To assess the possible influence of human immunodeficiency virus type 1 (HIV-1) infection on the clinical course of acute hepatitis A virus (HAV) infection, 15 HIV-1-infected homosexual men and 15 non-HIV-infected age-matched subjects were compared. HAV load was higher in HIV-1-infected than in non-HIV-infected patients (P<.001). Duration of viremia in HIV-1-infected patients (median, 53 days) was significantly (P<.05) longer than in non-HIV-infected patients (median, 22 days). HIV-1-infected patients had lower elevations in alanine aminotransferase levels than did non-HIV-infected patients (P<.01) but had higher elevations in alkaline phosphatase levels than did non-HIV-infected patients (P<.001). Some HIV-1-infected patients still had HAV viremia when clinical symptoms had disappeared and alanine aminotransferase levels had returned to normal (60-90 days after the onset of symptoms). HIV-1 infection was associated with prolongation of HAV viremia, which might cause a long-lasting outbreak of HAV infection in HIV-1-infected homosexual men.

We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4؉ cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 10 4 copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy.

HIV-1 genotype assay using plasma viruses has been widely applied for detection of resistant viruses in infected individuals, whereas there are only a few reports about proviral genotype in peripheral blood mononuclear cells (PBMCs). To determine which sample, plasma or PBMC, should be used for early detection of drug-resistant viruses during antiretroviral treatment, we analyzed 275 plasma-derived and 211 PBMC-derived HIV-1 protease sequences obtained from HIV-1-infected patients during protease inhibitor (PI) therapy. In 70 of 107 pairs (65.4%) of plasma and PBMC samples taken from the same blood draws, the numbers of PI resistance-associated mutations in the plasma-derived genotype were different from those in the PBMC-derived genotype. Plasma viruses had more PI resistance-associated mutations than PBMC proviruses (P = 0.0004). Analysis of serial samples showed that plasma-derived genotype assay could detect primary mutations about 425 days earlier than PBMC-derived genotype when the plasma viral load was less than 10(4 )copies/mL. Our data suggest that genetic turnover of PBMC proviruses is slower than that of plasma viruses and that time lag between emergence of mutations in plasma-derived and PBMC-derived genotypes correlates inversely with viral load. Plasma viruses should be the material of choice for early detection of drug resistance during antiretroviral treatment.

The central nervous system (CNS) is of particular importance in human immunodeficiency virus type 1 (HIV-1) infection. First, the CNS may be difficult to access for anti-retroviral treatment and may become a sanctuary for residual viruses. Second, HIV-1 infection may lead to AIDS dementia complex (ADC) culminating in HIV-1 encephalitis. In order to examine the pattern of drug resistance and the role of encephalitis in enhancing viral redistribution to the CNS, we compared pol gene quasispecies of the spleen and brain in two patients with and two patients without HIV-1 encephalitis, who had been treated with zidovudine (AZT). Although a variable degree of AZT resistance was noted in both the spleen and brain of all patients, phylogenetic analysis indicated that quasispecies developed rather independently in the systemic circulation (spleen) and CNS (brain) of patients without HIV-1 encephalitis, while similar pol gene sequences were obtained from the two compartments of patients with HIV-1 encephalitis. env gene V3 region of patients with HIV-1 encephalitis showed distinct quasispecies in the spleen and brain. Our results suggest that HIV-1 redistribution to CNS is more active in cases with encephalitis and that HIV-1 distributed late to CNS grow actively under certain selective pressure exerted on the V3 region of the env gene.

We encountered a case of HIV-1 infection in a previously healthy man, which was characterized by rapid progression to AIDS and death within 7 months in association with high levels of antigenemia throughout the clinical course and no humoral immune response for at least 6 months. Genetic changes of the third variable domain (V3) of the envelope gene of HIV-1 in serum samples were analyzed at four time points during his rapid clinical course. The nucleotide changes were confined to a maximum of three substitutions among 105 nucleotides of the V3 region. A major population of the viral clones in this patient showed one amino acid substitution from aspartic acid (a negatively charged amino acid) to lysine (a positively charged amino acid) at position 30 from the first cysteine of the V3 loop. This substitution was thought to be associated with phenotypic changes, and viruses with this sequence in the V3 region had a strong syncytium-inducing ability in MT-4 cells. It appears that the lack of a humoral immune response accelerated disease progression in our patient and a genetic change that appeared to produce a phenotypic change occurred at an early stage of the disease.

Human immunodeficiency virus type 1 circulates in vivo as a mixture of heterologous populations (quasispecies). We previously analyzed the quasispecies of the third hypervariable region (V3) in the viral envelope glycoprotein gpl20 in an infected individual and found that the species with a basic amino acid substitution (lysine for aspartic acid) at a particular position evolved and became a distinct population within a short period, followed by progression to the typical immunodeficiency stage (S. Oka et al., AIDS Res. Hum. Retroviruses 10: [271][272][273][274][275][276][277] 1994). In the present study, we examined the biological significance of this amino acid substitution by constructing recombinant viruses with specific point mutations and comparing their replication capabilities in different cell types. The results demonstrated that the single basic amino acid substitution was In a previous study, we have analyzed sequence changes of the V3 domain of HIV-1 in serum samples of a patient, which were obtained at four different times during the clinical course, which was characterized by rapid progression to AIDS and death within 8 months (26). We have also demonstrated striking temporal fluctuations of the viral quasispecies in this patient. Perhaps the most notable finding is that the sequence with a basic substitution, lysine for aspartic acid at position 323, one of the four key positions relevant to cell tropism and 7689 on July 4, 2020 by guest

We performed a population-based sequence analysis of the envelope V3 region of human immunodeficiency virus type 1 (HIV-1) in two infected hemophiliacs. The study was conducted over 6-9 years, extending from the asymptomatic phase to AIDS. In both patients, serial analysis showed that the V3 population at the initial stage consisted exclusively of putative non-syncytium-inducing (NSI) genotypes. Several of these clones continued to be present without change for many years until the terminal stage and often represented the dominant species in the population at each time interval. On the other hand, syncytium-inducing (SI) genotypes were initially absent but appeared shortly before severe depletion of CD4+ T cells and their proportion in the population appeared to correlate with the viral load. In sharp contrast to NSI genotypes, SI genotypes displayed a significantly shorter presence. Thus, rapid gross population changes were found in SI genotypes, which were particularly frequent in the asymptomatic phase and less frequent in the terminal stage. Furthermore, the ratio of nonsynonymous nucleotide substitutions per synonymous substitutions in the V3 region in SI genotypes was higher than the corresponding value of NSI genotypes and the phylogenetic tree analysis revealed that a longer branch length was observed in SI genotypes than in NSI genotypes. These results suggest that there might be a stronger pressure for selection on SI viruses than on NSI viruses during the high CD4 counts on the contrary to the fact that emergence of SI genotypes was well correlated with the rapid decline of CD4 count.

We investigated a new p24 antigen detection system by the chemiluminescence-enzyme-immuno-assay (CLEIA) and compared it with the conventional antigen-kits (A; Abbott, B; Coulter). The detection limit of CLEIA, A, and B were 4.3 pg/ml, 11.3 pg/ml, and 5.6 pg/ml, respectively. When the patient's sera and panel sera were examined by the three method, compatible results with the detection limit were obtained. CLEIA can be completed within 35 min, and is performed by an automatic system, which can treat many samples at one time. Therefore, this system is more suitable for screening of donor blood because of the shortening of the window period.