Human herpesvirus 8 and Kaposi's sarcoma in the Amsterdam cohort studies. Disease association, transmission and natural history - 1 Introduction

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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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Moritzz Kohn was born in Kaposvar, Hungary on 23 October,

1837.. He matriculated at the University of Vienna in 1856 and

graduatedd with the degrees of Doctor of Medicine in 1861 and

Doctorr of Surgery in 1862. His interest in dermatology began

whilee working in the Department of Syphilology at the

Allgemei-ness Krankenhaus and he transferred to Ferdinand von Hebra's

Departmentt of Dermatology in 1866. Kohn changed from the

Jewishh to the Roman Catholic faith in 1869, changed his surname

too Kaposi in 1871 and married von Hebra's daughter. Among his

manyy distinguished contributions to dermatology, Kaposi was the

firstfirst to describe xeroderma pigmentosum and idiopathic multiple

pigmentedd sarcoma. Moritz Kaposi died in 1902.

(Oberr W. Kaposi: The Man and the Sarcoma. In: Kaposi's

sar-coma:: A Text and Atlas. 1988. Edited by GJ Gottlieb, AB

Ackerman.. Lea and Febiger Publishers, Philadelphia, USA).

Introduction n

Epidemiologyy of Kaposi's sarcoma

Kaposi'ss sarcoma (PCS) is a vascular tumor that was first

de-scribedd as "idiopathic multiple pigmented sarcoma of the

skin"" by the Hungarian dermatologist, Dr Moritz Kaposi, in

18722 (1). Four epidemiological variants of KS are now

re-cognised;; the original report detailed the rare Classic variant

off KS which is found in elderly Mediterranean men and

Jewishh people born in Eastern Europe (2). Endemic KS has

beenn observed in children and young black adults in

equato-riall Africa and Transplant KS affects organ transplant

recip-ientss and patients who have previously received

immunosuppressivee therapy (3-7).*

AIDS-KSS is the most recent epidemiological variant to be

recognisedd and was first noted in immunocompromised

homosexuall men from New York and California (8). As

AIDS-KSS was found to be more common in persons who

acquiredd HIV-1 through homosexual contact than by

het-erosexuall contact, injection-drug use or contaminated

bloodd products, it was suggested that this vascular tumor is

causedd by an unidentified sexually transmitted agent (9).

Clinicall presentation of Kaposi's sarcoma

trointestinall tract with fatal consequence (1). The clinical

coursee of KS is, however, not always commensurate with

thatt of a malignant process; KS has been reported to regress

followingg the administration of antiretroviral therapy and to

remitt on reduction or cessation of immunosuppressive

therapyy (10,11).

Pathologicall features of Kaposi's sarcoma

Thee pathology of KS is similar for all variants; lesions tend

too progress through patch, plaque and nodular stages on the

skin.. Patch lesions are characterised by irregularly dilated

lymphatic-likee spaces lined by endothelial cells and

perivascularr infiltrates of lymphocytes and plasma cells. The

plaquee stage is notable for more extensive infiltration of

bloodd vessels and the presence of short fascicles of

spin-dle-shapedd cells. In the nodular stage, sheets of spindle cells,

withh ovoid but seldom mitotic nuclei, are seen and vascular

spacess are more prominent (12). Although spindle cells

ap-pearr to be the proliferative component of the KS lesion,

theree is considerable debate as to their endothelial origin

andd whether their proliferation represents a hyperplastic

polyclonall process rather than an oligo- or monoclonal

ma-lignancyy (13).

Thee original description of KS showed that this tumor not

onlyy affects the skin but also may involve the lungs and

Humann herpesvirus 8 (HHV8) / Kaposi's

sarcoma-associatedd herpesvirus (KSHV)

Discoveryy and classification

off HHV8 as a rhadinovirus

Twoo herpesviral D N A sequences were discovered using

Representationall Difference Analysis that preferentially

identifiedd the genomic differences between KS lesions and

normall skin (14). These sequences were present in 25 (93%)

off 27 AIDS-KS tissues compared with 6 (15%) of 39 lvmph

nodess and lymphomas from AIDS patients without KS.

