Serological and virological evidence of non-sexual transmission of human herpesvirus type 8 (HHV8).

Abstract

To evaluate whether or not human herpesvirus 8 (HHV8) can be transmitted through a non-sexual route a serological survey was carried out in a group of 51 catholic nuns. The seroprevalence rate and the geometrical mean antibody titre to anti-latent HHV8 antigen were similar in nuns and in a group of 60 women, matched by age, in the general population (27 vs. 24%; 1028 vs. 1575, respectively). Moreover, by using nested polymerase chain reaction (PCR), HHV8 DNA sequences were detected in 7 of 16 (43.8%) saliva and peripheral blood mononuclear cells (PBMC) from patients with classical Kaposi's sarcoma (KS) and in 3 out of 7 (42%) AIDS-KS patients. None of 5 HIV positive persons who did not have KS tested positive for HHV8 DNA. HHV8 DNA sequences were also detected in 2 of 12 (17%) saliva and 1 PBMC sample out of 12 healthy HHV8 positive individuals (age range: 30-80 years old). This paper suggests that non-sexual transmission of HHV8 is operating in our geographical setting and saliva may be a potential source of HHV8 spreading in the general population.

Full text

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Epidemiol. Infect. (2000), 125, 671–675.Printed in the United Kingdom
? Cambridge University Press
Serological and virological evidence of non-sexual
transmission of human herpesvirus type 8 (HHV8)
F. VITALE?, E. VIVIANO?, A. M. PERNA?, F. BONURA?, G. MAZZOLA?,
F. AJELLO?  N. ROMANO?*
?Dipartimento di Igiene e Microbiologia ‘G. D’Alessandro’ Uni?ersita ? degli Studi, Via del Vespro, 133,
90127 Palermo, Italy
?Azienda Unita ? Sanitaria Locale 6, Presidio Ospedaliero Malattie Infetti?e ‘Guadagna’, Palermo, Italy
(Accepted 17 April 2000)
SUMMARY
To evaluate whether or not human herpesvirus 8 (HHV8) can be transmitted through a non-
sexual route a serological survey was carried out in a group of 51 catholic nuns. The
seroprevalence rate and the geometrical mean antibody titre to anti-latent HHV8 antigen were
similar in nuns and in a group of 60 women, matched by age, in the general population (27 ?s.
24%; 1028 ?s. 1575, respectively). Moreover, by using nested polymerase chain reaction (PCR),
HHV8 DNA sequences were detected in 7 of 16 (43?8%) saliva and peripheral blood
mononuclear cells (PBMC) from patients with classical Kaposi’s sarcoma (KS) and in 3 out of
7 (42%) AIDS-KS patients. None of 5 HIV positive persons who did not have KS tested
positive for HHV8 DNA. HHV8 DNA sequences were also detected in 2 of 12 (17%) saliva
and 1 PBMC sample out of 12 healthy HHV8 positive individuals (age range: 30–80 years old).
This paper suggests that non-sexual transmission of HHV8 is operating in our geographical
setting and saliva may be a potential source of HHV8 spreading in the general population.
INTRODUCTION
Kaposi’s sarcoma-associated herpesvirus (KSHV) or
human herpesvirus 8 (HHV8), was initially discovered
in AIDS-related Kaposi’s sarcoma (KS) by repre-
sentational difference analysis [1]. Subsequently, by
polymerase chain reaction (PCR) and serology, it was
shown that HHV8 is strongly associated with all
epidemiological forms of KS (human immuno-
deficiency virus (HIV) associated, classic, endemic
and post-transplantation) [2].
A serological survey in Sicily, where the incidence
of classical KS was high before the AIDS epidemic,
showed that HHV8 infection is widespread in the
general population [3]. The definitive route(s) of
* Author for correspondence.
HHV8 transmission is still not well established, but
epidemiological and serological data suggest that
multiple modes may play a role.
Although HHV8 seropositivity has been shown to
be more closely linked to groups postulated a priori
to have a high risk of acquiring sexually transmitted
diseases (STD) [3, 4], antibodies to HHV8 were also
detected in prepuberal children in Sicily and in Africa,
where KS is endemic [3, 5].
To provide further information about a non-sexual
transmission of HHV8 infection, we carried out a
serological survey in a group of catholic nuns, living in
a religious rest house, in order to compare their
relative HHV8 antibodies prevalence with that
detected in a group of women, matched by age, in the
general population. We also conducted a molecular
study to detect HHV8 DNA in saliva and peripheral

