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

22 Abstracts 11198-11203

12th World AIDS Conference

S11198 Subtyping of HIV-1 in Mexico by envHMA and
gagPCR-FLP
Carmen Soler Claudin, Jose Carmen Gudiho Rosales. Unidad de Investigacion
En Retrovirus Fac. Quimica Unam/Indre SSA, Humanos Carpio 470 Col. Sto.
Tomas 11340, Mexico
Objectives: To compare the results of genetic subtyping of HIV by the env
heteroduplex mobility assay (envHMA) and by gagPCR-FLP.
Methods: DNA isolated from HIV-infected individuals PBMC's was used to amplify the gag and env regions. For envHMA, the Heteroduplex. Mobility Analysis
version 2 kit, donated by the NIH Reference Reagent Program was used. For
gag, two sets of primers were designed within conserved regions that amplify a
732 bp segment (positions 815-1547 from the HIV BRU strain). The amplicons
were analyzed by agarose gel separation after digestion with restriction enzimes
selected by computer analysis of HIV-1 reported sequences (Los Alamos Human
Retroviruses Database, 1995).
Results: Up to now, 236 DNA samples have been tested by envPCR, (all PCR
positive for the human p-globin gene amplification), but only 59 yield env amplicons (25%). Eleven of these were typed by HMA and shown to be B subtype. Two
samples gave hetroduplex bands of similar Rf with the B prototypes and the C1
reference strain. Eighty eight samples have been tested by gagPCR, and 55 gave
positive results (62%). Digestion of gag amplicons with Ddel in 41 samples gave
the common pattern of bands expected for the B subtype (bands of 125, 140 and
370 bp). The other 14 samples lack the 370 bp fragment but have bands between
500-700 bp in size, which are found within B, D and G subtype sequences.
Conclusion: In our study, a low percentage of samples were envPCR positive.
This could be due to problems with inhibitors in our PCR procedure, but we
cannot discard poor annealing of primers due to differences in sequence within
our DNA's. Although we found gagRFLP patterns of B subtype strains in most of
our samples, other gag genotypes may be present in our population.
|11199 Escape recombinants of HIV-1 in a dually-infected
transfusion recipient
Ricardo Sobhie Diaz1, P. Zanotto2, A. Mayer3, M.P. Busch4, E. Holmes5. 1Dipa,
EPM, Unifesp, R Botucata, 740, Sao Paulo, SP 04023; 2Federal University of
Sao Paulo (Unifes), Sao Paulo SP, Brazil; 3Aaron Diamond AIDS Research
Center, New York, NY; 4Blood Centers of the Pacific, San Francisco, CA, USA;
5Oxford University, Oxford, UK
Background: Before blood donations were routinely screened for HIV, two distinct
viral populations from unrelated blood donors (D1, D2) infected a multiply-transfused newborn infant (DR). This dual infection resulted in re-combination between
the two infecting viral strains (J Virol 1995; 69: 3273). Here, we continue the analysis of this epidemiologic cluster to address the issue of strain competition in the
context of selective correlates of recombination, in order to better understand the
nature of the processes at play.
Methods: cDNA from plasma was end-point diluted and amplified for the tat
and envV3-5 regions of HIV-1 by nested PCR, and sequenced. Sequences from
the members of the cluster were phylogenetically analyzed.
Results: The V3 region in DR genomes showed an intricate mosaic pattern
of recombination between parental D1- and D2-derived genomes. In the V4-5
region, however, all but one DR genome (20/21) had only D1-derived se-quences.
The D2-derived env regions were shown to be under positive selective pressure
(dn:ds >1, p < 0.001) but the D1-derived env regions were not. Tat region
sequences in DR showed D1 and D2-complexes without bias.
The bias in DR towards the absence of D2-derived V4-5 sequences can be
explained by a process of escape recombination if D2-motifs elicited a stronger
host response than D1-motifs, thereby conferring a selective advantage on recombinants carrying D-1 V4-5 sequences.
11200 1 Possible restrictions of G-A hypermutation on HIV-1
genome
Roberto Rabinovich, Silvia Marquina, D. Gutson, O. Libonatti. University of
Buenos Aires, Paraguay 2155 Piso 11, Buenos Aires, Argentina
Objective: To study possible restrictions of preferential mutations imposed by
overlapping open reading frames and the conservation of HIV-1 RNA secondary
structure stability.
Materials and Methods: All possible G-*A and A--G substitutions were simulated one at each time on nucleotide sequence of the env gen of HIVMN. For each
simulated sequence, the free energy folding of RNA molecule was calculated.
This energy was compared to the one of the wild type RNA molecule. Mfold
program designed by Zucker was used.
Results: Through the whole env gene, G-,A mutations resulted in a loss of
RNA stability in average per mutatin average per mutation of 1.20 Kcal/mol. Instead, A-G changes
lead to a gain of RNA stability of 0.96 Kcal/mol. When taking into account whether
the mutated nucleotide was paired or not, it happened that the loss of stability
was greater when the residue was paired in the case of G--A mutations, but in

the case of paired A mutated to G there was not any loss of energy, instead, the
gain of stability was lower than in the case of unpaired A.
The preferential usage of codons with A in the third position (NNA) was restrained in regions of overlapping open reading frames. Hence, in the region
where gag and pol overlap, the NNA/NNG ratio was 1 for the gag ORF and 2
for pol gene. These ratios were lower than those calculated for each whole gene
(gag: 2.5 and pol: 3.17).

