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Objective: To examine the epidemiological factors influencing the distribution and spread of HIV-1 subtypes among heterosexuals in the Netherlands., Method: A nationwide serosurveillance in 21 HIV/AIDS centres from 1997 to 1999 involved 200 individuals for whom the mode of HIV transmission was heterosexual contact or unknown. HIV-1 subtypes were determined by phylogenetic analysis of env V3 sequences and correlated with sociodemographic characteristics of the subjects and their sexual partners., Results: HIV-1 subtype B infection occurred in 121 subjects (60%). Non-B subtypes were identified in 31 (A), 24 (C), 10 (D), six (E), four (F) and three (G) individuals; one had an unclassified subtype. The proportion of subtype B was about 60% in four of the six regions of the Netherlands, but in the Northwest and Southwest regions these proportions were 76% and 46%, respectively. The Surinamese and Antilleans, large immigrant groups, were all infected with subtype B, as were almost all individuals with an unknown source. The proportions of non-B viruses did not change significantly over time in Amsterdam, where subtyping was available from 1988 onward, but a shift in the various subtype B strains was observed, suggesting introductions of new subtype B strains in Amsterdam., Conclusion: To date, HIV-1 non-B subtypes in the Netherlands are still found predominantly among heterosexuals with an epidemiological link with sub-Saharan Africa. Despite continuing introductions of non-B subtypes, the B/non-B distribution has been stable over time, most likely as a result of introductions of subtype B strains from Caribbean and South American countries.

Objective: To reconstruct the epidemiological relationships of the HIV epidemics among injecting drug users (IDU) in western Europe., Methods: HIV env V3 sequences of and epidemiological data were obtained from 145 IDU who seroconverted in three sequential periods: 1984-1988, 1989-1992 and 1993-1997. The sequences were phylogenetically analysed and examined for signature patterns characteristic of northern European IDU, including the conserved GGC codon in the V3 loop., Results: Subpopulations of genetically related HIV strains were observed in Italy, France, Scotland and Spain, in contrast to the Netherlands, Austria and Switzerland. This difference between the two groups of countries suggests that the HIV epidemics amongst IDU in the latter group was caused by multiple virus introductions. In Edinburgh and the surrounding area, most IDU were infected with the same GGC strain over the 12-year study period. The epidemic among IDU in north-western Europe started with GGC viruses, whereas in south-western Europe non-GGC viruses predominated. This geographical separation has faded during the course of the epidemic, most likely because of virus exchange among IDU populations.

Objective: To compare the performance of V3-loop peptide enzyme immunoassay (PEIA) methodologies from four different laboratories for subtyping HIV-1, and to determine the causes for the lack of correlation between V3-loop PEIA serotyping and subtyping by sequencing., Materials and Methods: Synthetic peptides derived from the amino-acid consensus sequences of the V3-loop of group M strains representing genetic subtypes A-F as well as reference strains were evaluated in PEIA by four different laboratories for their ability to accurately determine the subtype in a panel of 85 sera obtained from persons infected with known HIV-1 subtypes (28 subtype A, 34 subtype B, four subtype C, 10 subtype D, seven subtype F, one each of subtype H and G). Furthermore, the V3 loop of the corresponding virus was compared with the V3 loop of the peptides used in PEIA., Results: The correlation between HIV-1 subtyping by sequencing and V3-loop PEIA from the different laboratories varied considerably for the different HIV-1 subtypes: subtype A (46-68%), B (38-85%), C (75-100%), D (29-50%), and F (17-57%). A 70% agreement between PEIA and sequencing subtypes was observed for samples with the concordant presence of the same octameric sequences in the V3 loop of the virus and the V3 loop of the peptide used in PEIA; however, only 42% of specimens with different V3-loop octameric viral and peptide sequences yielded concordant results in V3-loop serotyping and genetic subtyping., Conclusion: Our results indicate that V3-loop PEIA methodologies used in different laboratories correlate poorly with genetic subtyping, and that their accuracy to predict HIV-1 subtypes in sera of Belgian individuals infected with different HIV-1 subtypes (A, B, C, D, F, G and H) vary considerably. The poor correlation between serotyping and genetic subtyping was partly due to the simultaneous occurrence of subtype-specific octameric sequences at the tip of the V3 loop of viruses belonging to different genetic subtypes.