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672F. Vitale and others
blood mononuclear cells (PBMC) of HHV8 anti-
bodies positive individuals with and without KS and
HIV infection.
METHODS
Patients and specimens
Between March and May 1999, 52 nuns who were
living in a religious retirement home in Palermo were
invited to participate in the study. All were elderly
(age range 70–90 years, median 78 years) and had
taken the veil as young adults (ages 18–22 years).
Fifty-one of the 52 gave their consent to participate to
the study; one declined because of ill health. Of the 51,
24 had been elementary school teachers, 12 had been
local nurses, 4 had worked in foreign missions, and 11
had provided general services to their convents (2
tailors, 5 gardeners, 4 cooks). None had received an
organ transplant, and only three had been transfused
in the previous 20 years. HHV8 seroprevalence in the
51 nuns was compared to that in 60 age-matched
women in the general population, whose residual sera
were obtained following the completion of routine
chemistry analyses. All gave their consent to be
included into the study.
Saliva and PBMC were collected from 16 classical
KS (mean CD4? cell count: 590?mm?), 7 HIV-
positive with KS (mean CD4? cell count: 261?mm?),
5 HIV-infected subjects without KS (mean CD4? cell
count: 288?mm?) and 12 adult blood donors (mean
CD4? cell count: 823?mm?). All subjects were HHV8
antibody positive as determined by an indirect
immunofluorescence assay using the BCBL1 cell line
as described [3]. None of the KS patients had oral
lesions.
IFA
Antibodies to latent and lytic antigens of HHV8, were
detected as previously described [3] by using BCBL-1
cell line. To induce HHV8 activation, the BCBL-1
were treated with 20 ng?ml tetradecanoyl phorbol-
ester acetate (TPA, Sigma Aldrich, Italy) and
200 U?mlhumanrecombinant
(Boehringer–Mannheim, Germany) for 5 days.
Uninduced and TPA-induced BCBL-1 cells were
collected, washed in PBS pH 7?4, spotted on slides, air
dried and fixed in cold acetone for 10 min. Fixed slides
were incubated with human sera diluted 1 in 120 and
interleukin6
then stained with rabbit anti human IgG-fluorescein
isothiocyanate conjugate (Sigma Aldrich, Italy).
PCR analysis
DNA from 100 µl of unstimulated whole saliva
samples was extracted by boiling with 25% w?v
Biorad Chelex 100 [6]. PBMCs, isolated by ficoll-
hypaque centrifugation of heparinized blood, were
processed for DNA extraction by phenol-chloroform
as described previously [7]. The presence of HHV8-
DNA was determined by PCR analyses: 1 µg and
15 µl of the DNA, respectively from PBMC and
saliva, were used for each PCR reaction. Samples were
screened for HHV8 open reading frame (ORF) 26 by
nested-PCR amplification [7]. All positive PCR
samples were also confirmed by second nested-PCR
that amplified a 197 bp fragment of ORF 72 (viral
cyclin D gene) [8].
To ensure that negative results were not due to non-
specific inhibition of PCR, an internal positive control
for human β-globin gene was included in each saliva
specimen and tested by PCR, using β-globin primers.
Statistical analysis
Comparison of geometrical mean titre (GMT) of
HHV8 antibodies was made with Student t test.
RESULTS
Table 1 shows the results of anti HHV8 antibodies
detected in sera of nuns and age-matched healthy
women.Fourteenofthe51(27%)nunshadantibodies
to HHV8 antigens.
This result is quite similar to that detected in the
age-matched women in the general population (15 of
60, 25%). Moreover, sera of nuns showed a GMT
(1029) to latent HHV8 antigen that was similar to the
general population (1575) (P?0?5).
In the molecular study, we found HHV8 DNA
sequences in saliva from 7 (43?8%) of 16 patients with
classical KS, and in 3 (42%) of 7 patients with AIDS-
KS, but not in the saliva of 5 HIV-positive individuals
without KS (Table 2).
We also analysed the saliva of 12 healthy HHV8
infected donors, divided in 2 age groups: 6 donors in
the age range (30–49 years) of the HIV positive
individuals with and without KS, and 6 in the age
range (50–80) of the classical KS patients. The same

