Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

12th World AIDS Conference

Abstracts 31197-31202 535

Conclusion: Approximately 18% of HIV infected women, and 14% of HIV
negatives at risk are infected by HHV8. At this time, no statistically significant correlation is apparent between HHV8 infection and presence of multiple historical,
immunologic, or clinical factors. Additional results in approximately 1400 women
will be presented.
S31197 The seroepidemiology of human herpesvirus 8 (HHV-8)
among clients attending sexually transmitted diseases
(STD) clinics in the United States, 1996
Edward Lew', H. Weinstock2, J. Black2, K. Kite-Powell2, J. Huong2, T. Woods2,
M. Gwinn2. 1600 Clifton Road NE, MS-E46 Atlanta, Georgia 30333; 2CDC
Atlanta GA, USA
Background: A sexually transmitted cofactor has been suggested in the pathogenesis of Kaposi's sarcoma (KS). HHV-8, the candidate etiological agent of
KS, is found in over 95 percent of KS lesions of all types. If HHV-8 is sexually
transmitted, one would expect a high prevalence of HHV-8 in sexually active
individuals. We therefore examined HHV-8 prevalence among clients attending
STD clinics.
Methods: Using a recombinant HHV-8 lytic antigen (Open Reading Frame
[ORF] 65, C-terminal portion residues 85 to 173) in a Western blot assay, 236
sera from patients attending 2 STD clinics in Denver and Phoenix in 1996 were
tested in a blinded fashion. A stratified sample was selected to include HIV-positive and negative men who have sex with men (MSM), HIV-negative heterosexual
men and HIV-negative women.
Results: 0/40 HIV-positive MSM, 3/38 (8%) HIV-negative MSM, 0/79 HIV-negative heterosexual men and 0/79 HIV-negative women tested positive for antibody
to the HHV-8 lytic antigen. The presence of antibody was associated with sexual
orientation when comparing MSM to heterosexual men and women (p = 0.04)
and HIV-negative MSM to heterosexual men (p = 0.03). The presence of antibody
was not associated with HIV status, race/ethnicity or clinic.
Conclusion: We have previously found this Western blot assay to have a
sensitivity of 50% in KS-positive patients. Here we found no antibody to the
HHV-8 lytic antigen among heterosexuals in this sexually active population. We
are currently screening this same sera using other newly developed assays. Our
finding that HIV status was not associated with antibody positivity suggests that
HIV infection itself may not increase the risk for HHV-8 but that sexual practices
among MSM may be an important determinant of HHV-8 infections.
31198 HHV-8 antibodies in AIDS-related Kaposi's sarcoma (KS)
patients in Russia
Vladimir E. Gurtsevitch, Elena L. Kadyrova', S.A. Galetsky', V.I. Shakhyildian2,
A.V. Kravtchenko2. Kashirskaya 24, Moscow; Cancer Research Center, RAMS,
Moscow, 2Russian Federal AIDS Center, Moscow, Russia
Objectives: a) To investigate the HHV-8 antibody presence in AIDS patients
before and after development of KS; and b) To study the anti-HHV-8 humoral
response in HIV infected individuals at different stages from the beginning of the
infection.
Design: Controlled study.
Method: Sensitive indirect immunofluorescence assay (IFA) with BCBL cell line
expressing HHV-8 (as a target cells) has been used for detection of antibodies to
capsid protein of this virus (HHV-8/VCA). Serum samples found to be reactive in
dilution >1: 160 were estimated as seropositive ones.
Results: Altogether, 25 serum samples from 14 AIDS patients have been investigated. Six samples were obtained from patients before KS development and
5 of them have been found to be positive to HHV-8/VCA. It is important to note
that there were no correlations between appearance of virus specific antibodies
and the time of KS development as well as clinical stage of the disease. Among
patients with clinical manifestations of KS HHV-8/VCA specific antibodies were
detected in 8 of 10. Interestingly, during observation time ranged from 2 to 29
months serological profile of KS patients was not practically changing: antibody
titers remained ether stable or increased no more than for one dilution. In control
group consisting of AIDS/HIV free patients with different pathologic states virus
specific antibodies in titers of 1: 160 have been unexpectedly detected in 3
patients with encephalopathy only.
Conclusions: The data obtained clearly demonstrated diagnostic value the
HHV-8/VCA specific antibodies. Their detection in HIV infected individuals may
indicate the beginning of KS development many months before the clinical manifestation of the disease. In some cases, however, above mentioned antibodies
could be detected in patients with other pathologies.

