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Human herpesvirus 8 and Kaposi's sarcoma in the Amsterdam cohort studies.
Disease association, transmission and natural history
Renwick, N.M.
Publication date
2001
Link to publication
Citation for published version (APA):
Renwick, N. M. (2001). Human herpesvirus 8 and Kaposi's sarcoma in the Amsterdam cohort
studies. Disease association, transmission and natural history.
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mi i
Twoo distinct gamma-2 herpesviruses in African
greenn monkeys: a second gamma-2 herpesvirus
lineagee among old world primates?
JulieJulie Greensill,JulieA. Sheldon, NeilM. Bjmwick, Brigitte E. Beer, SteveSteve Norky, Jaap Goudsmit, And Thomas F. Schulp
Abstract t
Primatee gamma-2 herpesviruses (rhadinoviruses) have so far been found in humans (Kaposi's sarcoma-associated herpesviruss [KSHY], also called human herpesvirus 8), macaques (Macaca Spp,) (rhesus rhadinovirus [RRV] and retroperitoneall fibromatosis herpesvirus [RFHV]), squirrel monkeys (Saimiri sciureus) (herpesvirus saimiri), and spider mon-keyss (Ateles spp.) (herpesvirus ateles). Using serological screening and degenerate consensus primer PCR for the viral DNA polymerasee gene, we have detected sequences from two distinct gamma-2 herpesviruses, termed Cblorocebus rhadinovirus 1 (ChRVi)) and ChRY2, in African green monkeys. ChRVl is more closely related to KSHY and RFHV, whereas ChRY2 is closestt to RRV. Our findings suggest the existence of two distinct rhadinovirus lineages, represented by the K S H V / R F H V / C h R V ll group and the RRY/ChRY2 group, respectively, in at least two Old World monkev species. Anti-bodiess to members of the RRY/ChRV2 lineage may cross-react in an immunofluorescence assav for earlv and late KSHY an-tigens. .
Introduction n
K a p o s i ' ss sarcomaassociated herpesvirus ( K S H V ) , o r h u -m a nn herpesvirus 8 ( H H V 8 ) (9), is found in all clinical for-ms o ff K a p o s i ' s s a r c o m a , in primary effusion l y m p h o m a s (7,8,9),, and in s o m e cases o f multicentric C a s t l e m a n ' s dis-easee (14, 27). K S H \7 is currently classified as a m e m b e r of t h ee r h a d i n o v i r u s s u b g r o u p of g a m m a h e r p e s v i r u s e s (9, 23). R h a d i n o v i r u s e ss have b e e n f o u n d in m a n y species, including cattle,, mice, a n d b o t h O l d W o r l d and N e w W o r l d p r i m a t e s ( 1 ,, 2, 3 , 9, 11, 12, 17, 2 1 , 22, 25). Viruses that infect N e w W o r l dd m o n k e y s include herpesvirus saimiri (HVS), w h i c h infectss the squirrel m o n k e y , and herpesvirus ateles ( H V A ) , w h i c hh infects t h e spider m o n k e y (1, 2, 3). O t h e r t h a n hu-m a n s ,, the hu-m a c a q u e s o f Asia are the only O l d W o r l d p r i hu-m a t e speciess d o c u m e n t e d thus far to h a r b o r r h a d i n o v i r u s e s . R h e suss r h a d i n o v i r u s (RRV) is w i d e s p r e a d a m o n g rhesus m a -c a q u e ss {Ma-ca-ca mulatto), g r o w s well in tissue -culture (11), andd has b e e n fully s e q u e n c e d (25). Its g e n o m e organization iss very similar t o t h a t o f K S H V , with h o m o l o g u e s o f several potentiallyy p a t h o g e n i c K S H V g e n e s , including t h o s e for K l ,, v I L - 6 , v I R F s , a D - t y p e cyclin, v F L I P , G - p r o t e i n - c o u p l e dd receptor, and t h e m e m b r a n e p r o t e i n K 1 55 (16, 25). H o w e v e r , of t h o s e K S H V g e n e s that have b e e nn tentatively linked to pathogenicity, R R V lacks a K 1 2
homologuee and has only o n e v M I P , in contrast to the three inn K S H V (25). Because o f this extensive similarity b e t w e e n RRVV a n d K S H V , R R V has b e e n p r o p o s e d as the m a c a q u e equivalentt of K S H V , and m a c a q u e s infected with R R V are potentiallyy a suitable animal m o d e l for K S H V infection. Re-cently,, coinfection o f rhesus m o n k e y s with RRV and simian immunodeficiencyy virus (SIVm!,C2.w) was implicated in t h e inductionn of multicentric lymphoproliferative disorder, reminiscentt o f multicentric Castleman's disease (31).
