World Journal of AIDS, 2013, 3, 221-230 Published Online September 2013 (
The Role of Human Herpesvirus 8 Molecular
Characterization in the Management of HIV Infected
Patients Diagnosed with Malignancies Associated
with Its Infection
Martínez Pedro Ariel1*, Kourí Vivian1*, Blanco Orestes1, Capó Virginia1, Abad Yoandra1,
Alemán Yoan1, Verdasquera Denis1, Jiménez Narciso1, Caballero Iraida2, Fleites Gilberto1,
Ugarte Yaumara1, Calderón Odalys1, Álvarez Alina1, Ulrich Hengge3
1Institute of Tropical Medicine “Pedro Kourí” (IPK), Autopista Novia del Mediodía, Habana, Cuba; 2“Hermanos Ameijeiras” Hospi-
tal, Havana, Cuba; 3Haut Zentrum (Skin Center), Dusseldorf, Germany.
Received April 16th, 2013; revised May 16th, 2013; accepted June 16th, 2013
Copyright © 2013 Martínez Pedro Ariel et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Despite the progress has been reached with Human herpesvirus 8 (HHV-8) research, there are gaps in the knowledge of
viral induced oncogenesis. The aim of the present study was to identify possible associations between HHV-8 subtypes,
HHV-8 loads and clinical manifestations of HIV infected patients diagnosed with different malignancies associated
with HHV-8 infection. Forty six HIV-1 infected individuals diagnosed with different HHV-8 associated diseases were
studied [37 epidemic Kaposi’s sarcoma (KS), 3 pleural effusion lymphoma (PEL); 5 peripheral lymphadenopathies (PL);
1 Hodgkin’s lymphoma (HL); 1 non Hodgkin’s lymphoma (NHL)]. HHV-8 loads were determined by quantitative real
time PCR (qRT-PCR) whilst HHV-8 subtypes were determined by open-reading frame (ORF)-K1 gen genotyping.
HHV-8 subtypes B, A, C, A5 and E were exhibited by 31.8%, 23.4%, 19.1%, 17% and 8.5% of the studied patients,
respectively. The median HHV-8 viral load did not differ between subtypes (p > 0.05) but HHV-8 viral loads were sig-
nificantly higher in PEL than in epidemic KS lesion or lymph nodes (p = 0.04). Subtype B was detected in 60% of pa-
tients with B cell lymphoma (NHL, PEL and HL) whereas subtype E was only detected in patients with epidemic KS
diagnosis. Our data suggest that HHV-8 DNA quantification instead of subtype identification could be used as a surro-
gate marker for monitoring its infection, not only in epidemic KS patients but also in HIV infected individuals with
lymphoproliferative disorders.
Keywords: Human Herpesvirus 8 or Kaposi’s Sarcoma-Associated Herpesvirus; Real Time PCR; Subtypes;
Lymphoproliferative Disorders; Cuban; HIV/AIDS
1. Introduction
HHV-8 also referred as Kaposi’s Sarcoma-Associated
Herpesvirus is the first known human Rhadinovirus which
belongs to the Gammaherpesvirinae subfamily, Herpes-
viridae family. It is implicated as the causative agent of
all clinical forms of Kaposi’s Sarcoma (classical, epide-
mic, endemic and iatrogenic), PEL and multicentric Cas-
tleman’s disease (MCD) [1] but its role in other lympho-
proliferative disorders has not been clearly established.
Although the HHV-8 genome is highly conserved along
most of the unique coding region (approx. 145 kbp), sev-
eral genomic regions display remarkable sequence vari-
ability making them useful markers of strain diversity
and potential epidemiologic patterns of HHV-8 spread.
Five major subtypes are recognized by K1 genotyping: A,
B, C, D and E. The K1 protein is highly variable in its
cysteine-rich N-terminal ectodomain and its amino acid
sequence has been shown to vary by up to 40%, with
changes concentrated in two hypervariable regions (VR1
and VR2) [2].
