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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 date2001

Link to publication

Citation for published version (APA):Renwick, N. M. (2001). Human herpesvirus 8 and Kaposi's sarcoma in the Amsterdam cohortstudies. Disease association, transmission and natural history.

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Download date:29 May 2021

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

JJ Virol 2000; 14: 1572-7

93 3

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 KSHV/RFHV/ChRVll 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

Kaposi 'ss sarcoma-associated herpesvirus (KSHV), or hu-

mann herpesvirus 8 (HHV8) (9), is found in all clinical forms

off Kaposi 's sarcoma, in primary effusion lymphomas

(7,8,9),, and in some cases of mult icentric Cast leman's dis-

easee (14, 27). K S H \7 is currently classified as a member of

thee rhadinovirus subgroup of gammaherpesviruses (9, 23).

Rhadinovirusess have been found in many species, including

cattle,, mice, and both Old World and New World primates

(1,, 2, 3, 9, 11, 12, 17, 21, 22, 25). Viruses that infect N ew

Wor ldd monkeys include herpesvirus saimiri (HVS), which

infectss the squirrel monkey, and herpesvirus ateles (HVA) ,

whichh infects the spider monkey (1, 2, 3). O ther than hu-

mans,, the macaques of Asia are the only Old Wor ld pr imate

speciess documented thus far to harbor rhadinoviruses. Rhe-

suss rhadinovirus (RRV) is widespread among rhesus ma-

caquess {Macaca mulatto), grows well in tissue culture (11),

andd has been fully sequenced (25). Its genome organization

iss very similar to that of KSHV, with homologues of several

potentiallyy pathogenic K S HV genes, including those for

K l ,, v IL-6 , v IRFs, a D- type cyclin, vFLIP,

G-prote in-coupledd receptor, and the membrane protein

K155 (16, 25). However, of those K S HV genes that have

beenn tentatively linked to pathogenicity, RRV lacks a K12

homologuee and has only one vMIP, in contrast to the three

inn KSHV (25). Because of this extensive similarity between

RRVV and KSHV, RRV has been proposed as the macaque

equivalentt of KSHV, and macaques infected with RRV are

potentiallyy a suitable animal model for KSHV infection. Re-

cently,, coinfection of rhesus monkeys with RRV and simian

immunodeficiencyy virus (SIVm!,C2.w) was implicated in the

inductionn of multicentric lymphoproliferative disorder,

reminiscentt of multicentric Castleman's disease (31).

However,, degenerate consensus primer PCR for the

herpesviruss D N A polymerase and glycoprotein B genes has

identifiedd other rhadinovirus sequences in macaques suffer-

ingg from retroperitoneal fibromatosis (RF) (21). RF is a

mesenchymall neoplasm with vascular components and is

alsoo associated with simian retrovirus 2, which causes

immunosuppressionn and an AIDS-lik e disease in macaques

(6,, 21, 28). Because of these features, RF in macaques had

previouslyy been suggested as an animal model for KS (28).

Bothh the rhesus macaque and the pigtailed macaque {Macaca

nemestrind)nemestrind) harbor RF herpesviruses (RFHVMm and

RFHVMn)) which appear to be more closely related to

KSHVV than K S HV is to RRV (5,21). In view of reports that

94 4

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-

Too 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 re-

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 DNA

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 GDTD1B and

DPCLNA,, followed by GDTD1B and DKMLE A [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

Results s

95 5

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

GDTD1BC C

DQAHNA" " DVYGAd d

DPCLNAd d

DKMLEA" " 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

Positio n n

Star t 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

i nn KSHV qenoi W

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 or C;; 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.

96 6

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/Llrev t t

L1R2 2

-0.5Kbp p

FIG.. 1. Positions of primers for consensus and virus-specific (ChRVl and ChRV2) PCR for DNA polymerase.

DQAHNAA 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 KSHV/RFHV/ 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-

97 7

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].)

bb ND, sequence not determined.