Thiss agent was named Kaposi's sarcoma-associated

herpes-viruss (KSHV) and is also known as Human Herpesvirus 8

(HHV8)) for taxonomie reasons. Preliminary phylogenetic

analysiss of a genomic subfragment placed HHV8 in the

Rhadinoviruss (gamma2) lineage of the gammaherpesvirinae

alongg with Herpesvirus Saimiri (HVS) (15). The closest

hu-mann relative is Epstein-Barr Virus (HBV), which belongs to

thee Lymphocryptovirus (gammal) herpesvirus subfamily

(Figuree 1).

Thee nucleotide sequence of HHV8 provides

insightss into KS pathogenesis

Thee genome of H H V 8 (based on two samples with a

differ-encee of 0.4°/o) has a 140.5-kb-iong unique region (LUR)

whichh is flanked by multiple 801bp terminal repeat

se-quencess (Figure 2) (16,17). Within the LUR, 81 potential

Openn Reading Frames (ORFs) with greater than 100 amino

acidss have been identified and several additional spliced

geness have since been added to this list. The overall G + C

contentt in the LUR is 53.5% and 84.5%> in the terminal

re-peatt sequence. The numbering of HHV8 ORFs is based on

positionall homologies with HVS due to substantial

col-linearityy between these genomes w

hereas those ORFs

with-outt positional homologues are numbered consecutively

withh a K prefix. The LUR consists of conserved herpesviral

geness that are involved in herpesvirus replication and

struc-turee and non-conserved genes that may provide insights

intoo the pathogenetic mechanisms of this virus (16,17). The

presencee in the viral genome of ORFs with significant

homologyy to mammalian genes involved in cellular growth

controll indicates that "molecular mimicry,..of cell cycle

reg-ulatoryy and signaling proteins is a prominent feature of this

virus"" (16). The type, function and expression of these viral

geness are discussed below.

Serologicall detection and seroepidemiology of

HHV8 8

Thee presence of HHV8 DNA sequences in all

epidemiolog-icall variants of KS implies that antibodies to HHV8 should

bee detectable at the time of KS diagnosis and sera from KS

patientss should thereby serve as positive reference material

(18).. Currently negative reference sera are chosen from a

populationn that has a low risk for KS. Sensitivity and

speci-ficityficity data for serological assays can be calculated from these

referencee materials.

Latentt antigens

Antibodiess to a high molecular weight (224-234kDa) latent

nuclearr antigen (LNA or LAN A) can be detected by

West-ernn Blot (WB) or immunofluoresence assays (I F A) on B cell

liness that were established from patients with body cavity

basedd lymphoma (BCBL)/ primary effusion lymphoma

(PEL)) with latent HHV8 infection (19-22). LAN A has been

shownn to be encoded by ORF73 following expression of

thiss gene in bacterial and mammalian expression systems

andd subsequent demonstration of the 222-234kDa doublet

B B

Neww World y2-herpesvirus s

Oldd World 2-herpesvirus s

Figuree 1a. DNA maximum likelihood tree for a 1802-bp fragment of herpesviral DNA polymerase. Sequences were aligned by using

CLUSTALXX and analysed by using the DNAML program (PHYLIP version 3.5c; J. Felsenstein and the University of

Washing-ton). .

b.. Neighbor-joining protein distance tree for a 454-bp fragment of herpesviral DNA polymerase. Sequences were aligned as

abovee and analysed by using the PROTDIST and NEIGHBOR programs in PHYLIP. These findings have been published

00 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 kb ll 1 1 1 1 1 1 1 1 1 i I I I l l ' l i

H H

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/

o nn

m a

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rr n

vlL66 vMIP-ll vBCK vMIP-l nut-1

UU -

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a a a a

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II HOT *Mf

/-FLIPP v-cyc LANA

Classs I transcripts Class II transcripts Class III transcripts Terminall repeat regions