The influence of particular subtypes on disease pro-
gression remains largely unknown. Therefore, it is im-
portent to identify biological markers related with HHV-
*These authors have equally contributed.
Copyright © 2013 SciRes. WJA
The Role of Human Herpesvirus 8 Molecular Characterization in the Management of HIV
Infected Patients Diagnosed with Malignancies Associated with Its Infection
8-associated diseases outcome. Some authors have alert-
ed that the risk of KS remains substantially increased in
HIV infected subjects and further decreases have not been
observed [3]; being KS recently diagnosed in patients
with controlled HIV infection and CD4 counts over 200
T cells [4]. Epidemic KS incidence has not been signifi-
cantly reduced in Cuba although locally produced anti-
retroviral drugs started being introduced as a form of
treatment since 2001. Previous studies have shown that,
with the exception of subtype D, the majority of HHV-8
subtypes were circulating among Cuban HIV population
[5,6]. However, a change in HHV-8 subtype distribution
was recently identified with an increase in subtype B
detection [7]. Thus, we decided to identify how viral fac-
tors (K1 subtype and viral load) may impact on the onco-
genesis induced by HHV-8 among HIV infected indivi-
duals diagnosed with different malignancies.
2. Materials and Methods
2.1. Patients and Samples
The study was approved by local and national ethics com-
mittees. All participants provided their written informed
consent to participate in the study. Forty six HIV-1 in-
fected individual diagnosed with HHV-8 associated dis-
eases between 2005 and 2011 inclusive were included
and different samples were collected (Table 1). One pa-
tient contributed with two samples since KS in the lymph
node was diagnosed six month after the initial diagnosis
of PEL in the pericardium. The diagnosis of vasoprolife-
rative lesion of the lymph node and the AIDS-related
complex lymphadenitis were based on the histopatholo-
gical diagnosis as described elsewhere [8]. Clinical, im-
munological and epidemiological data from each patient
were obtained throughout a retrospective review of indi-
vidual’s clinical records and are depicted in Table 2.
2.2. Sampling and DNA Extraction
DNA was extracted from frozen tissues samples by
QIAamp® DNA Mini Kit (QIAGEN, Germany), accord-
ing to the protocol for DNA purification from tissues de-
scribed by the manufacturer. In contrast, 200 µL of the
effusion fluid from PEL patients and the sample of saliva
obtained from the patient diagnosed with Hodgkin lym-
phoma were used to purify DNA using the same kit but
following the protocol for blood or body fluids. Genomic
DNA (gDNA) concentration was determined by spectro-
photometer (GeneQuant II, Pharmacia Biotech, USA)
and adjusted to 100 ng (10 µL).
2.3. qRT-PCR for HHV-8 DNA Quantification
An “in-house” Taqman methodology based qRT-PCR
was used for HHV-8 quantification in clinical samples.
The protocol conditions, primers and probe were de-
scribed by Watzinger et al. [9] with minor modifications
adapted for the LightCycler 1.5 [10]. The HHV-8 loads
were expressed as copies/100 ng of DNA. Human
bin was amplified by Real Time PCR using primers,
probes and the protocol previously described elsewhere
2.4. DNA Sequencing of HHV-8 ORF K1
A fragment of ORF-K1 gen was amplified by nested
PCR from the assayed samples following the protocol
published elsewhere [2]. PCR products from nested PCR
were purified using MiniEluteTM Purification Kit
(QIAGEN, Germany) following the manufacturer’s pro-
tocol and their final concentration were evaluated through-
out horizontal electrophoresis in 2% agarose gel stained
with ethidium bromide (0.5 μg/mL) and visualized through
a UV transilluminator.
