Abstracts Vol. 1 [International Conference on AIDS (11th: 1996: Vancouver, Canada)]

Track A: Basic Science

Tu.A.2073 - Tu.A.2077

equivalents (TLE) were determined using a commercially available [ELISA (TRAx,T Cell
Diagnostics).Virus was isolated by cocultivation of patient PBMC with CD8 - depleted PHA
- blasts from seronegative donors and characterized for in vitro biological properties by
cocultivation with cells from 3 established lines. Proviral DNA obtained from lysed PBMC
and/or by direct isolation from whole blood (DNA DIRECT Dynal) was anplifi'd using
nested primers flanking the V3 region. PCR amplificates were subjected to direct DNA
sequencing.
Results: CD4+/CD8+ TLE ratios ranged from 0.01 to 0.99.Viius was isolated from I14/29
(48%) and 8/9 (89%) samples from children and adults, respectively Five of the 8 adult isolates induced syncytia in MT- 2 cells; phenotypic characterization of the pediatric isolates is
in progress. Proviral V3 DNA was amplified from 22/22 (100%) isoltes; procedures to prepare genomic DNA directly from whole blood by biomagnetic separation are being optimized.V3 sequences have been generated directly from PCR products. T Ihe sequence
homology characteristic of HIV - I subtype F in Romanian orphans petrsists while several
HIV - I subtypes in addition to subtype F have been identified among the isolates from
Romanian adults.
Conclusions: The presence of different HIV - I subtypes in pediatric and adult Romanian
populations may reflect differences in the dynamics and modes of virus transmission characteristic of the two epidemics.The low frequency of virus isolation from children could be
due to stage of disease and/or the effects of AZT treatment. Phylogenetic analysis ofV3
sequences from the follow-up samples are compared to the original pediatric isolates. In
addition, analysis of the first adult isolates from Romania will be presented.
Carol Holm - Hansen, Centre for international Health, University of Bergen. N - 502!
Bergen, NorwayTel: 47 55974980 Fax: [phone] e - mail: carol.holmn -
[email protected]
Tu.A.2073
CLONING AND SEQUENCING OF A NON T-CELL LINE TROPIC HUMAN
IMMUNODEFICIENCY VIRUS TYPE 2 (HIV-2ALI).
Azevedo-Pereira J.M., Gonsalves, J.,Vital, J.,Vieira, A., Santos-Ferreira, M.O., Moniz-Pereira, J.
Dep. of Microbiology Faculty of Pharmacy University of Lisbon, Portugal.
Objectives: HIV-2ALI is a primary HIV-2 isolate, recovered from an individual from GuineaBissau. Biological characterization revealed that HIV-2ALI is a non-syncytium-inducing (NSI)
isolate, incapable to productively infects anyT-cell line, and with a low infectivity in vitro due
to some problems at virus entry In order to molecularly characterize this HIV-2 with exclusive tropism to primary CD4+ cells, we have cloned and sequenced the proviral DNA.
Methods: Chromosomal DNA was extracted from peripheral blood mononuclear cells
(PBMCs) infected with HIV-2ALI.This DNA was subjected to a long PCR protocol to
amplify full length viral genome.These PCR products were cloned in pCR3TM plasmid and
subcloned in M I 3mp I 8 phage. Single-stranded DNA was sequenced by the dideoxynucleotide-chain termination method.