Figuree 2. The HHV8 genome is 140.5kb long and is flanked by multiple terminal repeat sequences which are depicted as hatched

squares.. Three classes of transcript are recognised (136); class I transcripts are constitutively expressed in BCBL/PEL cells

andd these latent transcripts are coloured black, class II transcripts are expressed at low levels during latency but can be

in-ducedd chemically and are shaded grey and class III transcripts are only present following chemical induction and are

repre-sentedd in white. It should be noted that the nut-1 transcript is not translated. Blocks of structural genes that are conserved

betweenn most gammaherpesviruses are labelled l-V. This diagram shows "non-conserved" genes that are discussed in the

textt and the map is not to scale.

byy WB of the nuclear extract (23-25). LAN A WB and IFA

formatss have high sensitivity and specificity; HHV8

anti-bodiess are detected in the majority (71-100%) of AIDS-KS

andd Classic KS sera but only in the minority (0-4%) of

bloodd donors (19-22).

Lyticc antigens

Lyticc antigens have been identified to increase the

sensitiv-ityy of HHV8 immunoassays. Antibodies to lytic antigens

cann be detected by IFA on BCBL/PEL cell lines that have

beenn pre-treated with phorbol esters or sodium butyrate

(26-29).. Lytic antigens can be detected by WB or

radioim-munoprecipitationn of chemically induced BCBL/ PEL cell

liness and also by whole-virus enzyme immunoassay (ElA)

ass H H V 8 virions can be produced by treating the same cells

withh tetradecanoylphorbol acetate (TPA) (27-31). An EIA

thatt uses lysed HHV8 virion as the target antigen has high

sensitivityy (94%) and specificity (93%) (32). Recombinant

structurall antigens such as a capsid-related protein (ORF65)

andd membrane glycoprotein gp35-37 (ORFK8.1) are also

usefull for seroepidemiological purposes (22,30, 33-35).

Generally,, lytic IFAs have a sensitivity close to 100%

how-everr the specificity varies between 69%) and 100% in

differ-entt laboratories (36). Recombinant ORF65 proteins and

ORFK8.11 give sensitivities in the range of 75-90%, with

somee variability if different expression vectors or

purifica-tionn methods are used (22,30,33-35). The specificity of

re-combinantt ORF65 and ORFK8.1 based assays is generally

higherr than 80% and may approach 100% when assay

con-ditionss are varied (22,30, 33-35,37).

Geographicc distribution of HHV8

Itt is possible to demonstrate that certain geographic regions

havee comparatively high or low seroprevalences of HHV8

despitee the variety of HHV8 serological tests and blood

do-norss are a useful group in which to demonstrate this

varia-tion.. In Northern Europe, HHV8 appears to be rare in

bloodd donors, with seroprevalence rates using LAN A or

ORF655 less than 5% in the United Kingdom, Sweden and

Switzerlandd (22,38,39). HHV8 seroprevalence rates rise

dramatically,, using the same serological tests, in countries in

Southernn Europe; rates range from 2% to 32% in regions of

Italyy (19,40-42). Use of a lytic IFA also increases the

seroprevalencee in blood donors to 20% in Sweden and to

28%% in Italy (39,41).

Inn North America, less than 5% of blood donors have

anti-bodiess to ORF65 and/or LAN A (19-22,26). This

seropre-valencee rate ranges from 0-14% when seven IFAs and EI As

aree used to study interlaboratory and interassay variation on

aa panel of aliquoted sera (36). Anti-LANA antibodies were

virtuallyy undetectable in other studies on US blood donors

(23,31,33).. Use of a lytic IFA to detect antibodies against an

undefinedd antigen increases the seroprevalence (range:

8-29%)) among US blood donors (26,33,36). ORFK8.1 WB

andd whole-virus EIA detect similar proportions (8-11%>) of

bloodd donors (31,33). Only 4% blood donors were

seropos-itivee with the whole-virus EIA or lytic/ LAN A IFA in

Ja-maicaa (43). HHV8 antibodies were detected by whole-virus

EIAA in 5%) or less blood donors from the United States,

Ja-maicaa and Trinidad (44).