The Kit Dye labeled dideoxy Terminator Cycle Se-
quencing from Beckman Coulter (USA) was used for nu-
cleotide sequence analysis, following the manufacturer
recommendations. One hundred fentomoles of PCR pro-
ducts were added to a mixture containing 1 µl (5 pmol)
of either forward or reverse primer (LGH2090 and
LGH2508), 8 µl of sequence reaction mixture (DTCS
Quick Star Master Mix) and water up to a final volume
of 20 µl. The sequencing reaction was conducted by 50
cycles of two minutes at 96˚C for 20 seconds, 50˚C for
20 seconds and finally 60˚C for 4 minutes. The obtained
fragments were purified following the manufacturer in-
structions. Finally, the purified products underwent elec-
trophoresis on a Beckman Coulter CEQ8800 sequencer.
2.5. Sequence Analysis
Initial evaluation of each sequence was performed using
the nucleotide search engine BLAST at NCBI (USA), to
confirm that the amplified product was K1. Then, nucle-
otide sequences were manually edited with both forward
and reverse primers using MEGA version 4 [12]. To de-
termine the K1 subtype of each subject’s strain(s), nu-
cleotide sequences were aligned by Muscle in Jalview
version 2.4 [13] along with the following reference strains
obtained from Genetic sequence database (Gen-Bank) at
the National Center for Biotechnology Information (NCBI)
[AF133038 (A1); AF130305 (A2); U86667 (A3);
AF133039 (A4); AF178823 (A5); AF133040 (B1);
AF130259 (B2); AF133041 (C1); AF133042 (C3);
AF133043 (D1) AF133044 (D2); AF220292 (E)].
Maximum likelihood (ML) trees were estimated using
PAUP 4.0 beta under the best-fit substitution model cal-
culated by Modeltest 3.7 [14] using the Akaike informa-
tion criterion. New HHV-8 nucleotide sequences and the
Copyright © 2013 SciRes. WJA
The Role of Human Herpesvirus 8 Molecular Characterization in the Management of HIV
Infected Patients Diagnosed with Malignancies Associated with Its Infection
Copyright © 2013 SciRes. WJA
Table 1. HHV-8 subtypes identified by ORF-K1 genotyping from HIV-1 infected individuals in which HHV-8 associated dis-
eases were diagnosed between 2005 and 2011 inclusive.
Specimen ID Age Gender KSHV
subtype Diagnosis materialHistopathological diagnosis Accession
Cub-B06-576 22 Female A5 Tissue Kaposi’s sarcoma FJ986113
Cub-81/07 31 Male A5 Tissue Kaposi’s sarcoma FJ986114
Cub-209/06 35 Male A Tissue Kaposi’s sarcoma FJ986115
Cub-551/06 46 Male A5 Tissue Kaposi’s sarcoma FJ986116
Cub-2033/05 29 Male A5 Tissue Kaposi’s sarcoma FJ986117
Cub-758/06 36 Male A5 Tissue Kaposi’s sarcoma FJ986118
Cub-310/07 44 Male A Tissue Kaposi’s sarcoma FJ986119
Cub-B06-603 22 Male A Tissue Kaposi’s sarcoma FJ986120
Cub-473/07 35 Male A Tissue Kaposi’s sarcoma FJ986121
Cub-1064/06 30 Male A Tissue Kaposi’s sarcoma FJ986122
Cub-2032/05 45 Male B Tissue Kaposi’s sarcoma FJ986123
Cub-553/06 41 Male B Tissue Kaposi’s sarcoma FJ986124
Cub-737/06 46 Male B Tissue Kaposi’s sarcoma FJ986126