However,, degenerate c o n s e n s u s primer PCR for the herpesviruss D N A polymerase and glycoprotein B genes has identifiedd o t h e r rhadinovirus sequences in macaques suffer-ingg from retroperitoneal fibromatosis (RF) (21). R F is a mesenchymall n e o p l a s m with vascular c o m p o n e n t s and is alsoo associated with simian retrovirus 2, which causes i m m u n o s u p p r e s s i o nn and an AIDS-like disease in m a c a q u e s (6,, 21, 28). Because of these features, RF in macaques had previouslyy b e e n suggested as an animal model for K S (28). Bothh t h e rhesus m a c a q u e and the pigtailed m a c a q u e {Macaca
nemestrind)nemestrind) h a r b o r R F herpesviruses ( R F H V M m and R F H V M n )) which a p p e a r to be m o r e closely related t o
KSHVV than K S H V is to R R V (5,21). In view o f reports t h a t
somee currently circulating KSHV strains may have under-gonee a recombination event with a related rhadinovirus (15, 18,, 19, 32), and the high prevalence of KSHV in sub-Saha-rann Africa (reviewed in reference 24), we decided to
investi-T oo determine the seroprevalence of KSHV-related viruses, wee studied sera from 78 captive African green monkeys (AGMs),, housed at the Paul-Ehrüch-Institut, Langen, Ger-many,, including three subspecies of Chlorocebus aethiops: C.
aetbiopsaetbiops aethiops (grivet), C. aethiopspygerytbrus (vervet), and C. aetbiopsaetbiops sabaeus (sabaeus). Some animals were naturally
in-fectedd with SIVAGM and simian T-lymphotrophic virus type 1.. None of the animals had been housed with species other thann AGMs at the institute. The sera were tested for cross-reactivityy with KSHV orf65/VP19 by enzyme-linked immunosorbentt assay (26) and for unspecified early and late antigenss by immunofluorescent antibody assay (IFA) (Ad-vancedd Biotechnology Inc., Columbia, Md.) (10). Sera from sixx animals (7.8%) reacted against orf65/VP19 alone, and seraa from 37 (47.4%) reacted in the lytic IFA. Sera from onlyy two (2.5%) animals reacted in both assays. Fifty sera weree also tested for antibodies to the latency-associated nu-clearr antigen (LANA) by IFA (13, 26), but none were posi-tive. .
Inn a separate study carried out in the Department of Human Retrovirologyy at the University of Amsterdam (N. Renwick ett al., unpublished data), 201 plasma or serum samples from
Cercopithecus,Cercopithecus, Chlorocebus, Miopithews, Cercocebus, Alacaca, Mandrillus^Mandrillus^ Colobus, Presbytis, Pan, Pongp, Callithrh, Cebus, luigothrix,luigothrix, Saguinus and Saimiri species were screened for
an-tibodiess to orf73 (LANA) or orf65/VP19 as previously de-scribedd (20). Three were found to have antibodies to combinantt orf65/VP19, seven had antibodies against
re-gatee the existence of KSHV-related viruses in African pri-mates. .
combinantt orf73 (LANA), and three had antibodies in both assays.. Positive animals included Chlorocebus aethiops,
ChlorocebusChlorocebus aethiops pygerytbrus, Cercopithecus albogularis, Cercopi-thecusthecus ascanius (two), Cercopithecus cephus, Cercopithecus mona, CercopithecusCercopithecus nictitans, Cercopithecus pogonias, Colobus guere^a, MacacaMacaca nemestrina, Macaca sylvanus, and Papio cynocephalus anubis. anubis.