Seroprevalencee data are limited for blood donors from

otherr regions. The seroprevalence of HHV8 among former

bloodd donors in Israel was 22% using LAN A IFA and only

22 (0.2%) of 1000 blood donors were LAN A IFA positive in

Japann (45,46).The seroprevalence of HHV8, using LAN A

IFA,, was 20% in black blood donors and 5% in white blood

donorss in South Africa (47). It is salient to note that HHV8

iss considered an endemic infection in regions of Italy, Africa

andd South America (48-50).

Diseasee Association

HHV88 in populations with or at risk of AIDS-KS

Inn the United States of America and Europe, antibodies to

LANN A and/or ORF65 are more common among HIV-1

infectedd homosexual men (23-39%) than among HIV-1

in-fectedd drug users, heterosexuals, transfusion recipients or

personss with hemophilia (0-7%) (19-22, 26, 41,51). Higher

seropp re valence rates are seen using lytic IFA however the

distributionn pattern remains the same; over 90% HIV-1

in-fectedd homosexual men have HHV8 antibodies compared

too 2 3 % HIV-1 infected injection-drug users and 2 1 %

HIV-11 infected women (26). These studies have all

demon-stratedd high prevalences of HHV8 antibodies in patients

withh AIDS- KS (range: 52-85%) compared with

HIV-in-fectedd controls without KS (range:18-35%). As a result,

sig-nificantt odds ratios (range: 2-19) have been found, where

calculable,, for the presence of HHV8 antibodies between

KSS cases and controls (19-22,26,38).

HHV88 in populations with or at risk for Classic KS

HHV88 seroprevalences using LANA or ORF65 range from

94-100%% among persons with Classic KS and from 4-19%

Sexuall transmission

Inn a cohort of Danish homosexual males, the presence of

antibodiess to HHV8 LANA and ORF65 at study entry was

independentlyy associated with the number of receptive anal

intercoursess and sex with men from the United States of

Americaa (57). HHV8 seroconversion in this study was

inde-pendentlyy associated with visits to homosexual

communi-forr matched or blood donor controls with odds ratios,

whenn calculable, ranging from 130-257 (19,22,40,51).

HHV88 in populations with or

att risk for Transplant KS

Seroconversionn against HHV8 ORF65 occurred within the

firstfirst year after transplantation in Switzerland (52).

Case-controll studies from different geographic regions

havee again demonstrated a strong association between

HHV88 antibodies and Transplant KS. In Italy, 10 (91%) of

111 transplantation recipients with KS were seropositive to

antibodiess to LANA and ORF65 compared to 2 (12%) of

177 organ recipients who served as controls (OR=75) (53).

Highh odds ratios were also seen in case-control studies from

Francee using LANA and ORF65 (OR=28) and Saudi

Ara-biaa using the lytic antigens, p40 and sVCA (OR=34) (54,55).

HHV88 in populations with or at risk fo Endemic KS

Dataa on Endemic KS are scarce; a combined total of 44

pa-tientss with Endemic KS were all positive by lytic/LANA

IFAA (19,26,56).

tiess in the United States of America and HIV-1

seropositivity. .

Inn San Francisco, anti-LANA antibodies were detected in

40%% exclusively homosexual men, 13% men who reported

mostlyy homosexual activity and were not detected in

exclu-sivelyy heterosexual men (58). HHV8 seropositivity was

stronglyy associated with HIV-1 infection and self-reported

historyy o f sexually t r a n s m i t t e d disease in this study and

H H V 88 s e r o p r e v a l e n c e increased linearly with the n u m b e r

o ff male i n t e r c o u r s e p a r t n e r s in t h e preceding two years.