Cub-557/06 41 Male B Tissue Kaposi’s sarcoma FJ986128
Cub-1373/07 42 Male B Tissue Kaposi’s sarcoma FJ986129
Cub-626/06 39 Male B Tissue Kaposi’s sarcoma FJ986131
Cub-1109/06 37 Male B Tissue Kaposi’s sarcoma FJ986132
Cub-1835/06 36 Male B Tissue Kaposi’s sarcoma FJ986133
Cub-427/07 41 Male B PEL pleural Kaposi’s sarcoma FJ986134
Cub-9/07 41 Male E Tissue Kaposi’s sarcoma FJ986135
Cub-1105/06 50 Male E Tissue Kaposi’s sarcoma FJ986136
Cub-127/07 29 Male E Tissue Kaposi’s sarcoma FJ986137
Cub-286/07 43 Male C Tissue Kaposi’s sarcoma FJ986138
Cub-296/09 a 25 Male C PEL pericardium PEL FJ986139
Cub-134/06 29 Male C Tissue Kaposi’s sarcoma FJ986140
Cub-234/06 57 Male C Tissue Kaposi’s sarcoma FJ986141
Cub-1106/06 36 Male A Tissue Kaposi’s sarcoma FJ986142
Cub-556I/06 30 Male A Tissue Kaposi’s sarcoma FJ986143
Cub-375/07 35 Male C Tissue Kaposi’s sarcoma FJ986144
1T/2009 32 Male A5 Lymph node AIDS-related complex lymphadenitis GU475457
17T/2009 34 Male B Lymph node KS in the lymph node GU475458
32T/2009 25 Male A Lymph node Vasoproliferative lesion of the lymph node GU475459
50T/2009a 25 Male C Lymph node KS in the lymph node GU475460
Cub-58LN/2009 43 Male B Lymph node non Hodgkin’s lymphoma JF979530
The Role of Human Herpesvirus 8 Molecular Characterization in the Management of HIV
Infected Patients Diagnosed with Malignancies Associated with Its Infection
Cub-59S/2009 19 Male B Saliva Hodgkin’s lymphoma JF979531
Cub-62LN/2009 37 Male A5 Lymph node AIDS-related complex lymphadenitis JF979532
Cub-147T/2011 55 Male B Tissue Kaposi’s sarcoma JF979533
Cub-151T/2010 29 Male B Tissue Kaposi’s sarcoma JF979534
Cub-213T/2010 43 Male A5 Tissue Kaposi’s sarcoma JF979535
Cub-275T/2011 46 Male A PEL peritoneal PEL JF979536
Cub-516T/2009 33 Male E Tissue Kaposi’s sarcoma JF979537
Cub-762T/10 47 Female B Tissue Kaposi’s sarcoma JF979538
Cub-763T/2010 38 Male A Tissue Kaposi’s sarcoma JF979539
Cub-1426T/2009 28 Male A Tissue Kaposi’s sarcoma JF979540
Cub-1629T/2009 34 Male A Tissue Kaposi’s sarcoma JF979541
Cub-1501T/2009 44 Male C Tissue Kaposi’s sarcoma JF979542
Cub-1680T/2010 58 Male C Tissue Kaposi’s sarcoma JF979543
a. nucleotide sequences belongs to the same patient.
Table 2. Descriptive data of HIV-1 infected individuals di-
agnosed with HHV-8 associated diseases.
Variables Studied population
N = 46
Median age 37.4 years
(Range 22 - 58)
Gender Female 2 (4.3%)
Male 44 (95.7%)
Race White 32 (69.6%)
Mulatto 10 (21.7%)
Black 4 (8.7%)
Sexual behavior Heterosexual 5 (10.9%)
Men who have sex
with men (MSM) 41 (89.1%)
Median HIV viral
load* (copies/mL) 90,634
(Range 50 - 580,000)
Median CD4+ T cell
count ( c e l l s / m m 3) 241 (Range = 8 - 884)
CD4+ T cell count <200 21 (45.7%)
200 - 499 25 (54.3%)
>500 -
*Data not available for six patients.
derived aminoacid (aa) sequences were deposited in Gen-
Bank using the National Center for Biotechnology Infor-
mation (Bethesda, MD).