Conclusion: In lentiviruses, the A richness has been associated with a preferred
G-A substitution. Although amino acid sequence conservation can restrain the
accumulation of A, as it was demonstrated in overlapping genes, conservation
of RNA secondary structure stability would be an aditional restriction of G-aA
hypermutation.
11201 Determination of genetic subtypes of HIV-1 from Korean
patients with advanced AIDS
Kang-Won Choe1, M.D. Oh1, J.W. Kim1, D.H. Shin1, S.W. Park1, H.S. Shin2
K.H. Park3. 1Dept. Internal Medicine, Seoul National University Hospital,
Yongon-Dong 28, Seoul; 2Chungbuk University College of Medicine, Choengju;
3Clinical Research Institute, SNUH, South Korea
Background: To determine the subtypes of HIV-1 in Korean patients with advanced AIDS, we analyzed the nucleotide sequences of HIV proviral DNA and
constructed a phylogenetic tree.
Methods: Peripheral blood mononuclear cells (PBMCs) were collected from
19 patients with AIDS who were referred from all over the country. DNA was extracted and proviral V3 env sequence was amplified by polymerase chain reaction
(PCR). DNA sequences of PCR product were determined by cycle sequencer
with fluorescence labeled dye terminators and aligned with a set of reference
sequences. A phylogenetic tree was constructed by neighbor joining method.
Results: Enrolled patients were in the advanced stage of AIDS with CD4 counts
ranging form 2 to 40 (median 17). Sequencing analyses of the PCR products
(ca 330 bp) revealed eighteen cases (95%) of subtype B and one case (5%)
of subtype A. The tetrameric motifs at the tip of the V3 loop were comprised of
GPGR, GPGS (six cases, 32%, respectively), GPGK, GPGG (two cases, 11%,
respectively), and GPGQ (one case, 5%).
Conclusions: Subtype B of HIV-1 was predominant and only one case was of
different clade (subtype A) in advanced AIDS patients in Korea.
S11202 Seroreactivity to HIV-1 peptides in pregnant women in
Porto Alegre, RS State, Brazil
Regina Loureiro1, J.S. Casseb2, V. Bongertz3, S. Asquidanini4, E. Bet5,
V.L.B. Silveira5, C.S. Dias5. 1Rua Humberto De Campos 50, Porto Alegre; 2Sao
Paulo State University, Sao Paulo, SP; 30svaldo Cruz Foundation, Rio De
Janeiro, RJ; 4Santa Casa of Porto Alegre Hospital, Porto Alegre, RS; 5Public
Health Secretariat of RS State, Porto Alegre, RS, Brazil
Background: Brazil is the largest country in the South America and reports the
majority of AIDS cases reported in the region (116389 - 1980 to 08/30/1997).
Rio Grande do Sul is the southeast state in Brazil and has the third number of
AIDS cases and the most prevalence of HIV/AIDS among women of all Country.
The large land mass cultural, economical, and climatologic diversity are also
reflected into the HIV-1 genetic variation in Brazil. Recent studies have shown
that three subtypes (B, F and C) circulate in Brazil. Some investigators believe certain subtypes of HIV-1 are preferentially transmitted by the heterosexual
route.
Methods: A total of 1800 pregnant women who were admitted to Maternal
Hospital in Porto Alegre were invited to participate of this study during 1995.
Fifty-two (52/1800; 2.9%) of the sera samples were confirmed to be HIV-1 positive by Western blot and twenty-six gave indeterminate samples were tested by
synthetic peptides representing subtypes which are circulating in Brazil.
Results: We find in this samples: subtype B Us/European strain (7-13.5%), subtype B-Brazilian strain (6-11.5%), subtype F (5-9.6%), and subtype C (18-34.6%);
nineteen percent (10 samples) was indeterminate and 6 (11.5%) negative. Of the
26 HIV-1 indeterminate sera samples, two typed as subtype C, two were not
available for subtyping, and the rest were negative for all peptides tested. The
two subtype C sera also were positive for a synthetic peptide representing gp41
of MN strain, suggesting that these women were in seroconversion period.
Conclusions: We observed that HIV-1 subtype C was the most prevalent
subtype in this sample, contrasting with that seen in other regions of the Brazil..
The presence of the subtype C in this population appears to be geographically
restricted. It is possible that the presence of subtype C may be association with
high prevalence heterosexually acquired of HIV-1 in low-income women of Porto
Alegre. These findings will be relevant in the design of intervention programs,
such as vaccine development in Brazil.
S11203 HIV-1 variability in Morocco
Rajae El Aquad'2, Elmir El Harti2, S. Amzazi3, H. Himmich4, J.C. Gluckman5,
F. Simon6, A. Benjouad3. 2Laboratoire D'lmmunologie; 1 lnstitut National
D'Hygiene, 27 Avenue, IBN Batouta B.P 769 Rabat; 3Laboratoire De Biochimie,
Faculte des Sciences, Rabat; 4Service Des Maladies Infectieuses, Hospital IBN
Rochd, Casablanca, Morocco; 5Laboratoire D'lmmunologie - Hospital
Pitier-Salpetriere, Paris; 6Laboratoire De Virologie - Hospital Bichat, Paris,
France

Objectives: To investigate the HIV1 subtypes circulating in Morocco, wich is
located between Western Europe countries and South African countries where
the patterns of HIV1 subtypes are different.
Methods: 200 sera HIV1 positive of Moroccan patients living in representative
geographic areas of Morocco, were studied by using a peptide specific enzyme
immunoassay, wich is based on in vitro competition for antibodies binding between
peptides of V3 consensus sequences of clades A-F.