H H V 88 a n t i b o d i e s , as d e t e c t e d using L A N A / O R F 6 5 or lytic

I F A ,, h a v e b e e n linked to p a r a m e t e r s of sexual b e h a v i o u r

suchh as n u m b e r o f p a r t n e r s a n d oroanal c o n t a c t in Sydney

andd San F r a n c i s c o (59,60).

Seraa f r o m patients in a L o n d o n sexually transmitted disease

clinicc w e r e tested with latent I F A and 198 (7%) o f 2718

pa-tientss w e r e s e r o p o s i t i v e ; i n d e p e n d e n t risk factors w e r e

h o m o -- a n d bisexuality, b i r t h in Africa, a history of syphilis,

H S V - 22 and H I V - i n f e c t i o n (61). H H V 8 seroposttivity, as

de-t e r m i n e dd by lyde-tic I F A , was linked de-to de-the n u m b e r o f sexual

p a r t n e r ss and a history o f o t h e r sexually transmitted diseases

inn Swedish w o m e n (62). Similar associations with sexually

t r a n s m i t t e dd diseases w e r e seen in N o r t h American w o m e n

withh o r at risk o f H I V - 1 infection using either O R F 6 5 and

O R F K 8 . 11 F.I A o r a c o m b i n a t i o n o f lytic/latent IFA (63,64).

Inn W e s t e r n Sicily, H H V 8 seroprevalences using a lytic IFA

w e r ee h i g h e r in H I V - 1 positive and negative h o m o s e x u a l

m e n ,, m e n w h o h a d sex w i t h prostitutes, female prostitutes

a n dd clients at a sexually t r a n s m i t t e d disease clinic than in the

generall p o p u l a t i o n (65). T h e seroprevalence o f H H V 8 was

h i g h e rr in female c o m m e r c i a l sex w o r k e r s than control p o p

-ulationss in C a m e r o o n a n d H o n d u r a s (66-68).

Childhoodd transmission

T r a n s m i s s i o nn b e f o r e p u b e r t y a p p e a r s to be rare in the

U n i t e dd States b u t d o e s o c c u r in countries w h e r e H H V 8 is

m o r ee w i d e s p r e a d ; H H V 8 a n t i b o d i e s are detectable in

chil-d r e nn from C a m e r o o n , Flgypt, S o u t h Africa, U g a n chil-d a anchil-d

It-alyy (40, 6 7 , 69-77). Similarly H H V 8 antibodies are found in

c h i l d r e nn o f individuals o f African origin in F r e n c h G u i a n a

a n dd also in Brazilian A m e r i n d i a n s (49,76). Age distribution

o ff H H V 8 a n t i b o d i e s s h o w e d that the H H V 8

seroprevalencee for adults was reached well before puberty

inn Ugandan children but that H H V 8 infection was also rare

beforee t h e age o f 2 (72). Similar results w e r e found in

Cam-eroonn w h e r e there was a steady increase in seroprevalence

fromm 2 8 % at 4 years o f age to 4 8 % a b o v e 15 years (74).

Cor-relationn with hepatitis B infection suggests that H H V 8 is

transmittedd horizontally in c o n d i t i o n s of close contact and

crowdingg (72). I I H V 8 seroprevalence was almost equal in

spouses,, children and siblings of K S patients in Sardinia and

indicatess intrafamilial transmission (70). H H V 8 appears to

bee transmitted within families in Israel and significant

mother-childd and sib-sib correlations t o r H H V 8

seropositivityy were seen in I Tench G u i a n a (45,76).

Inn South Africa, the majority (88%) of H H V 8 seropositive

childrenn had a seropositive m o t h e r indicating that

mother-childd transmission exists (71). A similar conclusion

wass reached in Italy and 5 children with KS all had H H V 8

seropositivee m o t h e r s in Z a m b i a (78,79). Vertical

transmis-sionn of H H V 8 was n o t seen in m o t h e r - i n f a n t pairs from

Haiti,, N o r t h e r n Italy o r Africa (80,81).