Wee initially chose five animals (one vervet, three grivets, andd one unknown subspecies) from the Paul-Ehrlich-Institutt AGMs with cross-reactive antibodies against ORF65/VP199 and attempted to amplify herpesvirus D N A polymerasee sequences from peripheral blood mononuclear celll (PBMC) DNA (extracted with the Qiagen whole-blood kit)) by consensus PCR based on the method previously de-scribedd (21). We modified the reported primers after includ-ingg sequence from RFHVMm, RFHVMn, and RRV (11, 21, 25)) and designed an additional primer (SVGA) to allow am-plificationn of a longer DNA polymerase sequence fragment (Fig.. 1; Table 1). PCR cycling conditions were 94°C for 1 min,, 55°C for 1 min, and 72°C for 1 min, for 30 cycles. Af-terr three rounds of heminested PCR (GDTD1B and DVYGAA in the initial round, followed by G D T D 1 B and DPCLNA,, followed by GDTD1B and DKMLEA [Fig. 1; Tablee 1]), a fragment of 88 bp (excluding primer sequence) withh 48.9% nucleotide identity with KSHV was obtained fromm animal Z8, a vervet monkey. Specific reverse primers (Z8revll and Z8rev2 [Fig. 1; Table 1]) were designed from thiss sequence and used in nested PCRs with the primer
TABLEE 1. Sequences of primers used for consensus and virus (ChRV1 and ChRV2)-specific PCR for DNA polymerase
Primer r Orientationn 5' 3' sequence3
Degenerate e
GDTD1B
C CDQAHNA" "
DVYGA
d dDPCLNA
d dDKMLEA" "
SVCA A
ChRV11 specific Z8F1 1 Z8F2 2 Z8R1 1 Z8R2 2 Z8rev1 1 Z8rev2 2 Z8rev3 3 ChRV22 specific L1F1 1 L1F2 2 L1R1 1 L1R2 2 L1rev1 1 CGGCATGCGACAAACACGGAGTCNGTRTCNCCRTA A CCCAGTATCATNCARGCRCACAA A ACMTGTAACGCGGTKTACGGSTTYACVGG G GTCGCCTCTGGCATCCTVCCDTGCMTNAA A CAGGGCAGGAARATGCTGGARACRTCNMAGGC C CCAGYGTNTGYGTKAACG G ACGATGTTATTTCTACACCCGCG G GAATCTAACGTCGACGCGACCAG G CTCATGGACCTCAAACACGGATC C GCTGGAGGTCTTCCCATCCGC C TGAAGTGGGCTTCTGTCC C TCTGTCTTTGCAATAGGTGGC C AGTGTGGAGTAACAGAGG G CCGGACGGGACCTGCACCTG G TACGAAACGTTCGCGCTCAGCG G ACGCGGAATCTCGCGCCGGG G GCAGCTCGTCCGGGGTCAGG G ACGACGCGGAATCTCGCGCCGGG G Position n Start t 13643 3 13133 3 13409 9 13439 9 13490 0 11784 4 11812 2 12050 0 12607 7 12200 0 13602 2 13573 3 13171 1 13178 8 13221 1 13605 5 13552 2 13602 2 inn KSHV qenoiW End d 13610 0 13154 4 13437 7 13467 7 13521 1 11801 1 11834 4 12072 2 12584 4 12180 0 13584 4 13562 2 13155 5 13197 7 13242 2 13585 5 13533 3 13585 5 NN indicates A, G, C, or T; R indicates A or G; M indicates A orC;; K indicates G or T; S indicates G or C; Y indicates C or T; V indicatess C, G, or A.
Nucleotidee numbers as in reference 23. Positions for
virus-specificc primers correspond to equivalent residues in the KSHVV genome.
Describedd in reference 21.
Modifiedd from primers described in reference 21.
Degenerate e primers s SCVA A AVYGA A APCLNA A AQAHNAA AKMLEA GDTD1B ChRVl l specificc primers Z8F1 1 Z8F2 2 Z8R1 1 Z8rev3 3 Z8R2 2 Z8revl l <--Z8rev2 2 ChRV2 2 specificc primers L1F1 1 L1F2 2 LlRl/Llrevt t L1R2 2 -0.5Kbp p
FIG.. 1. Positions of primers for consensus and virus-specific (ChRVl and ChRV2) PCR for DNA polymerase.