Throughout Sequin Application version 11.0. The
GenBank accession numbers for 46 new sequences ob-
tained in this manuscript are: FJ986113 to FJ986144,
GU475457 to GU475460, JF979530, JF979532 to
2.6. Statistical Methods
IBM SPSS Statistics package version 19 and Epidat ver-
sion 3.1 were used to process all the data. ANOVA test
was performed in order to compare the values of HHV-8
loads among identified subtypes, histopathological diag-
nosis and different compartments in which the tumor
arise. Contingency tables were constructed and Chi-
square test or Fisher’s exact test were used for comparing
data. Odds ratios (OR) and their 95% confidence inter-
vals (CI) were used to assess the association between va-
riables. HHV-8 sequences obtained from the same indi-
vidual were included separately for statistical analysis
since they were amplified at different times from diffe-
rent diseases. The value of HHV-8 loads in saliva from
the patient diagnosed with HL was not included in the
3. Results
The phylogenetic characterization allowed the identifica-
tion of different HHV-8 subtypes among the studied in-
dividuals (Figure 1). HHV-8 subtypes B, A, C, A5 and E
were exhibited by 31.8%, 23.4%, 19.1%, 17% and 8.5%
of the studied patients, respectively. Overall, no statisti-
cal associations were discovered between HHV-8 sub-
types and the following variables: CD4+ T cell counts,
HIV-1 loads, sex and age (p > 0.05).
Copyright © 2013 SciRes. WJA
The Role of Human Herpesvirus 8 Molecular Characterization in the Management of HIV
Infected Patients Diagnosed with Malignancies Associated with Its Infection
Figure 1. Unrooted maximum likelihood tree generated by PAUP 4.0 beta under the best-fit substitution model calculated by
Modeltest 3.7 using the Akaike information criterion with a fragment of the K1 gene of HHV-8. Branch lengths are drawn to
scale, with the bar indicating 0.03-nt re placement per site. Numbers on each node indicate the p value by which the cluster is
supported. References strains were labeled with circles ().
There were no significant differences in subtypes fre-
quencies among HHV-8 associated diseases (p > 0.05)
(Table 3). No statistical differences were found among
K1 subtypes when the median elapsed time between HIV
diagnosis and HHV-8 associated diseases appearance
were compared (p = 0.444). Nevertheless, individuals in-
fected with subtype E and A5 seem to progress slower to
HHV-8 associated diseases than individuals infected by
subtypes A, B or C. No differences were detected be-
tween the median copy number of each identified HHV-8
subtypes irrespective the histhopathological diagnosis
(p > 0.05) (Figure 2). However, the median HHV-8 viral
load was significantly higher in PEL than in epidemic KS
lesion or lymph nodes (p = 0.04) (Figure 3).
3.1. Epidemic KS
Patients diagnosed with epidemic KS showed a wide
range of subtypes although strains belonged to subtype E
were only identified in the tissue of these patients (Table
3). Nodular stage of epidemic KS were identified in 12/37
(patients 32.4%) whilst 8/37 (21.6%) and 2/37 (5.4%)
were considered to be in macular and patch stage;
Copyright © 2013 SciRes. WJA
The Role of Human Herpesvirus 8 Molecular Characterization in the Management of HIV
Infected Patients Diagnosed with Malignancies Associated with Its Infection
Figure 2. Logarithmic HHV-8 viral loads between subtypes
identified from HIV-1 infected individuals diagnosed with
different HHV-8-associated diseases (2005-2011).
Figure 3. Logarithmic HHV-8 viral loads between different
anatomic sites in which samples were collected from Cuban
HIV-1 infected individuals diagnosed with different HHV-
8-associated diseases (2005-2011).
respectively. No associations were identified between the
histological stages, HHV-8-subtypes and their DNA copy
number in the affected tissue (p > 0.05). However, 15 of
37 obtained biopsies (40.5%) were not classified by his-
topathological analysis.