Parenterall transmission

Inn Switzerland, a country w h e r e H H V 8 is not e n d e m i c , the

appearancee o f IgM and IgG antibodies to H I 1 V 8 lytic

capsidd ( O R F 6 5 ) antigen within m o n t h s or transplantation

indicatess that H11V8 is transmitted t h r o u g h renal allografts

orr blood transfusion (52). H H V 8 is m o r e likely t o be

reacti-vatedd in transplant recipients w h o orginate in countries

wheree H H V 8 is endemic (53,54,82-85). F v i d e n c e for both

transplantation-associatedd transmission and reactivation

hass been seen in a c o h o r t or renal transplant recipients in

Belgiumm (86).

H H V 88 has been transmitted experimentally from the C D 19

cellss of an H H V 8 seropositive b l o o d d o n o r to H H V 8

nega-tivee CD 19 cells (87). Transmission through contaminated

bloodd products appears to be rare; of 14 individuals who

re-ceivedd blood products from HIV-1 and HHV8 co-infected

donors,, 10 developed antibodies to HIV-1 but none

seroconvertedd to HHV8 (88). Similarly in Jamaica,

recipi-entss of blood products from HHV8 seropositive donors

re-mainedd seronegative (89). Anti-LANA antibodies were

foundd in the minority (1%) of Romanian children who were

HIV-11 infected as a result of parenteral transmission (90).

Seroprevalencee rates in haemophiliacs vary from 0-28%

de-pendingg on whether a lytic or latent IFA is used (22,26,91).

Nucleicc acid based detection of HHV8

Nucleicc acid based studies have not been sensitive enough

too determine the prevalence of HHV8 infection within

pop-ulations.. Detection of HHV8 by polymerase chain reaction

(PCR)) may also depend on the severity of clinical disease

(92-95).. Nonetheless, PCR studies have demonstrated the

strongg association between HHV8 and KS; HHV8 is

de-tectablee by PCR in peripheral blood mononuclear cells

(PBMCs)) of AIDS-KS patients (range: 35-91%) and

HIV-infectedd controls (range 0-19%)) with odds ratios

rang-ingg from 2-440 (96-100). Sequence variation in the ORF26,

ORFK1,, ORF73 and ORF75 genes can be used to study the

molecularr epidemiology of HHV8, however this

burgeon-ingg field is not covered in this thesis.

HHV88 nucleic acid sequences

inn cells and body fluids

PCRR studies have also been invaluable in determining the

distributionn of HHV8 in cells and body fluids. Viral DNA

sequencess have been detected in the following cells;

endo-theliall and spindle cells of KS lesions, B cells, CD8+ T cells,

macrophages,, monocytes, prostatic giandular epithelium,

circulatingg endothelial cells, brain tissue, lung tissue, skin,

bonee marrow, non-neoplastic lymph nodes and

gastrointes-tinall mucosa from HIV-1 seropositive individuals

(101-113).. HHV8 sequences were not detected in the faeces

off HIV-1 infected individuals (96).

HHV88 DNA sequences have been detected in body fluids

andd provide some insight into the mode(s) of HHV8

trans-mission;; HHV8 has been detected in saliva from HIV-1

in-fectedd persons with or without KS (96, 114-118). HHV8

hass been detected by PCR in saliva and nasal secretions of

HIV-11 infected individuals and HHV8 seropositive persons

butt not in control subjects (119, 120). HHV8 is also

detect-ablee in bronchoalveolar lavage samples from AIDS-KS

pa-tientss (121-124). Reports on the proportions of HHV8 in

semenn and prostate glands of HIV-1 infected and

unin-fectedd individuals range dramatically from 0-100% (125).

Thesee differences are probably due to variations in the assay

orr reflect geographic and population differences; a recent

multicenterr study has shown that it is easy to contaminate

thee HHV8 PCR and in those that were not contaminated,

HHV88 DNA was detected infrequently (8%)) in semen from

HIV-11 infected and uninfected subjects (126). HHV8 is

rarelyy detectable in cervical and vaginal secretions (127).

