Naturelle,
Clères, France
Suggested citation
for this article:
Duprez R, Boulanger E, Roman Y, Gessain A. Novel γ-2-herpesvirus
of the Rhadinovirus 2 lineage in gibbons. Emerg Infect Dis [serial
on the Internet]. 2004 May [date cited]. Available from: http://www.cdc.gov/ncidod/EID/vol10no5/03-0964.htm
We obtained 475
nucleotides of the DNA polymerase gene of a novel human herpesvirus
8 homolog sequence in a gibbon. The finding of this new gibbon virus,
which clusters with a related chimpanzee virus in the rhadinovirus 2
genogroup, suggests the existence of a novel γ-2-herpesvirus in
humans.
Among Gammaherpesvirinae, Epstein-Barr virus (EBV) and Kaposi
sarcoma–associated herpesvirus or human herpesvirus 8 (KSHV/HHV-8) represent
human prototypes of the Lymphocryptovirus (γ-1-herpesvirus)
and Rhadinovirus (γ-2-herpesvirus) genuses, respectively
(1). Both viruses play an important role in human multistep
carcinogenesis, especially in immunodeficient patients. Indeed, Kaposi
sarcoma and EBV-associated Burkitt lymphoma currently represent the most
frequent cancers in some geographic areas, especially in Africa.
Several γ-1-herpesviruses have been isolated from nonhuman primates,
especially African and Asian apes, since the 1970s (2,3).
However, most of the sequences of these viruses, which allow phylogenetic
studies, have only been recently obtained (4).
Rhadinoviruses (or γ-2-herpesviruses) have also been found in many
animal species including New and Old World primates. Among the latter,
recent comparison and phylogenetic analyses of all available sequences
support the existence of two distinct genogroups called RV1 and RV2 for
Rhadinovirus genogroups 1 and 2 (5–9).
Among African great apes, four γ-2-herpesviruses have recently
been discovered. These are PanRHV1a/PtRV1 (Pan rhadinovirus 1a/Pan
troglodytes rhadinovirus 1), PanRHV1b (Pan rhadinovirus 1b) and GorRHV1
(gorilla rhadinovirus 1) from chimpanzees and gorillas respectively, in
the RV1 group and PanRHV2 (Pan rhadinovirus 2) from chimpanzees in the
RV2 group (7,10,11). By contrast, γ-2-herpesvirus
has never been found, so far, in Asian apes. The goal of this study was
therefore to search for γ-2-herpesviruses in Asian apes.
To look for KSHV-related viruses, we first performed a serologic analysis.
Plasma of 38 captive Asian apes, including 30 orangutans (25 Pongo
pygmaeus pygmaeus and 5 P. p. abelii) and 8 gibbons (including
1 Hylobates gabriellae and 7 H. leucogenys), originating
from different zoos and primate centers mainly from France, were tested
by two different immunofluorescence assays, as previously described (12,13).
Briefly, the first method, which uses KSHV-infected human KS-1 cells as
source of antigens, allows mainly the detection of antibodies directed
against lytic KSHV antigens, while the second, using KSHV-infected BC3
cells, allows only for the detection of antibodies directed against the
latent nuclear antigen. Results demonstrated a clear antilytic cross-seroreactivity
in plasma samples from 10 orangutans and 3 gibbons with titers from 1/20
to 1/160, while seroreactivity against the latent nuclear antigen was
more rarely detected (7 orangutans and 2 gibbons), mostly with very faint
patterns.
We then performed a polymerase chain reaction (PCR)-based study using
high molecular weight DNA extracted either from peripheral blood mononuclear
cells or from buffy-coat of 35 of these Asian great apes (8 gibbons and
27 orangutans for which DNA was available). We attempted to amplify a
small fragment of the highly conserved herpesvirus DNA polymerase gene
by heminested PCR, using previously described consensus-degenerate primers
(7,8,14). Among the 35 DNA samples,
18 scored positive (5 gibbons and 13 orangutans) on the ethidium bromide
gel, with a band of the expected size (237 bp), corresponding to the KSHV-positive
control. Purifying, cloning, and sequencing of these products indicated
the presence, in 8 orangutans and 4 gibbons, of two species-specific lymphocryptovirus
sequences, that were slightly different from each other but very closely
related to EBV, as recently reported (4). However, in
1 H. leucogenys (gibbon 7), database searches, using BLAST web
server (available from http://www.ncbi.nlm.nih.gov/blast/Blast.cgi),
indicated a novel γ-herpesviral DNA polymerase sequence that was
closely related to the Rhadinovirus genus strains. No other novel
herpesviral sequence was detected in orangutans or gibbons.
