The results of the present study show that M89I/V is associated with PI experience in subtypes C, F and G but not in subtype B. M89I/V should be considered a secondary PI mutation with an important effect on nelfinavir susceptibility in the presence of L90M.

Tenofovir disoproxil fumarate (TDF) genotypic resistance defined by K65R/N and/or K70E/Q/G occurs in 20% to 60% of individuals with virological failure (VF) on a WHO-recommended TDF-containing first-line regimen. However, the full spectrum of reverse transcriptase (RT) mutations selected in individuals with VF on such a regimen is not known. To identify TDF regimen-associated mutations (TRAMs), we compared the proportion of each RT mutation in 2873 individuals with VF on a WHO-recommended first-line TDF-containing regimen to its proportion in a cohort of 50,803 antiretroviral-naïve individuals. To identify TRAMs specifically associated with TDF-selection pressure, we compared the proportion of each TRAM to its proportion in a cohort of 5805 individuals with VF on a first-line thymidine analog-containing regimen. We identified 83 TRAMs including 33 NRTI-associated, 40 NNRTI-associated, and 10 uncommon mutations of uncertain provenance. Of the 33 NRTI-associated TRAMs, 12 – A62V, K65R/N, S68G/N/D, K70E/Q/T, L74I, V75L, and Y115F – were more common among individuals receiving a first-line TDF-containing compared to a first-line thymidine analog-containing regimen. These 12 TDF-selected TRAMs will be important for monitoring TDF-associated transmitted drug-resistance and for determining the extent of reduced TDF susceptibility in individuals with VF on a TDF-containing regimen.

The human immunodeficiency virus type 2 (HIV-2) is responsible for 4. 5% of AIDS cases in Portugal. Six HIV-2 subtypes have been described so far, subtype A being proposed as more pathogenic than the rest. The relationship between the clinical status and levels of both cellular and plasma HIV-2 viraemia is not well known, nor their modifications under antiretroviral therapy. Thirty-two consecutive HIV-2 infected persons (17 men, 15 women) attending two different hospitals in Lisbon in 1997 were enrolled prospectively in the study. All but 4 individuals most likely acquired the infection through heterosexual contact. More than half of the study population was of African origin, mainly from Guinea-Bissau. Eleven (34.4%) patients had developed clinical manifestations included within the B or C groups of the CDC classification system for HIV infection, with the rest being asymptomatic. Half of the population was undergoing antiretroviral treatment at the time of the study. HIV-2 subtypes were investigated using a new Nef-based restriction fragment length polymorphism (RFLP) method that allows differentiation of the main two variants, A and B. Plasma viral load was quantified using a new quantitative competitive reverse transcriptase polymerase chain reaction (QcRT-PCR) procedure as well as the Amp-RT assay. Virus isolation was attempted from peripheral blood mononuclear cells. All but one person carried HIV-2 subtype A. Plasma viraemia examined by QcRT-PCR was measurable in 15 (50%) of 30 subjects, yielding in all instances values below 20,000 HIV-2 RNA copies per ml. Plasma RT activity could be detected in only 10 (33%) of 30 subjects, a rate much lower than that seen in HIV-1 infection. Virus was isolated from 16 (53.3%) of 30 patients. A significant correlation was found between CD4+ counts, clinical status, rate of virus isolation, and plasma viral load by both QcRT-PCR and Amp-RT. In conclusion, HIV-2 subtype A is the predominant variant circulating in Portugal among both natives and immigrants. A lower cellular and plasma viral load with respect to HIV-1 was seen in persons without immunosuppression, from whom the rate of virus recovery was extremely low.

Viremia is significantly lower in HIV-2 than in HIV-1 infection, irrespective of disease stage. Nevertheless, the comparable proviral DNA burdens observed for these two infections indicate similar numbers of infected cells. Here we investigated this apparent paradox by assessing cell-associated viral replication. We found that untreated HIV-1-positive (HIV-1 ؉ ) and HIV-2 ؉ individuals, matched for CD4 T cell depletion, exhibited similar gag mRNA levels, indicating that significant viral transcription is occurring in untreated HIV-2 ؉ patients, despite the reduced viremia (undetectable to 2.6 ؋ 10 4 RNA copies/ml). However, tat mRNA transcripts were observed at significantly lower levels in HIV-2 ؉ patients, suggesting that the rate of de novo infection is decreased in these patients. Our data also reveal a direct relationship of gag and tat transcripts with CD4 and CD8 T cell activation, respectively. Antiretroviral therapy (ART)-treated HIV-2 ؉ patients showed persistent viral replication, irrespective of plasma viremia, possibly contributing to the emergence of drug resistance mutations, persistent hyperimmune activation, and poor CD4 T cell recovery that we observed with these individuals. In conclusion, we provide here evidence of significant ongoing viral replication in HIV-2 ؉ patients, further emphasizing the dichotomy between amount of plasma virus and cell-associated viral burden and stressing the need for antiretroviral trials and the definition of therapeutic guidelines for HIV-2 infection.