quencee motifs shared by ChRV2 and RRV or the

KSHV/RFHV// 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

DNAA 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,

99 9

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 - V V A AA* -

QVV A K V-A-T-V-- T

-G-K II V K V-E-G-I-Y L MK- -

-G-- NN V K V-E-G-I-Y L IK- -

-G-Q AA A S LDVEFA VLS -

KNTA* *

NEKL* *

NEK-* *

K-STF F

** A

*-*PR-SYQ --

*S*P--AYQ --

DR-TP-RV- V V

RR-N-R--K T T

-L- KK S R

-A- KK S R

-K-TRRSIM G G

--VAEHP-T EE GS-L S

--PDEHE-F -- V- I HSVY

--PEEHD-F -- V- V LSV Y

-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 26 0

IIQISCVLH TT VGNDKPYTR* MLLGLGTCDP LPGVEVFEFP SEYDMLAAFL SMLRDYNVEF ITGYNIANF D LPYIIARAT Q VYDFKLQDFT

- LL F — A-DK S Q * I-- S E - -E-T-I L T-- F A D L T — NID-KN -

-F- TT TREGA-NPPN I-FS V I-DTD- L

-FWQQ --TPDT*-K N S A A VEDS-- Y

-FWHH A-ALDT*-- N S S A VENT-- Y

--W SS T-EEAGRY- R I-- T E D I E Y

VEDS-- YY A-I--FE-D - L S- -

—AA HG- F T I F 1 S- -

—— I HG- F -LI-- F 1 S- -

—— L Y-- F QLI--LS-- I V V

L-T --

L-D --

L-D --

-LD --

-S --

— — — — -RH H

—NLR-NEY--

I-NI—S-Y S S

I-NI—S-Y S S

I-SINPASL G 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 44 0

YCVIDSVLVMM DLLLRFQTHV EISEIAKLA K IPTRRVLTD G QQIRVFSCLL EAAATEGYIL PVPKGDAVSG YQGATVISPS PGFYDDPVLV

__!! R M — ft R K — R — T G- 1

KM-MI--

K — M F - K— —

K--KF-MI --

NH-VI --

AVYGA. .

-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

,Z8rev 2 2 531 1

LDKQQLAIK VV TCNAVYGFTG VASGILPCLN IAETVTLQGR KMLERSQAFV EAISPERLAG LLRRP*

SG-SHH - - Q - Q*

TT -G KD-SD - I Q - - *

TT -G T A - - D — Q —*

LL 1 - R -- T M-K - - L T - D E - QT R - S - A*

LL 1- R -- T M-KSY- — L T T - D - RT R - G - E*

LL I S T K - K - - I - E M T - V - - QE IVPHK *

LL XS T K - K I - I —MT-DT-QE I V P H I *

- Z 8 r e vll 6io 'I DD VSPDARFKVI

*- TT AGTE- H

* —— A - - E

*V TT AD-G R-V

*V TT ARHG V

*L NN H E H E - K - R --

*V KK HE K - R - -

„GDTD1B B

- N - L F -- S S -AK -- -AN-QAA -APS-DAWAP LN-EGQLR—

100 0

Z8F2 2

811 ^

GCEVFESNVDD AIRRFVLDHG FSTFGWYECS NPAPRTQARD

- TT R E A- T H- E — LSG —

- VV - T S- A RAT-- L

I-- TT II-N N T- K SACF--T N —

TT I-N D T- K SAC—ITN- -

--RI-- AA - T N D - V S- R RAI—L-H- -

Z8R2 2

^^ 17 0

SWTELEFDCSS WEDLKFIPER TEWPPYSILS FDIECMGEKG FPNATQDEDM

QQ G QL R Q RM- - A - --R-- R V

AR-AA SVQAD- S D R-V A T--A - --C--R-G- A

-Y-D II G YN--E-HA — N-M A 1 --C-KNEG- L

-H-D II G YY--E-HAD - N-M - 1 --C-KNEG- L

-Y AA Y-- E VG--SVRR- D SS--S-QA- A L — E- —T--NEA- L

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-E- TT — **- - G- L A

11 - E **- - G V--I - - A

RV-SS — I-Q - -M-KDM**- N G V--I - I A

RVV I-Q - -T-KDT**- N G V--I - I A

--RA-G-C —— RR-HDA**- K G-L-ANT— R -T-LI -

44 11 AQAHNA Z8rev 3 L1F 1

VDFASLYPSII IQAHNLCYST LIPGDSLHL H PHLSP*DDY:

QGA-PA-- -E-R-* -

—Q-NAILS-- -E-T-* N

—T-SA--G -- -E-T-* -

RDD -D-T-* -

M_H-RDD -N-T-* -

HNN N Y -Y-K-* S

H H A__ Nyy K _* s

M-TPGEE-RLL AG-R-G E

350 0

QVCREKLSLSS DYKLDTVAKQ CLGRQKDDIS YKDIPPLFK S GPDGRAKVGN

DD K - R R- -A A 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 -

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- I - A K - E - V --

L - - A K - E - V H H

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- SS AT — S

- SS T—T

- SS T

-GRR S

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- SS S

-SRR S

- - SS S

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LL M-

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— E — R — AA A

KRKEIRKTL A A

D—R R

N - --

R A - --

— R A — R R

A - -QK — —

A—QK — —

A - K - L - --

—— G S

—— E I - R

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— E - - R R L -M M

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— A — T M R - --

— G — T M R ---

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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.

101 1

B B p-herpesvirus s

P R UU I y1-herpesvirus

Neww World y2-herpesvirus s

Oldd World 2-herpesvirus s

Figuree 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). No 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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104 4