Using the same PCR approach with a specific reverse primer for the second
PCR (HyloRHVas: CAT CGT GCG TCC CTG CAG CG), we amplified, from the DNA
sample of gibbon 7, a second overlapping fragment, resulting in a 475-bp
final fragment of a new herpesviral DNA polymerase gene (GenBank accession
no. AY465375). Nucleotide comparison of a 451-bp fragment, corresponding
to the best alignment of all the γ-herpesviruses available sequences,
indicated that the novel gibbon rhadinovirus sequence was more closely
related to the corresponding sequences of the RV2 genogroup viruses (76%,
73%, and 71% of nucleotide identity with ChRV2 (Chlorocebus rhadinovirus
2), MndRHV2 (Mandrillus rhadinovirus 2) and PanRHV2, respectively), than
to the corresponding sequences of the RV1 genogroup viruses (70%, 69 %,
and 63% of nucleotide identity with KSHV, PanRHV1a, and PanRHV1b fragments,
respectively). Similar results were obtained when the comparison was done
on the amino acid sequences.
Phylogenetic analyses, by using two different methods (neighbor joining
and DNA maximum parsimony), were performed with two different sets of
sequences. The first set comprises all available primate γ-herpesvirus
DNA polymerase gene sequences. The second set comprises most of the available
corresponding herpesvirus sequences, including the g-2-herpesviruses originating
from nonprimates species. Nearly identical tree topologies were obtained
for the two phylogenetic methods when the analyses were restricted to
the primate sequences (Figure 1; data not shown).
The addition of the γ-2-nonprimate herpesviruses (Figure
2) modified the positioning of some sequences within the γ-2
herpesviruses and some bootstrap values as compared to the analysis restricted
to primate viruses. However, all analyses clearly localized, with bootstrap
values from 83% to 85%, the novel gibbon viral sequence (HyloRHV2) within
the Rhadinovirus genus in the RV2 genogroup (Figures
1 and 2; data not shown). All together, these
studies as well as previous works, demonstrate the existence, among the
primate γ-2-herpesviruses, of three distinct separate lineages,
as seen on Figure 1. The first lineage corresponds
to the New World group, including rhadinoviruses of spider and squirrel
monkeys. The second lineage, the RV1 group, comprises the rhadinoviruses
of Old World primates, including those of humans, chimpanzees, gorillas,
African green monkeys, mandrills, and macaques. The third lineage, which
corresponds to the RV2 group, also contains rhadinoviruses of Old World
nonhuman primates (chimpanzees, African green monkeys, macaques, baboons,
mandrills, and our novel gibbon HyloRHV2). The novel HyloRHV2 sequence
clearly localized with the PanRHV2 strain, a recently reported strain
from chimpanzees, and formed a distinct genogroup within the RV2 clade,
supported by a bootstrap value of 85% (Figure 1).
These two sequences, which branch off alone in the RV2 genogroup, independently
of all other Old World monkey viral strains, may represent the prototype
strains of a great apes lineage similar to that found in the RV1 genogroup.
Based on the established view that herpesviruses have diverged from a
common ancestor, in a manner mediating cospeciation of herpesviruses with
their host species through latent infection, such findings reinforce the
hypothesis of a putative RV2-related herpesvirus in humans.
The prevalence of HyloRHV2 infection, in our series of gibbons, was determined
by Southern blot hybridization of heminested PCR products, with a specific
oligonucleotide probe (HyloRHVpr: TTA CGG CTT TAC TGG GGT GGC GAG). Only
one sample (gibbon 7) scored positive. Furthermore, we also developed
a nested PCR assay using specific primers targeted to the new HyloRHV2
strain (HyloRHVs: GCA TCC CTC CCT GAC AGA GAA TG, HyloRHVas: CAT CGT GCG
TCC CTG CAG CG). PCR products hybridization with the specific internal
oligonucleotide probe (HyloRHVpr), only detected the same positive sample.
We also tried to specifically amplify an RV2-related virus in our series
of DNA samples from orangutans using novel and specific primers targeted
to RV2 genogroup strains. None of the 27 DNA scored positive, a result
which might be explained either by the absence of an orangutan RV2-related
virus in these DNA samples or by its presence at a level not detectable
by our method, a situation similar to that observed for KSHV infection
in humans. Indeed, in PBMC DNA, PCR analyses detect KSHV viral fragments
in only 10% to 20% of KSHV-seropositive healthy persons (15).
In conclusion, our data demonstrated for the first time the existence
of a gibbon rhadinovirus. Furthermore, after the recent demonstration
of two distinct Rhadinovirus lineages within the common chimpanzees
(7), and based on the known molecular evolution of herpesviruses,
our findings of a novel RV2 virus in gibbon may suggest the possible existence
of a novel γ-2-herpesvirus in humans, belonging to the RV2 genogroup.
Acknowledgments
We thank Ernst Verschoor,
Klaus Eulenberger, Thierry Petit, and Jacques Rigoulet for their great
help in obtaining some of the orangutan blood samples; Vincent Lacoste
for critical review of this manuscript and continuous interest in this
work; and Laurent Meertens for advice regarding phylogenetic studies.
Renan Duprez and
Emmanuelle Boulanger are financially supported by la Ligue Nationale
Contre le Cancer and l’Agence Nationale de Recherche sur le SIDA (ANRS),
respectively.
Mr. Duprez is a
Ph.D. candidate with primary research interests in the clinical and
molecular epidemiology and physiopathology of γ-herpesviruses
in humans, particularly Kaposi sarcoma–associated herpesvirus clonality.
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