Methods: Vaccinia virus recombinants (rVV) expressing HCMV UL97 genes
carrying the following mutations: M460 - V, H520 -~ Q, ACR590-593del, C592
- G, A594 - V, L595 - S, L595 -, F, L595del, G598 - S and C607 -, Y were
generated following a reported protocol (Metzger C. et al., J Virol 68: 8423-7,
1994). The HCMV UL97 gene expression was verified by Western blot analysis
and GCV phosphorylation levels in rVVinfected 143B cells were determined by
HPLC analysis. Finally, data were subjected to statistical analysis.
Results: Intracellular GCV phosphorylation levels induced by rVV expressing
the mutated UL97 genes were greatly reduced compared to a rVV expressing
the wild type UL97 gene. In fact, a reduction of 43.6-89.7% was documented.
In addition, significant differences in the GCV phosphorylation by rVV with mutations in different UL97 regions homologous to different domains of protein kinases
were observed: compared to wild type UL97, mutation H520 - Q or M460 - V
were consistently responsible for high levels of inhibition of GCV phosphorylation
(89.7% and 84.5%, respectively), whereas different levels of GCV phosphorylation were observed in rVV with mutations in a region homologous to domain
IX (43.6-86.0%). In particular, deletion of L595 greatly impaired GCV phosphorylation (86.5%), while changes in position 592 and 598 had a minor effect on
GCV anabolism (43.6-57.3%). In contrast, autophosphorylation of pUL97 was
not affected in the mutated UL97 recombinants with respect to UL97 wild type
rVV.
Conclusion: i) generation of HCMV UL97 rVV is a valid alternative to the classical marker transfer technique (Sullivan V. et al., Nature 358: 162-4, 1992); ii)
the relevance of two additional UL97 mutations (C592 -- G, G598 - S) in determining GCV-resistance was confirmed using the new technique; iii) the gradient
of intracellular GCV-phosphorylation by different rVV may help in elucidating the
role of different UL97 mutations in conferring GCV-resistance to clinical isolates.
31201 Primary infection of human herpesvirus 6 in children with
perinatal HIV-1 infection
Uraiwan Kositanont', C. Wasi', N. Wanprapar', P. Bowonkiratikachorn2,
R. Sutthent', K. Chockephaibulkit', K. Yamanishi3. 'Department of Microbiology,
Faculty of Medicine, Siriraj Hospital, Mahidol Univ, Bangkok,
2Charoenkrung-Pracharak Hospital, Bangkok, Thailand; 3 Osaka University
Medical School, Osaka, Japan
Objectives: To investigate the potential in vivo immunological and virologic
relations between HHV-6 and HIV-1 in children with perinatal HIV-1 infection.
Design: Prospective study.
Methods: Two hundred and twenty-seven infants born to HIV-1 seropositive
mothers were enrolled. The infants were followed prospectively with visit at 1-3,
6, 9, 12, 13-18 and > 18 months. All EDTA -anticoagulated blood samples from
each subject were tested for HHV-6 and HIV serology and DNA detection as
well as T-cell lymphocyte categories. All the infants in this cohort were bottle-fed.
Antibody to HHV-6 were tested by indirect immunofluorescence assay using
antigen prepared from HST strain. Antibody to HIV-1 was detected by enzymelinked immunoassay and/or gelatin particle agglutination. Nested PCR for HIV
proviral DNA was performed in samples using two pairs of primers specific to
HIV-1 gag sequences. Nested PCR for HHV-6 DNA was performed using primers
derived from the immediate early gene locus of HHV-6. T-lymphocyte subsets
were determined by using dual label monoclonal antibodies and measured by
flow cytometric analysis.
Results: HIV-1 DNA was detected in 50 (22%) of 227 infants born to HIVseropositive mothers. The seropositivity rate increased to 84% (71/85 cases) by
one year of life among HIV-uninfected children whearas that among HIV-infected
children was 32% (6/19 cases). The earliest detection of HHV-6 infection was 1
month of age. The cumulative infection rate in HIV-infected children at 12 months
of age (33%) was lower significantly than that in uninfected (78%) (p < 0.05).
Among 19 HIV-infected children, all 9 cases (100%) showed decrease in CD4+
cells after HHV-6 infection within 5 months.
Conclusion: The earliest detection of HHV-6 infection at 1 month of age
implied that HHV-6 may infect in utero. The cumulative infection rate of HHV-6
among HIV-infected children was lower significantly than that among uninfected
children. There was an inverse correlation observed between lower degree of
immunosuppression and acquisition of HHV-6 infection.
31202 Kinetics of cytomegalovirus in vivo in HIV infected
patients versus transplant recipients under Ganciclovir
therapy
Christopher Payan', S. Kouyoudjiam1, J.M. Chennebault2, F. Cogny3, N. Ifrah4,
E. Pichard3, F. Lunel'. 'Laboraitoire de Virologie Chu Angers 4 Rue Larrey,
Angers Cedex; 2Service de Maladies Infectieuses Chu Angers, Angers; 3Service
de Nephrologie chu Angers, Angers; 4Service D'Hematogie Chu Angers, France
Objective: To analyse the short-term kinetics of cytomegalovirus (CMV) in patients after ganciclovir (GCV) treatment.
Methods: Peripheral white blood cells (WBC) of 10 HIV-infected patients
(AIDS), 6 kidney (KT) and 2 bone marrow (BMT) transplant recipients were

assessed by a CMV DNA quantitative system (HCS Murex kit), as previously
discribed (Payan et al. J. Virol. Methods 1997). These WBC were recovered from
the whole blood specimens of these patients every day up to 7 days and at days
10, 15, 20, 30 of GCV therapy.
Results: The medianes of initial CMV DNA (i) concentration in WBC, of viral
half-life and turnover calculated from the viral curves obtained for each patients
in each group are shown in the next table.

1311991

Ganciclovir (GCV) phosphorylation levels using vaccinia
virus recombinants expressing human cytomegalovirus
UL97 genes from GCV-resistant clinical isolates from
AIDS patients

Giuseppe Gerna, Fausto Baldini1, L. Samoncini1, D. Michel2, A. Zimmermann2,
M. Heuschmid2, T. Mertens2. 1 Servizio di Virologia IRCCS Policlinico, San
Matteo 27100 Pavia, Italy; 2lnst. Mikrobiol. Immunol. University of Ulm, Ulm,
Germany
Objectives: To quantitatively investigate the biological role of mutations in the
human cytomegalovirus UL97 encoded phosphotransferase detected in ganciclovir-resistant HCMV strains recovered from AIDS patients.