Results and Conclusions: Complete nucleotide sequence has been determined and compared to other HIV-2 and SIV isolates. Phylogenetic analysis reveals that this virus belongs to
HIV-2 subgroup A. Comparative sequence analysis and construction of chimeric viruses
could contribute to clarify the molecular determinants responsible for the low infectivity
and the non T-cell line phenotype exhibited by HIV-2ALI
Jos M. Azevedo Pereira, Dep. of Microbiology Faculty of Pharmacy, University of Lisbon,
Portugal, Fax: 35 I. 1.7934212
Tu.A.2074
ANALYSIS OF THE TEMPORAL RELATIONSHIP BETWEEN HUMAN
IMMUNODEFICIENCY VIRUS TYPE I QUASISPECIES IN SEQUENTIAL BLOOD
SAMPLES AND VARIOUS ORGANS OBTAINED AT AUTOPSY
Van 't Wout. Angelique B*, Ran LJ*, Kootstra NA*, Pals ST**, Schuitemaker Ho. Clin.ViroImmunology, Central Lab. Netherlands Red Cross Blood Transf. Serv. and Lab. for Exp. &
Clin. Immunology of the University of Amsterdam; **Academic Medical Centre; Amsterdam,
The Netherlands
Objectives: To gain a better insight into the temporal relationship between HIV-I quasispecies in tissues and in peripheral blood mononuclear cells (PBMC) of HIV- I infected individuals.
Methods: Sequential PBMC samples and various organs obtained at autopsy from 3
patients, who died of AIDS related complications, were available for analysis. A 300 basepair
region encompassing the third variable domain (V3) of the virus envelop, known to influence the biological properties of HIV- I was studied. Biological HIV- I clones were isolated
from the PBMC samples and cellular tropism and syncytium inducing (SI) capacity were
determined. In addition, virus DNA was amplified and sequenced. Genomic DNA was isolated from I cm3 of organ tissue and virus DNA was amplified by means of PCR and
cloned and sequenced using the PGEM vector system.
Results: HIV could be amplified from all organs and PBMC samples. The 3 patients showed
different distributions of HIV- I in tissues and periphery Both NSI and SI genotypes could be
detected in the different tissues.Tissue specific quasispecies were observed in brain, lung
and testis. Lymphoid tissues, such as bone marrow, lymph node and spleen harboured several different variants similar to those detected i, blood just before death. Extensive phylogenetic analysis is currently being performed or e obtained V3 sequences.
Conclusions: These preliminary results show tiss e and time specific distribution of HIV- I
quasispecies. Combining our results with the availible clinical data may a'so elucidate
whether the presence of HIV- I in tissues is cause or consequence of the pathology seen in
-lV I infection.
AB v-in t Wout, CLB, Plesmanlaan 125, 1066 CX AmsterdamThe Nethelands tel: +3 I 20
512 3679, fax: [phone],
Tu.A.2075
LIMITED HIV-I SUBTYPE B EVOLUTION SUGGESTS THAT SUBTYPE B IS A
DISTINCT VIRAL QUASISPECIES
ViadinirV. Lukashov Jaap Goudsmit. Department of Human Retrovrology. Academic
Medical Cente, University of Amsterdam, the Netherlands
In the course of the AIDS epidemic population-wide genetic variation of the pp I120 V3
region of HIV- I subtype B was shown to increase, while its consensu~s sequence is stable

