HIV-1 entry into human intestinal cells involves both GalCer and CXCR4/fusin. HIV-1 isolates such as 89.6 that are able to use CXCR4/fusin as coreceptor, but do not bind to GalCer, do not infect these cells. These data raise the possibility that CXCR4/fusin may function as a coreceptor for HIV-1 entry into CD4-/GalCer+ intestinal epithelial cells.

The susceptibility of human immunodeficiency virus type 2 (HIV-2) to protease inhibitors (PI) is largely unknown. We studied HIV-2 protease genes from 21 HIV-2-infected patients who were exposed or not exposed to PI. The aim of this study was (i) to characterize the polymorphism of HIV-2 protease in the absence of drug, (ii) to know whether the HIV-2 protease gene naturally harbors HIV-1 drug resistance codons, and (iii) to identify mutations emerging under PI-selective pressure. Sixty-five HIV-2 RNA or proviral DNA samples were directly sequenced from the plasma or peripheral blood mononuclear cells of 8 patients who had received PI and 13 patients who had never received any antiretroviral. In untreated patients, the highest amino acid variability in HIV-2 protease was observed at positions 14, 40, 43, 46, 65 and 70, and seven codons (10V, 32I, 36I, 46I, 47V, 71V, and 73A) associated with drug resistance in HIV-1 were highly prevalent. In addition, at six positions (positions 7, 46, 62, 71, 90, and 99), the amino acid variability or the amino acid frequencies or both differed significantly in PI-treated and untreated patients, suggesting that mutations 7K3R, 46V3I, 62V3A/T, 71V3I, 90L3M and 99L3F were occurring under PI-selective pressure. At these positions, at least one sample simultaneously harbored both wild-type and mutated codons, while substitutions at positions 62, 71, 90, and 99 were confirmed in a longitudinal analysis. Moreover, the presence of codons 46I and 99F in the absence of drug in HIV-2 subtype B proteases may reflect natural resistance to PI. In conclusion, the present study revealed that HIV-2 strains harbor specific patterns of natural polymorphism and resistance.

Multiple nucleoside resistance involves specific mutational patterns of the HIV-1 pol gene that are independent of the classic mutations conferring resistance to individual dideoxynucleosides. These include a cluster of five mutations in the reverse-transcriptase (RT) coding region (A62V, V75I, F77L, F116Y, and Q151M) generally referred to as multidrug resistance (MDR) mutations, and insertions of one or several amino acid residues between codons 67 and 70 of RT, a flexible region joining two antiparrallel beta sheets (beta3-beta4 insertions). The objectives of this study were (i) to determine the prevalence of multidrug resistance genotypes (MDR mutations and beta3-beta4 insertions) in a cohort of 632 patients who were extensively pretreated with anti-HIV drugs and not responding to their current antiretroviral therapy, and (ii) to analyze the association of multidrug resistance genotypes with other resistance mutations in the RT and protease genes. Among viruses sequenced from these patients, 15 (2.4%) of them contained an insertion and 2 (0.3%) contained a deletion in the beta3-beta4 finger subdomain of RT. In 9 cases, the insertion was associated with a D67S, G, or E mutation. In addition, we identified 13 (2.1%) viruses harboring specific MDR mutations (mainly Q151M and/or A62V, V75I, F116Y). Interestingly, the A62V mutation was found in 6 of the 15 strains with an insertion, whereas the other MDR mutations were not observed in insertion mutant strains. Especially high levels of resistance to zidovudine were observed for viruses with a beta3-beta4 insertion in the background of A62V, L210W, and T215Y. Otherwise, MDR mutations and beta3-beta4 insertions were found in association with the classic mutations conferring resistance to zidovudine, lamivudine, nonnucleoside RT inhibitors, and protease inhibitors, according to treatment history. Finally, we observed a genome with a deletion of codon 70 associated with a Q151M MDR mutation. These data suggest that the emergence of HIV-1 multidrug resistance, which may occur in various genetic contexts, poses a challenging problem in formulating treatment strategies.

The present study examines the distribution of Hepatitis C virus (HCV) genotypes in Marseille, France in 2001-2002 and evaluates the efficiency of two in house direct sequence PCR protocols based on 5'NC analysis or NS5B analysis. By 5'NC sequencing, the distribution of 535 HCV strains derived from patients attending gastroenterology and AIDS referral centers, or dialysis units was as follows: 33% were infected by genotype 1a; 26% by 1b; 7% by 2; 22% by 3a; 10.7% by 4. In univariate analysis, HCV distribution was associated with age and source of infection, whereas in multivariate analysis only injecting drug use was an independent determinant for genotype distribution. Among the 535 specimens submitted to 5'NC direct sequencing, 18% could not be classified accurately into subtypes. A subset of 187 samples was amplified efficiently and sequenced by targeting the NS5B region of the viral genome. The two methods yielded concordant results in 70% of cases. Specimens unsubtypeable or misclassified most frequently by 5'NC analysis were type 1b and subtypes 2a/2c and 4a/4c. The data show that 5'NC direct sequence analysis is a sensitive method to identify genotypes in all cases, but that it can lead to subtyping misclassification (in particular, subtype 1b and 1a) or doubtful results (in particular subtypes 2a/2c and 4a/4c). Conversely, NS5B direct sequence assay, based on phylogenetic analysis, allowed better discrimination between subtypes. These two approaches are complementary and should be made available in clinical laboratories to ensure a reliable survey of HCV strains.

To assess the molecular epidemiology of human immunodeficiency virus type 1 (HIV-1), a screening method was developed for identification of non-B subtypes from sequence data obtained for resistance testing. The method is based on the evaluation of the percentage of divergence of a given sequence from the reference B subtype HXB2. Analysis of 1720 reverse-transcriptase (RT) and 1824 protease sequences stored in a database allowed for the determination of a threshold level of divergence from HXB2 above which a non-B subtype could be unambiguously characterized regardless of the pattern of resistance mutations (>8.6% for RT; >10.8% for protease). This conclusion was validated by phylogenetic analysis of RT, protease, and env genes. Overall, 72 (4.2%) and 73 (4.0%) non-B sequences were identified in the RT and protease coding regions, respectively. This method allows for the rapid detection of non-B subtypes among retrospective, recent, and future RT and/or protease sequence databases.