In-terestinglyy the number of HHV-8 copies per microgram of

tissuee or body fluid from KS patients was highest in PBMCs

followedd by saliva and semen and undetectable in faeces

(128).. HHV8 is also detectable, albeit rarely, in

cerebrospinall fluid and urine (118,129,130).

Presencee and location of HHV8 and expression of

HHV8-encodedd genes Kaposi's sarcoma lesions

HHV88 DNA sequences have been demonstrated by in situ

hybridisationn and/or amplification in spindle cells and

en-dotheliall cells that line the vascular spaces of patch, plaque

andd nodular KS lesions, however these sequences are not

presentt in normal endothelial cells (131-134). In early KS

le-sions,, HHV8 is present in less than 10% of cells forming the

ectaticc vessels whereas it is present in greater than 90% of

spindlee cells in nodular KS (135). HHV8 transcripts can be

groupedd in the following three classes; Class I and Class II

transcriptss are constitutively produced in BCBL/PEL cells

howeverr Class II transcripts are induced by TPA treatment

whereass Class 111 transcripts are only produced following

TPAA treatment (136). Class I and III transcripts correspond

too the latent and lytic phases of the herpesviral lifecycle

whereass Class II transcripts do not conveniently fit this

par-adigm,, ("lass 1 transcripts are expressed in KS spindle cells,

ass judged by in situ hybridisation or immunohistochemistry

andd indicate latent persistence of HHV8 in the majority of

KSS spindle cells (137-149).

Pathogenesiss of Kaposi's sarcoma

KSS used to be considered solely as a "cytokine-driven"

pro-liferationn because IL-6 enhances the proliferation of spindle

cellss in culture and IFN-gamma induces endothelial cells to

acquiree the phenotypic features of spindle cells

(150-152).Thee pathogenesis of KS can now also be

consid-eredd in terms of the functions of HHV8-encoded proteins,

whichh are grouped below on the basis of their

correspond-ingg transcript (136, 153).

Proteinss that are translated

fromm Class I transcripts

Cellss that express viral Fas-ligand interleukin converting

en-zyme-likee caspase (v-FLIP, encoded by ORFK13/ORF71)

aree protected against apoptosis induced by the CD95 death

receptorr and other related death receptors (154).

Virall cyclin (v-cyclin, encoded by ORF72) binds to

cyclin-dependentt kinase (cdk) 6 and results in phosphorylation of

thee retinoblastoma-tumor suppressor protein and histone

HII (155-157). It should be noted that phosphorylated

reti-noblastomaa protein is unable to repress transcription

fac-torss such as E2F which regulate cellular DNA synthesis

(153).. Viral cyclin/cdk6 is also resistant to inhibition by

cel-lularr CDK inhibitors such as pi6, p21 and p27 (158).

Latencyy associated nuclear antigen (LANA-1, encoded by

ORF73)) is abundantly expressed in KS spindle cells (25,

144,159).. LANA-1 colocalizes with the viral episome in

interphasee nuclei and along mitotic chromosomes

indicat-ingg that this protein is involved in episome maintenance

du-ringg cell division (160). This protein also inhibits the ability

off the p53 tumor-suppressor protein to induce cell death

(161). .

Proteinss that are translated

fromm Class II transcripts

Virall IL-6 (vIL-6, encoded by ORFK2) activates the Janus

tyrosinee kinase (JAK)-signal transducers and activators of

transcriptionn (STAT) pathway that is known to have

multi-plee downstream effects on cellular survival, proliferation,

differentiationn and apoptosis (162,163). Viral IL-6 induces

B-celll proliferation and prevents apopotosis in susceptible

celll lines (164-166). This cytokine also promotes

haemato-poiesis,, plasmacvtosis and angiogenesis in athvmic mice

(167). .

Virall Bcl-2 (vBcl-2, encoded by ORF16) has anti-apoptotic

propertiess in yeast and baby hamster kidney cells although

thee ability of vBcl-2 to form a dimeric protein with human

Bcl-22 is disputed (168,169).