3.2. B Cell Lymphoma
Subtype B was detected in 3/5 studied patients diagnosed
with B cell lymphoma (NHL, PEL and HL) but PEL pa-
tients exhibited different HHV-8 subtypes (Table 3).
EBV DNA was also detected in all the studied effusion
fluid by qRT-PCR (data not shown). Subtype B (58LN/
2009) was detected in an HIV infected patient diagnosed
with NHL of immunoblastic subtype which had no prior
history of KS but he was markedly immunosuppressed
(CD4+ T cell count 112 cells/mm3). In contrast, Cub
59S/09 (subtype B) was isolated from a mixed cellular
subtype of a Hodgkin lymphoma patient’s saliva. Al-
though HHV-8 DNA was not amplified in the lymph
node 22,980 copies/100 ng of DNA were found in saliva
by qRT-PCR Higher EBV DNA copies number was also
quantified in the saliva and lymph node collected both
from NHL and Hodgkin lymphoma patients (data not
3.3. Peripheral Lymphadenopathies
Different subtypes were exhibited by patients diagnosed
with peripheral lymphadenopathies (Table 3). No history
of KS lesions on the skin or mucous surface neither on
chest X-rays, abdominal scan and upper digestive tract
endoscopy revealed signs of visceral KS. Patients diag-
nosed with AIDS-related complex lymphadenitis exhib-
ited subtype A5 and they showed an enlargement of the
lymph node as the only clinical sign (1T/2009: cervical
area; Cub-62LN/2009: axillary area). HIV-1 infection was
diagnosed few months the beginning of lymph node swol-
len. They had a CD4 T cells counts over 200 cells
whilst their HIV-1 loads were 27,000 and 30 copies/mL;
respectively. The detected copy numbers of HHV-8 DNA
in the lymph node were 79 and 495 copies/100 ng of
gDNA, respectively. Subtypes A5 were significantly as-
sociated with the diagnosis of AIDS-related complex lym-
phadenitis (p = 0.0271).
KS was diagnosed in two lymph nodes fragments of
the studied HIV-1 infected individuals. The first one ex-
hibited subtype C (50T/2009) which was identified in the
lymph node of patient who complained of a swollen of
the cervical area with no other symptoms at the time by
which KS diagnosis was made. He had a previous history
of a PEL at the pericardium (Cub-296/09). Both aa se-
quences grouped in the same branch of the tree (p =
0.032) (Figure 1) however visual comparison of the ali-
gnment showed specific changes (50T/2009: G191A,
L197S, L213I, P254H). His CD4 T cell count was 142
cells/mm3 and the copy number of HHV-8 DNA in the
lymph node was 215,100 copies/100 ng of gDNA. The
second individual in whom KS was diagnosed resulted to
be infected by subtype B (17T/2009) and complained a
localized enlargement of lymph nodes in the inguinal
area, accompanied by fever. His CD4+ T cell count was
300 cells/mm3 and the HIV-1 viral load was 14,000 cop-
ies/mL. The determined value of HHV-8 DNA in the
lymph node was 3,300,000 copies/100 ng of gDNA.
The vasoproliferative lesion of the lymph node was
diagnosed in an AIDS rapid progressor admitted due to a
three month history of fever, weight loss, cervical mass,
fatigue and dyspnea. On the physical examination a re-
markable paleness of the mucous surface and skin were
noted, accompanied by spleen enlargement. Laboratory
examination showed severe anemia (4.5 g/L) and in-
creased erythrocyte sedimentation rate (130 mm). The
patient was severe immune depressed (CD4 T cell
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The Role of Human Herpesvirus 8 Molecular Characterization in the Management of HIV
Infected Patients Diagnosed with Malignancies Associated with Its Infection
Copyright © 2013 SciRes. WJA
Table 3. Distribution of HHV-8 subtypes according the histopathological diagnosis of the studied samples (2005-2011).