D Q A H N AA to obtain 454 bp of viral sequence (excluding primers).. Another heminested PCR, using Z8rev2, a new specificc primer Z8rev3, and SVCA (Fig. 1; Table 1) then yieldedd a further 1,348 bp of viral sequence. After all se-quencess were assembled, 1,802 bp (excluding primers) were obtainedd for this virus, termed Chlorocebus rhadinovirus 1 (ChRVl).. A similar screen by consensus PCR of another 21 PBMCC DNA samples from animals with cross-reactive an-tibodiess in the IFA and 7 with no evidence of cross-reactiv-ityy yielded a markedly different herpesvirus DNA polymer-asee sequence from a seronegative monkey, LI. The virus wass termed ChRV2. By using a PCR strategy similar to that usedd for ChRVl, but with ChRV2-specific primers (Llrevl andd L1R2 [Fig. 1; Table 1]), 454 bp of sequence (excluding primers)) was determined for ChRV2.
AA comparison of nucleotide and amino acid identities amongg primate rhadinoviruses (Table 2) indicates that
ChRVll is most closely related to RFHVMn, RFHVMm, andd KSHV (72.2, 68.5, and 70.9% nucleotide identity, re-spectively).. It appears to be least closely related to the two Neww World rhadinoviruses HVS and HVA (60.6 and 61.3% nucleotidee identity), with an intermediate degree of related-nesss to RRV and ChRV2 (63.5 and 65.4% identity) (Table 2).. When ChRV2 is compared with all these viruses, the samee three groupings are apparent, with ChRV2 being clos-estt to RRV (84.1% nucleotide identity), most distant from HVSS and HVA (55.7 and 52.9%o nucleotide identity), and thee K S H V / R F H V / ChRVl group in an intermediate posi-tionn (60.6 to 65.9%o nucleotide identities). The same three groupingss are also seen when the GC content is tabulated and,, to some extent, also forthe CpG ratios of individual vi-rusess (Table 3).
Ann alignment of the amino acid sequence corresponding to thee 1,802-bp fragment is shown in Fig. 2 and reveals
se-Tablee 2. Nucleotide and amino acid identities between ChRV1, ChRV2, and other gammaherpesviruses. .
%% Identity with3
Viruss QhRVI ChRV2 Nucleotidee Protein Nucleotide Protein
KSHV V ChRV1 1 ChRV2 2 RFHVMm m RFHVMn n RRV V HVS S HVA A EBV V 70.9 9 65.4 4 68.5 5 72.2 2 63.5 5 60.6 6 61.3 3 59.1 1 (66.6) ) <ND) ) <ND) ) (ND) ) (59.9) ) (50.6) ) (49.5) ) (50.1) ) 79.5 5 70.9 9 81.5 5 82.8 8 68.2 2 64.2 2 64.9 9 57.6 6 (78.3) ) (ND) ) (ND) ) (ND) ) (67.7) ) (61.9) ) (61.6) ) (67.6) ) 65.9 9 60.6 6 60.8 8 63.0 0 84.1 1 55.7 7 52.9 9 60.1 1 76.2 2 70.9 9 70.2 2 72.2 2 88.7 7 70.9 9 68.2 2 57.6 6
aa Numbers refer to values obtained in a comparison of the 454-bp fragment, which is
availablee for all viruses. Numbers in parentheses indicate identity for the 1,802-bp fragment,, which is not available for ChRV2, RFHVMm, and RFHVMn.
ND,, sequence not determined.
Tablee 3. GC-content and CpG dinucleotide frequencies in DNA polymerase fragments from gammaherpesviruses. . GCC content (%) CpG ratio3 Virus s KSHV V ChRV1 1 ChRV2 2 RFHVMm m RFHVMn n RRV V HVA A HVS S EBV V 1,802-bp p equivalent t 53.9 9 54.1 1 NDÖ Ö ND D ND D 59.0 0 36.2 2 35.2 2 62.6 6 454-bp p equivalent t 54.3 3 54.5 5 63.8 8 51.0 0 55.9 9 59.8 8 38.9 9 40.0 0 64.9 9 1,802-bp p equivalent t 0.80 0 0.96 6 ND D ND D ND D 1.18 8 0.45 5 0.29 9 0.70 0 454-bp p equivalent t 0.91 1 0.93 3 1.15 5 1.13 3 0.96 6 1.25 5 0.29 9 0.28 8 0.73 3 aa
Observed frequency/expected frequency of CpG dinucleotides for the mono-nucleotide composition.. (Expected frequency = proportion C x proportion G x
[sequencee length -1].)