Threee viral macrophage inflammatory proteins (vMIPs)

havee been identified namely; vMlP-I (ORFK4), vMIP-II

(ORF4.1)) andvMIP-III (ORF6) (16,17,164,165). All three

proteinss belong to the CC chemokine family and are

agonistss of the chemokine receptors CCR3, CCR4 and

CCR88 which selectively chemoattract Th2 lymphocytes

(170-174).. These chemokines are highly angiogenic in the

chorioallantoicc assay and also block the entry of HIV-1 into

cellss that express CCR3 amd CCR5 (164, 167, 170, 173,

175). .

Virall interferon regulatory factor (vIRF-1, encoded by

ORFK9)) inhibits interferon-responsive gene expression

andd prevents interferon-induced growth arrest (176-179).

Virall IRF-1 also transforms NIH3T3 cells and induces

tu-morss when injected into nude mice (176,177). Two other

vIRFss are recognised but further functional analysis is

re-quiredd (180,181).

O R F K ll 2 encodes a small protein termed Kaposin whose

transcriptt (TO.7) is expressed in abundance in unstimulated

BCBL/PELL cells and is detectable in the majority of KS

tu-morss of differing epidemiological variants (139, 182).

Ex-pressionn of this protein in Rat-3 cells results in focal

trans-formationformation and subcutaneous injection into nude mice

pro-ducess highly vascular and undifferentiated sarcomas (183).

Theree are two variants of the latency associated membrane

proteinn (LAMP, encoded by ORFK15) which have several

srcc homology 2-like motifs and potential tumor necrosis

factorr receptor-associated factor binding sites in its

cyto-plasmicc tail. It is postulated that this protein is similar to the

EBVV latent membrane proteins which are involved in

cellu-larr transformation and maintenance of viral latency in B

cellss (184,185).

Thee epidemiology of AIDS-KS suggested that this vascular

tumorr was caused by an unidentified sexually transmitted

infection.. Following the identification of HHV8 DNA

se-quencess in AIDS-KS tissue, an argument rapidly ensued as

Proteinss that are translated

fromm Class III transcripts

O R F K ll posseses an immunoreceptor tyrosine activation

motiff involved in B-cell and T-cell antigen receptor

signal-ingg and is the positional analogue of saimiri transforming

proteinn (STP) and one of the EBV latent membrane

pro-teinss (186). A role in cellular transformation is surmised

fol-lowingg the demonstration that rodent fibroblasts that

ex-presss ORK1 show evidence of transformation and that

O R F K ll can replace STP in the HVS genome and induce

lymphomaa in the common marmoset (187).

Virall G protein-coupled receptor (vGPCR, encoded by

ORF74)) signals through the

phosphoinositide-inositol-triphosphate-proteinn kinase C pathway and stimulates rat

kidneyy fibroblasts to proliferate (188). Viral GPCR appears

too be expressed only in a subpopulation of KS spindle cells,

whichh presumably undergo lytic replication (189).

Expres-sionn of vGPCR in a focus-formation assay led to focal

transformationn of NIH3T3 cells and subsequent injection

off transformed cells into nude mice caused tumors that

consistedd of spindle-shaped cells (190). Transgenic mice

thatt express v-GPCR within haematopoietic cells develop

angioproliferativee lesions (191).

ORFK33 and ORFK5 are proteins encoded by Class III and

Classs II transcripts respectively that inhibit the expression

off MHC class I antigens by enhancing endocytosis of

sur-facee MHC molecules (192).

too whether HHV8 was the cause of KS or a "passenger"

vi-rus.. We developed an Enzyme Immunoassay for the rapid

detectionn of antibodies to recombinant HHV8 encoded

lytic-phasee capsid (ORF65) and latent phase nuclear

(ORF

73)) antigens. In this thesis, we examined the disease

association,, transmission and natural history of HHV8

in-fectionn in the homosexual and drug user cohorts of the

Am-sterdamm Cohort Studies on HIV-1 infection and AIDS.

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