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673Non-sexual transmission of human herpesvirus 8
Table 1. Pre?alence of HHV8 antibodies in a community of nuns matched
by age (70–90 years old) to healthy women of general population
Group No. tested
No. (%) with HHV8
antibodies
GMT* latent
HHV8 antibodies
Nuns
Healthy women
51
60
14 (27?4%)
15 (25?0%)
1028?9†
1575?0†
* Geometric mean titre.
† Student t test. t?0?46 P?0?5.
Table 2. HHV8-DNA detection by PCR in sali?a and peripheral blood
mononuclear cells from HHV8 antibodies positi?e subjects with and
without Kaposi’s sarcoma and HIV infection
Clinical status
HHV8-DNA in saliva
HHV8-DNA in PBMC
Number
positive?tested%
Number
positive?tested%
HIV-negative with KS
HIV-positive with KS
HIV-positive without KS
Healthy donors
Age range: 30–49
Age range: 50–80
7?16
3?7
0?5
43?8
42
0
7?16
3?7
0?5
43?8
42
0
1?6
1?6
16?6
16?6
0?6
1?6
0
16?6
frequency of HHV8 DNA detection (16?6%) was
observed in the saliva of these healthy HHV8-infected
donors regardless of age (Table 2). HHV8 DNA
sequences also were detected in PBMC of the HHV8
infected individuals. Although nearly half of the
subjects with classical KS or AIDS-KS had HHV8
sequences in their PBMC, compared to only 1 of the
17 subjects without KS lesions.
The presence of virus in saliva did not always
correlate with its detection in PBMC. In fact only
three subjects with classical KS and one with AIDS-
KS had HHV8 DNA detected in both saliva and
PBMC. There was no association between the CD4?
cell count and detection of HHV8-DNA either in
saliva or PBMC in HIV infected individuals.
DISCUSSION
Serological assays have revealed great geographic
variability in the prevalence of HHV8 infection.
Although to date there is no serological ‘gold
standard,’ an international pattern of HHV8 sero-
prevalence is apparent. There are substantial studies
showing a very low viral circulation in the general
populations of the United States and northern Europe
[9, 10] whereas in the countries of the Mediterranean,
HHV8 infection begins in childhood and increases
steadily with age, reaching approximately one-quarter
of the elderly populations [3]. In Africa, HHV8
infection also begins in childhood, increasing steeply
with age such that the majority of young adults are
antibody positive [5].
What it is not yet well established are the modes of
transmission of virus throughout the general popu-
lation. In a previous paper [3], we found that high
seroprevalence rates in Sicily were observed in the
older individuals, reaching 22?3% of those who were
51–70 years old. Among younger subjects, sero-
prevalence also was significantly increased among
homosexual men and female prostitutes, suggesting
that sexual contact may be a major mechanism for
HHV8 transmission. However, HHV8 antibodies also
were present in prepuberal children.
In this study we have sought HHV8 antibodies in a
group of Catholic nuns as an indirect effort to assess
whether or not HHV8 can be transmitted through a
non-sexual route. These elderly women are assumed
to have had little or no sexual activity. They had lived
their entire adult lives in convents and had worked
with children and in hospitals. Our data demonstrate
that HHV8 seroprevalence was as high in the nuns as
it was among presumably sexual active women in the

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674F. Vitale and others
general population. This suggests that non-sexual
transmission is operating in Sicily.
Several reports have cited saliva as the main vehicle
of transmission [11–14]. In our study, HHV8 DNA
was detected in saliva from nearly half (43?8%) of
patients with classical and AIDS-associated KS. The
source of viral production is unknown. No patient
had an oral lesion, and therefore it is unlikely that the
viral shedding was due to the local viral production by
the spindle cells.
The detection of the virus in saliva might be due to
infected PBMC in the mouth. We found, however,
that the virus was notalways present in an individual’s
circulating PBMC and saliva. Therefore, HHV8
detection in saliva does not merely reflect its presence
in PBMCs. Perhaps, like Epstein–Barr virus, HHV8
replicates in oropharynx epithelial cells.
The presence of the virus in the saliva may be due
to its primary replication or its reactivation as occurs
in other herpesviruses infections. As we noted direct
correlations of age with both antibody seroprevalence
and antibody titres to HHV8 latent antigen [3, 15] we
suggested that HHV8 reactivation may be common in
elderly healthy HHV8 infected individuals.
This hypothesis was not supported by our current
results, detection of the virus in saliva was unrelated
to age. However, we cannot exclude the possibility
that the virus may undergo reactivation and rep-
lication in other sites. The virus, indeed, was present
in PBMC. HHV8 has been previously identified in
PBMC of AIDS-KS and classical KS patients, and its
detection in HIV patients was suggested to be
predictive for their eventual development of KS [16].
Nevertheless, the main reservoir of the virus has yet to
be determined.
Although this and other studies demonstrated the
presence of virus in saliva [11–14] the epidemiological
and medical implications of salivary shedding are at
present difficult to evaluate. One recent study, how-
ever, detected a higher prevalence and copy number of
HHV8 in saliva compared with semen [14], suggesting
that oral contact might carry more of a risk for
transmission than intercourse [17]. Larger and well
designed longitudinal studies are need to assess the
role of HHV8 at different sites for the transmission of
the virus in the general population.
ACKNOWLEDGEMENTS
We thank James J. Goedert for careful review of the
manuscript and helpful comments. This paper was
supported in part by a grant from Ministero
dell’Universita ?
edella
Tecnologica, Italy.
RicercaScientificae
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