quencee motifs shared by ChRV2 and RRV or the K S H V / R F H V // ChRVl group. By maximum Hkelihood analysiss of the 1,802-bp DNA sequence (which is not avail-ablee for ChRV2 and RFHVMm/RFHVMn) (Fig. 3a), ChRVll appears most closely related to KSHV, followed by RRVV and, in a separate branch, by the New World rhadinovirusess HVS and HVA. The same branching order wass obtained with other phylogenetic methods, including neighborr joining analysis and parsimony (not shown), and supportedd by high bootstrap values (Fig. 3a). Similar analy-sess were carried out on the 454-bp DNA polymerase frag-mentt available for other rhadinoviruses, including ChRV2 andd RFHVMm/RFHVMn. A neighbor joining tree of the correspondingg 151-amino-acid protein sequence (Fig. 3b) illustratess the existence of three separate lineages among rhadinoviruses,, one for the New World viruses HVS and HVAA and two for the Old World viruses. In spite of being derivedd from different species living, respectively, in Asia andd Africa, RRV and ChRV2 cluster together, as do KSHV, ChRVl,, and RFHVMm/RFHVMn. Furthermore, viruses fromm the same species (ChRVl and ChRV2 from AGMs andd RRV and RFHVMm from rhesus macaques) group separatelyy from each other. This strongly suggests that at leastt two Old World primate species harbor two fairly dis-tinctt rhadinovirus lineages. In humans, one of these lin-eagess is represented by KSHV, whereas the other lineage currentlyy lacks a known representative.
Furtherr specific primers were designed (Z8F1, Z8F2, Z8R1,, and Z8R2 for ChRVl and L1F1, L1F2, and L1R1 [withh L1R2] for ChRV2 [Fig. 1; Table 1]) and used to screen D N AA from PBMCs of 68 AGMs for the presence of either virus.. PCRs were repeated to ensure the veracity of results, inn some cases on new aliquots of PBMCs. Six other mon-keyss were found to be infected with an ChRVl -like virus; an animall from the Amsterdam panel (a redtailed monkey,
CercopithecusCercopithecus ascanius) and five AGMs from the
Paul-Ehrlich-Institutt (two grivets and three unknown sub-species).. Hence, ChRVl-like viruses appear to be infre-quentt among captive African monkeys, or they are difficult too detect because of low viral load. In this, they resemble the otherr members of this lineage, KSHV and RFHVMm/RFHVMn;; KSHV is detected in only approxi-matelyy 10 to 20% of serologically reactive individuals (4, 24, 30),, although concurrent human immunodeficiency virus typee 1 infection, or iatrogenic immunosuppression, mark-edlyy increases the risk of developing Kaposi's sarcoma in KSHV-infectedd individuals (4, 26, 30), and RFHVMm-RFHVMnn is detected only in animals with RF orr after immune suppression (6). Sequence analysis of the PCRR products obtained from these animals with the ChRVll diagnostic primers (Z8F2 and Z8R2; 107 bp of se-quence,, excluding primers) indicated that the sequences ob-tainedd