HHV-8 subtyp es
A B C E A5
Epidemic KS 9 19.2% 11 23.4% 7 15% 4 8.5% 6 12.8%
B cell lymphoma PEL 1 2.1% 1 2.1% 1 2.1% -
- 1 2.1%
- 1 2.1% - -
KS in the lymph node - 1 2.1% 1 2.1% - -
AIDS-related complex lymphadenitis - - - - 2 4.2%
Vasoproliferative lesion of the
lymph node 1 2.1% - -
Total 11 23.4% 15 31.8% 9 19.1% 4 8.5% 8 17%
PEL: pleural effusion lymphoma; HL: Hodgkin’s lymphoma; NHL: non Hodgkin’s lymphoma.
count: 163 cells/mm3) and the HIV-1 viral load was
160,000 copies/mL. Hepatomegaly and splenomegaly
were found on the abdominal scan. Subtype A (32T/2009)
was identified by nucleotide sequence analysis with a
total amount of 900,000 copies/100 ng of gDNA in the
lymph node.
4. Discussion
Although some authors have pointed that viral factors are
not likely to play an important role in HHV-8 oncogene-
sis [15], the available evidence is not conclusive and they
need to be explored thoroughly. So far, differences in the
replication rates by HHV-8 subtypes have not been re-
ported. The possible impact of HHV-8 subtypes and their
viral load in disease pathogenesis have been difficult to
evaluate before. Perhaps, the low incidence of HHV-8
associated diseases in populations at risk and also the
restricted geographical subtype’s distribution have con-
fined its assessment. Present findings suggest that HHV-
8 oncogenicity is not related to specific subtype demon-
strating that there are not differences between their rep-
licative capacities.
In contrast, it was known that HHV-8 viral loads in
affected tissue should be considered as an important mar-
ker for monitoring its infection. In agreement with others
[16-18], the obtained data highlight that HHV-8 load va-
ried among anatomic sites and it would be related with
the histhopathological diagnosis being higher in PEL.
Those findings support the usefulness of qRT-PCR in
HHV-8 diagnosis and underline the existence of not well
defined viral characteristic that allows HHV-8 mainte-
nance, replication and also its capacity to transform tar-
get cells into a malignant one among different neoplasms
[19]. Unfortunately, those mechanisms are not well un-
derstood yet.
Interestingly, like the previous report from Cassar and
colleagues [20], Cuban E subtypes were detected in epi-
demic KS lesions whereas it seems to be rarely associ-
ated with KS in Brazilian Amerindians [21]. Thus, it
would indicate that the Cuban subtype E has evolved to a
more invasive variant which can transform endothelial
cells or maybe the lower frequency of Amerindian’s al-
leles among Cuban population [22] has predisposed the
appearance of KS, not only the epidemic but also the
classical variant [23].
The development of a variety of lymphoproliferative
disorders due to B cells infection by HHV-8 have been
previously reported by others [24]. Nevertheless, it was
not previously demonstrated in Cuba. Present data have
expanded the spectrum of HHV-8 associated diseases
among HIV population; maybe, the role of HHV-8 in ma-
lignant lymphoproliferation was underestimated before.
It reveals the significance of HHV-8 diagnosis for the
management of those malignancies arising in HIV popu-
lation which may be predominantly caused by subtype B.
Now, there is a controversy around the participation of
HHV-8 in NHL. In this sense, some authors have also
agreed that HHV-8 DNA detection in the lymph node
would be considered not only as opportunistic infection
but also as an agent involved in malignant lymphoprolif-
eration [25]. Furthermore, rare HHV-8 positive solid
lymphomas have been described as extra-cavitary PELs
forms [26] and Engels and colleagues have previously re-
ported a similar case of NHL from Ugandan pediatric pa-
tient [27]. On the other hand, it is possible that HHV-8
may be shed in saliva from HIV infected patient diagno-
sed with Hodgkin lymphoma, its role in this malignancy
has not been established before. Accordingly, it has to be
pointed that the number of studied patients with lympho-
ma diagnosis is limited. Therefore, the participation of
HHV-8 in lymphoproliferative disorders will need to be
The Role of Human Herpesvirus 8 Molecular Characterization in the Management of HIV
Infected Patients Diagnosed with Malignancies Associated with Its Infection
established in the future.