Objective: To evaluate sequence evolution in relation to different rates of disease progression in infants infected with HIV-1., Design: Variability in the gp120 V3 region was analysed in HIV-1-infected children with different clinical courses, slow progression (n = 2) versus progressive disease (n = 3)., Methods: Cloning and sequencing of virus-derived DNA from uncultured peripheral blood mononuclear cells was performed at two to three timepoints from birth and up to the fifth year of life. Sequence variability was estimated by calculating the genetic distance and the proportion and ratio of synonymous and non-synonymous nucleotide substitutions over time., Results: Genetic distances were significantly shorter in children with fast progression to disease, a predominance of synonymous nucleotide substitutions also being detected at later timepoints. Conversely, a preferential accumulation of non-synonymous nucleotide substitutions was apparent in children with slow disease progression. Furthermore, a positive correlation between a decreased ratio of synonymous/non-synonymous nucleotide substitutions and the ability of children's sera to react with synthetic peptides representing the autologous virus sequence was determined., Conclusion: Data suggest that an antigenically more diverse virus population emerges in infected children with slower progression to disease as a result of a stronger immune pressure.

Objective: To address the question of whether T-cell-line adaptation of the original LAI and MN (NM) HIV-1 populations biased the interpretation of the intraindividual and population-wide virus distributions., Patients and Methods: HIV-1 genomic RNA coding for the gp120 C2V3 region was obtained from serum samples of patients LAI and MN and compared to the proviral DNA derived from simultaneously sampled peripheral blood mononuclear cells (PBMC) as well as B-and T-cell lines., Results: Two (10%) of 20 clones of HIV-1 LAI RNA and none of 16 clones of the HIV-1 MN RNA carried syncytium-inducing (SI)-determining amino-acid changes. HIV-1 LAI RNA formed on SI and two non-SI (NSI) phylogenetic clusters. The HIV-1 LAI DNA in PBMC included both SI and NSI clones but lacked one NSI cluster and contributed NSI clones to the SI cluster as well as an SI clone to the NSI cluster, indicating the existence of an intermediate SI/NSI genotype. In vitro culture using either primary cells or B-cell lines yielded only SI clones, distributed over two SI/NSI mixed clusters. Long-term propagation in T-cell lines further restricted the clonality and yielded SI clones belonging to only one cluster. On the population level, HIV-1 LAI, MN and BRU sequences all clustered according to the individual host and apart from each other and separate from the epidemiological controls without notable influence of SI/NSI distinction or cell-culture adaptation., Conclusion: Our data demonstrate a selection bias during the cell-line adaptation of HIV-1 strains LAI and MN with more impact on phenotypic than on genotypic characteristics.

Objective: To carry out, within France, a large-scale molecular epidemiological investigation on the principal neutralizing determinant of HIV-1, located in the third variable region (V3) of the envelope protein. Such investigations are of the utmost importance in the identification and monitoring of the distribution and spread of different viral strains internationally., Design: Using polymerase chain reaction (PCR), we examined the genetic variation of the V3 region sequences of 28 HIV-infected patients from Paris, France., Results: Comparison of the Parisian V3 loop sequences with other published data indicates that the range of diversity in France is included within that of a large group that contains sequences from North America, the rest of Europe, Japan, India and Africa. Variability appears to be lower in the V3 loop than in its flanking regions. Five out of the six putative N-linked glycosylation sites show preferential alterations to charged amino acids. We report two motifs at the tip of the loop that have not been described previously., Conclusions: The structural homogeneity and the wide geographic representation of the major V3 group suggests that a common strategy could be applied to a large proportion of isolates in the development of a broad-spectrum HIV vaccine.