from the six Chlorocebus aethiops animals, Z8 and five otherr positive animals, were identical. PCRs were repeated forr these animals, with the same sequence being deter-mined.. The sequence from the more distantly related redtailedd monkey {Cercopithecus ascanius) (29) differed from ChRVll by 2 nucleotides (98.1% nucleotide identity), with thee substitutions A-G (KSHV nucleotide 12093 [23]), and G-AA (KSHV nucleoSVCAtide 12147), leading to Glu-Gly andd Gly-Asp substitutions, respectively. While more exten-sivee sequence comparisons among different cercopithecine speciess are required, this could indicate a higher degree of sequencee conservation among ChRVl-like viruses than amongg RFHVs in different macaque species (21). However, wee cannot currently exclude transmission of ChRVl among cercopithecinee monkeys in captivity. As determined with thee ChRV2 diagnostic primers (L1F1, L1F2, L1R1, and L1R22 [Fig. 1; Table 1]), 22 AGMs (33.3%) were PCR posi-tivetive for the ChRV2-type virus. This is again reminiscent of RRV,, the other member of the second lineage (Fig. 3), whichh is easily isolated from different macaques (11, 25); serologicall studies, using purified RRV as the antigen,
KSHV V ChRVl l RFHVMm m RFHVMn n ChRV2 2 RRV V HVA A HVS S EBV V SVCA A
• •
Z8F1 1 11•
FRQRCYFYTLL APQGVNLTHV LQQALQAGF* RR --P L-- V--AY*
*GRASCGFSTT EPVRKKILRA YDTQQYAVQK ITLSSSPMMR TLSDRLTT*C *—TP-A-T-- -L-K V KT-D-Y- VV A
AA*-QVV AK V-A-T-V--T -G-KII VK V-E-G-I-YL MK---G--NN VK V-E-G-I-YL IK---G-QAA AS LDVEFA VLS-KNTA* * NEKL* * NEK-* * K-STF F ** A *-*PR-SYQ-- *S*P--AYQ--DR-TP-RV-V V RR-N-R--KT T -L-KK SR -A-KK SR -K-TRRSIMG G --VAEHP-TEE GS-LS --PDEHE-F-- V-I HSVY --PEEHD-F-- V-V LSVY -GNHAGDYHKK HPNSVC VA*--KV—Y-VA*N N KI—S-VS*N N HVATW-QDKH H KSHV V ChRVl l RFHVMm m RFHVMn n ChRV2 2 RRV V HVA A HVS S EBV V 1711 260 IIQISCVLHTT VGNDKPYTR* MLLGLGTCDP LPGVEVFEFP SEYDMLAAFL SMLRDYNVEF ITGYNIANFD LPYIIARATQ VYDFKLQDFT
-LL F — A-DKS Q* I--S E- -E-T-IL T--F A D L T —
NID-KN--F-TT TREGA-NPPN I-FSV I-DTD-L -FWQQ --TPDT*-KN S AA VEDS--Y -FWHH A-ALDT*--N S SA VENT--Y --WSS T-EEAGRY-R I--T ED IE Y VEDYY A-I--FE-D- L — AA HG-F TI F 1 —— I HG-F -LI--F 1 S--—— L Y--F QLI--LS--I V V L-T-- L-D-- -LD--
-S--— -S--—
— —
-RH H —NLR-NEY--I-NI—S-YS S I-NI—S-YS S I-SINPASLG G KSHV V ChRVl l RFHVMm m RFHVMn n ChRV2 2 RRV V HVA A HVS S EBV V KSHV V ChRVl l RFHVMm m RFHVMn n ChRV2 2 RRV V HVA A HVS S EBV V 3511 440 YCVIDSVLVMM DLLLRFQTHV EISEIAKLAK IPTRRVLTDG QQIRVFSCLL EAAATEGYIL PVPKGDAVSG YQGATVISPS PGFYDDPVLV__!! R M — ft R K — R — T G - 1 KM-MI--K — M F - KM-MI--K — — K--KF-MI-- NH-VI--A V Y G NH-VI--A . . -QA--APCLNAA . -D---AKMLEAA , --R-NF— — -RA-N N -RA-N N -QK-NF---X1R2 2 TPEGQG-- —SNNVNTD-- --SNDVNAD-- -M-SASDRD--N-II E--T-II NNA-N_ jj N N A -Q-LL S N S — ,L1R1/Llrevl l ,Z8rev2 2 531 1