over time (Kuiken et al: PNAS, 1993, 90:906 I).These observations prompted us to investigate the biological significance of HIV- I subtypes and their genetic integrityTo anticipate
future HIV- I evolution two scenarios can be envisioned.The first scenario is based on the
premise that intrasubtype HIV- I variability did not yet reach its outbound and will continuously expand over time leading to merging of the HIV- I subtypes.The second scenario
hypothesizes that the level of HIV- I subtype B variability is reaching the limit of virus
sequence distance to the consensus that is compatible with virus survival or fitness.This
scenario is based on the premise that HIV- I subtypes represent self-sustained populations,
which are able to maintain their integrity over time.We addressed this issue by analyzing
the population-wide V3 evolution relative to the subtype B consensus in 124 seroconverters (1985-1992) as well as the intrapatient evolution relative to the consensus in 55 individuals over a period of 5 years from seroconversion.
At the population level, the mean nucleotide distance of the V3 sequences to the consensus
increased significantly over time (1985 - 0.057~0.021, 1992 - 0.077+~0.029, p=0.04).The
increase of the nucleotide distance to the consensus over time was due to the accumulation of synonymous substitutions (1985 - 0.04 I~0.021, 1992 - 0.084~0.038, p<0.00 I). In
contrast, the mean nonsynonymous distance to the consensus did not change significantly
(1985 - 0.058~0.02 1, 1992 - 0.067~0.029, p>0. I), indicating selective pressure against nonsynonymous evolution away from the consensus.
For the intrapatient evolution, a significant negative correlation was found between the
direction of the evolution of the V3 sequences over the 5 year period relative to the consensus and their distances from the consensus at seroconversion. Large nucleotide
sequence distance to the consensus at seroconversion was associated with evolution
towards the consensus.
Our results indicate that HIV- I subtype B V3 region evolution is limited to an area with a
set genetic distance to the subtype B consensus sequence.This suggests that HIV- I genetic
subtypes are phenotypic entities spreading as distinct virus populations and are unlikely to
merge during progression of the AIDS epidemics. Neither the size of the subtype B quasispecies nor its center (the consensus sequence) are substantially changing in time suggesting
that HIV- I subtype B is optimally adapted to the environment.
Vladimir V. Lukashov, Department of Human Retrovirology, Academic Medical Center;
University of Amsterdam, Meibergdreef I 5, I 105 AZ Amsterdam, the Netherlands tel 31 -
20-5664522; fax 1-20-69 I 653 I
Tu.A.2076
EVIDENCE FOR IN VIVO RECOMBINATION AS A SOURCE OF HIV- I GENETIC
DIVERSITY.
Ait-Khaled Mounir*, Crandall KA#Templeton AR#, EmeryVC*. * Royal Free Hospital,
London UK; # University of Texas, Austin, USA.
Objectives:To investigate the phylogenetic relationship between HIV- I LTR proviral variants
isolated from lymph node biopsies and peripheral blood samples obtained at the time of
biopsy and subsequent to that time in 4 HIV- I infected individuals. For one individual, postmortem samples were also available.To investigate the extent of recombination events in
the variants using a newly developed method.
Methods: Phylogenetic reconstruction methods on sequence data are based on the
assumption that no recombination occurred in the set of aligned sequences. HIV- I has been
shown to undergo significant recombination events during its life cycle, however; there is a
paucity of evidence for in vivo recombination in the infected host for the generation of HIVI quasispecies. Here, we describe a new method for reconstructing intraspecific phylogenies
with the power to detect recombination in aligned sequences.
Results: At least 30 HIV- I LTR variants from each of the 4 HIV- I infected individuals were
analyzed using this new phylogenetic method.The analysis confirmed the role of secondary
lymphoid organs as a source of new variants and the direct association between the lymphoid organ histological architecture and the sequestration of HIV-I infected cells in these
organs. Moreover, we detected evidence of recombination among sequences from 3 of the
4 patients.
Conclusions: HIV- I undergoes a "fast-forward" evolution in its host, the main site of evolution are the secondary lymphoid organs where HIV- I de novo replication is high at all stages
of disease.The reverse transcriptase misincorporations and the high in vivo replication rate
of HIV- I are known to be the source of genetic diversity, here we present evidence for an
in vivo role of recombination events as another important source of this diversity
M Ait-Khaled,Virology Department, Rowland Hill Street, London NW3 2PFTel: 017 I 794
0500 x495 I Fax: 017 I 830 2854, email: [email protected]
Tu.A.2077
BREAKPOINTS IN THE FULL-LENGTH GENOMES OF INTERGENOTYPIC
RECOMBINANTS OF HIV-I AND THEIR FUNCTIONAL IMPLICATIONS
McCutchan, Francin E, Salminen MO, Carr JK, Hahn BH2-, Robertson DL3., Sharp
PM, Burke DS4 I. Henry M. Jackson Foundation, Rockville, MD: 2. University of Alabama,
Birmingham, AL; 3University of Nottingham, Nottingham. UK; 4'Walter Reed Army Institute
of Research, Rockvilie, MD.
Introduction: Inter-genotypic recombinants of HIV- I are known to arise at appreciable frequency in the global pandemic and may be of epidemiological importance: an A/E recombinant strain is largely responsible for the HIV- I epidemic in Southeast Asia. Complete
genomic sequence has been unavailable for most recombinant strains. Here we report the
full sequence of two A/D recombinants from Uganda, an A/C recombinant from Zambia, an
A/E recombinant from Thailand, and an A/G recombinant of Kenyan origin. Recombination
breakpoints between genetic segments from different clades have been mapped, permitting,
for the flrst time, an assessment of the predicted virion structures.
Methods: DNA from primary virus cultures on donor peripheral blood mononuclear cells
was used as template for long PCR amplification of virtually full-length HIV- I genomes. PCR
products were molecularly cloned and fully sequenced using fluorescent dye terminators
and an Applied Biosystems 373A automated DNA sequencer Analyses of recombination
breakpoints were performed by bootscanning, by distance scanning, or by the distribution of
phylogenetcaly informative sites.
Results: Multiple breakpoints between genetic segments fl-om different clades have been
found in all isoltes.Within mapping precision, recurrent breakpoints were found at the
osembuane-spunning domain ofgp41, in a region near the T'end of gag and in the t.J3

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