The studied PEL cases were the first to be diagnosed
since the beginning of Cuban AIDS epidemic in 1986. At
present, it is revealed that PEL can be caused by different
HHV-8 subtypes. In addition, the K1 amino acid changes
that were identified in the strain isolated from the same
individual are in agreement with the results obtained by
Lacoste and colleagues [28]. However, others have ob-
tained contrasting results [29] suggesting that K1 varia-
tion did not occur over the lifetime of a single infected
host. Even though ORF-K1 mutation rates were identi-
fied to be similar to genes in other human pathogens [e.g.
gen env from HIV-1 [2], there are neither evidences of
error-prone replication mechanism that would permit
HHV-8 positive selection not specialized mechanisms to
rapidly generate a high level of diversity [30]. The causes
of this extreme variability are not well understood but
host immune pressure seems to be one of the possible ex-
planations. However, the effects of Taq polymerase error
prone during PCR amplification would not be definitely
The clinical characteristic of HHV-8 primary infection
has not been well defined. The first report was done by
Oksenhendler and colleagues in an HIV subject [31] and
it has been latter described in renal and bone marrow
transplant recipients [32,33] as in children from Egypt
and Africa [34,35]. Conversely, its occurrence has been
difficult to prove since previous serological status is dif-
ficult to establish. HHV-8 DNA detection at lymph node
fragments obtained from Cuban patients diagnosed with
AIDS-related complex lymphadenopathy and vasoprolif-
erative lesion of the lymph node would be indicative of
its primary infection, since neither signs nor symptoms of
KS or other HHV-8 associated diseases were identified.
Unfortunately, the seroconversion to HHV-8 was impos-
sible to identify since no serum samples collected before
the onset of symptoms were available. Thus, it would be
impossible to arrive to this diagnosis in the Cuban stud-
ied patients. However, a closer follow up was recommen-
ded to clinicians since those individuals were recogniz-
ed at risk of developing malignancies associated with this
Gammaherpesvirus. The association between subtype A5
with AIDS-related complex lymphadenitis as a form of
HHV-8 primary infection will need to be elucidated in
the future since a small number of patients were included.
Overall, our findings suggest that although different
HHV-8 subtypes circulate among Cuban HIV-1 popula-
tion, it is not necessary to determine the infecting subtype
for their clinical management. In contrast, HHV-8 viral
load could be used as a surrogate marker for monitoring
its infection, not only in epidemic KS patients but also in
those diagnosed with different lymphoproliferative dis-
orders. Moreover, it was elucidated that the spectrum of
HHV-8 associated diseases has expanded among Cuban
HIV population irrespective the K1 subtypes. Others host
factors will need to be explored in the future if HHV-8
transmission would need to be limited among Cuban
HIV population.
5. Acknowledgements
The corresponding author is grateful for the training re-
ceived at the 16th International Bioinformatics Work-
shop on Virus Evolution and Molecular Epidemiology in
Rockville, USA, September 2010 (http://www.rega.ku-; and for the travel grant sup-
ported by Vlaamse Interuniversitaire Raad (VLIR)
(ZEIN2008PR358). Likewise, he would like to thank the
professors working on the Computational Molecular Evo-
lution course that he attended 10 - 21 April 2011 run by
Wellcome Trust Advanced Courses. We are also indebt-
ed to Dr. Mariana Varela from the Department of Vete-
rinary Medicine, University of Cambridge, Cambridge,
UK for the support in data analysis and also to Dr. Maria
Nascimento, PhD, from the London School of Hygiene
and Tropical Medicine for her suggestions in the manu-
script writing.
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