LDKQQLAIKVV TCNAVYGFTG VASGILPCLN IAETVTLQGR KMLERSQAFV EAISPERLAG LLRRP* S G - S HH - - Q - Q * TT - G KD-SD - I Q - - * TT - G T A - - D — Q — * LL 1 - R - - T M-K - - L T - D E - Q T R - S - A * LL 1 - R - - T M-KSY- — L T T - D - R T R - G - E * LL I S T K - K - - I - E M T - V - - Q E IVPHK* LL XS T K - K I - I — M T - D T - Q E I V P H I * - Z 8 r e v ll 6io 'IDD VSPDARFKVI *-TT AGTE-H * —— A - - E *VTT AD-G R-V *VTT ARHG V *LNN H E H E K R *VKK HE K R -„GDTD1B B
- N - L F -- S S -AK-- -AN-QAA -APS-DAWAP LN-EGQLR—
Z8F2 2
811 ^
GCEVFESNVDD AIRRFVLDHG FSTFGWYECS NPAPRTQARD -TT RE A-T H-E — L S G —
-VV -T S-A RAT--L I--TT II-NN T-K SACF--TN —
TT I-ND T-K SAC—ITN----RI--AA -T ND -V S-R
RAI—L-H--Z8R2 2
^^ 170 SWTELEFDCSS WEDLKFIPER TEWPPYSILS FDIECMGEKG FPNATQDEDM
QQ G QLR Q RM-- A- --R--R V
AR-AA SVQAD- SD R-VA T--A- --C--R-G-A -Y-DII G YN--E-HA— N-MA 1 --C-KNEG-L -H-DII G YY--E-HAD- N-M- 1 --C-KNEG-L -YAA Y--E VG--SVRR-D SS--S-QA-A L — E - —T--NEA-L
2611 < ^ 5 k KIKTGSVFEVV HQPRGGSDGG NFMRSQSKVK ISGIVPIDMY -E-TT — **-- G-L A 11 -E **-- G V--I- -A RV-SS — I-Q- -M-KDM**-N G V--I- IA RVV I-Q- -T-KDT**-N G V--I- IA --RA-G-C—— RR-HDA**-K G-L-ANT—R
-T-LI-4 -T-LI-4 11 AQAHNA Z8rev3 L1F1
VDFASLYPSII IQAHNLCYST LIPGDSLHLH PHLSP*DDY: QGA-PA-- -E-R-*-— Q - N A I L S -- -E-T-*N —T-SA--G-- -E-T-*-RDD -D-T-*-M_H-RDD -N-T-*-HNN NY -Y-K-*S H H A_ _Nyy K_ *s M-TPGEE-RLL AG-R-GE 350 0 QVCREKLSLSS DYKLDTVAKQ CLGRQKDDIS YKDIPPLFKS GPDGRAKVGN
DD K -R R- -AA I-R I - - K D D I — K D D -- A D L1F2 2 :: TFVLSGGPVH - - H H - - HH T — — M — SS — I M — S — I SS R T — V Y -R --NN DH NN NH RH H FVKKHKRESL L
~ ~ ^ ~ ~ ~
IQTT — I Q A - --V H - - F F G K - E - V - I - A K - E - V - I - A K - E - V --L - - A K - E - V H H LAKLLTVWLA A - SS AT — S - SS T — T - SS T -GRR S -GRR E - SS S - S RR S - - SS S R--LL MM M-— E M-— R M-— A A A KRKEIRKTLA A D—R R N - R A - --— R A --— R R A - - Q K — — A — Q K — — A - K - L - --—— G S —— E I - R —— E I - L — E - - R R L - M M 5 3 ( ( SCTDPALKTI I — A — T M R - — G — T M R - — T — T M R - --A - D — S S A - D — S S QQ QDT EE LDT AARQR-Figure.. 2. Alignment of a 599- to 608-amino-acid fragment (primers SCVA and GDTD1B), using CLUSTALX (EMBL, Heidel-berg,, Germany), of ChRV1 with published rhadinovirus and Epstein-Barr virus sequences. The shorter sequences of ChRV2,, RFHVMm, and RFHVMn are also shown (primers AQAHNA and GDTD1B). Gaps for optimal alignment are indicatedd by asterisks. Amino acid residues identical to the sequence of KSHV are indicated by dashes. Undeter-minedd sequence is indicated by dots. Residues that aree conserved within the KSHV/RFHV/ChRVI lineage and within thee RRV/ChRV2 lineage are shown in bold. In addition, ChRV1 and KSHV both have a deletion relative to the other sequencess at position 190, where the sequence for the RFHVs is undetermined. Positions of degenerate and vi-rus-specificc primers are shown. The origin of sequences is as described in the legend to Fig. 3a.
B B
p-herpesvirus s P R UU I y1-herpesvirus Neww World y2-herpesvirus s Oldd World 2-herpesvirus sFiguree 3a. DNA maximum likelihood tree for the 1,802-bp fragment (primers SCVA and GDTD1B) of DNA polymerase. Sequences weree aligned by using CLUSTALX and analyzed by using the DNAML program (PHYLIP version 3.5c; J. Felsenstein and thee University of Washington). One hundred replica samplings were subjected to bootstrap analysis (SEQBOOT). The tree iss unrooted. Other sequences included and their EMBL accession numbers are as follows: HHV1/HSV1 (X04771), HHV2/HSV22 (M16321), HHV3/VZV (X04370), HHV4/Epstein-Barr virus (V01555), HHV5/HCMV (AF133589), HHV6A (X83413),, HHV7 (U43400), HHV8/KSHV (U75698), HVS (X64346), HVA (HVA3) (AF083424), RFHVMm (AF005479), RFHVMnn (AF005478), RRV (AF029302).
Figuree 3b. Neighbor-joining protein distance tree for the 151 amino acid residues encoded by the 454-bp fragment (primers DQAHNA andd GDTD1B) of DNA polymerase. Sequences were aligned by using CLUSTALX and analyzed by using the PROTDIST andd NEIGHBOR programs in PHYLIP. One hundred replica samplings were analyzed. The tree is unrooted.
foundd evidence for a high prevalence of the virus (~50% positivityy rate) (11). We have also recently detected ChRV2-likee sequences in 3 of 11 wild-caught Cercopithecus
monamona animals, which are quite closely related to C aethiops
andd Cercopithecus ascanius species (29; Greensill et al., unpub-lishedd data).
Wee investigated whether detection, by PCR, of ChRVl or ChRV22 correlated with the presence of cross-reacting anti-bodiess to KSHV, as determined by orf65/vpl9 ELISA or lyticc IFA. Among 66 AGMs (18 grivets, 4 vervets, 2 sabaeus,, and 42 unknown) also tested by PCR, reactivity in
lyticc IFA was wide spread (37 of 66; 56%) but particularly highh in those in which ChRV2 could be detected by PCR (166 of 22; 73.7%), compared to those in which it was not (21 off 44; 47.8%; P = 0.054). N o such correlation was found be-tweenn reactivity in orf65/vpl9 ELISA and ChRV2 detec-tionn (0 of 22 ChRV2 PCR-positive animals had antibodies too orf65/VP19, whereas 5 of the remaining ChRV2 PCR-negativee animals had orf65/VP19 antibodies). ChRVll was detected in only seven animals in our study, so anyy correlation between antibody reactivity and infection withh the virus is difficult to resolve. However, of the seven ChRVll PCR positive animals, two had antibodies to
102 2
orf65/VP19,, three had antibodies detected in the lytic IFA, andd two were nonseroreactive. Of the remaining 73 ChRVl PCR-negativee animals, 10 had antibodies to orf65/VP19. Thee existence of two rhadinovirus lineages in at least two Oldd World monkey species could suggest the existence of a similarr situation in the great apes, and perhaps in humans. Thee correlation of PCR-detectable ChRV2 with reactivity in lyticc IFA is in accord with the concept that this assay, in par-ticularr when carried out at low serum dilutions, may detect antibodiess against a related virus. Whether this explains somee cases of lytic IFA reactivity in humans where KSHV infectionn could not be confirmed by other, more specific as-sayss remains to be seen. Evidence for recombination of the righthandd end of the KSHV genome with a related rhadinoviruss has recently been reported (15,18,19,32). It is conceivablee that this could have occurred as the result of a coinfectionn in humans with two related viruses. In this studyy we have found two examples, with an AGM from Paul-Ehrlich-Institutt colony and the Amsterdam red-tailed
monkey,, of coinfection with representatives of the ChRVl andd ChRV2 groups.
Iff the existence of two separate lineages of gamma-2 herpesvirusess in different primate species is confirmed by moree extensive sequence analysis it may be necessary to de-rivee a nomenclature that distinguishes between these two branches. .
Nucleotidee sequence accession numbers. The 1,802-bp sequencee of ChRVl and the 454-bp sequence of ChRV2 havee been deposited in GenBank under accession no. AJ2515733 and AJ251574, respectively.
Acknowledgement t
Wee are grateful to Helen Williams for technical assistance andd to D. Ablashi of Advanced Biotechnologies Inc. for providingg the lytic IFA kits.
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