Journal of human virology最新文献

The antigenic diversity, rapid genetic integration into host cell DNA, and immune evasion tactics of human immunodeficiency virus type 1 (HIV-1) create formidable obstacles to the development of an effective vaccine against it. In spite of this, the advent of conformationally constrained HIV-1 Env and gp120 immunogens has made it feasible to formulate HIV-1 vaccines that induce broadly cross-reactive neutralizing antibodies and afford protection through humoral mechanisms. This paper reviews recent advances made by the authors toward the development of an HIV-1 vaccine that elicits such antibodies in both the mucosal and systemic immune compartments.

Objectives: Human immunodeficiency virus type 2 (HIV-2) progression to disease is significantly slower than that of human immunodeficiency virus type 1 (HIV-1). Genetic determinants for susceptibility to disease progression were hypothesized to play a more significant role in this infection compared with HIV-1. We sought to identify common human lymphocyte antigen (HLA) alleles in the Senegalese population and to compare HLA profiles between HIV-2-infected individuals with low and high risk for disease progression.

Study design/methods: We conducted a case-control study investigating possible associations between MHC class I genes and the risk of disease progression in HIV-2-infected individuals. The MHC class I genotype was molecularly defined using polymerase chain reaction with sequence specific primers (PCR-SSP) in 62 female sex workers from the Dakar, Senegal cohort. Lack of antibodies to the HIV-2 antigen p26 has been previously shown to predict disease progression and was used in this study as a surrogate marker. Twenty-one cases were identified lacking antibodies to p26, therefore at a higher risk of disease progression, and were compared with 41 p26 antibody-positive, randomly selected controls.

Results: Statistical analysis showed that HLA B35 was significantly associated with lack of p26 antibodies, and higher risk of disease progression ( < 0.05). The same association was found for the self-defined class I haplotypes B35-Cw4 and A23-Cw 7 ( < 0.05). The HLA B 53 allele was associated with slower disease progression; however, this association was not statistically significant. We observed a trend whereby heterozygotes were at lower risk for HIV-2 disease progression, as previously reported in HIV-1 disease.

Conclusions: In this West African population, a distinct profile of HLA class I alleles was observed, and many of these appear to influence disease progression in HIV-2 infection.

Objectives: To determine the role of the hepatitis C virus 3' untranslated region in viral mRNA translation in transfected cells and in cell extracts.

Study design/methods: Noninfectious hepatitis C virus mini-genome RNAs with various deletions in the viral 3' untranslated region were transfected into cells or translated in vitro, and the translation efficiency was determined.

Results: We have found that the presence of the hepatitis C virus 3' untranslated region modestly increases mRNA translation. The positive effect correlated with the binding of a 45-kDa cytoplasmic factor to the hepatitis C virus 3' untranslated region. Furthermore, the U-rich sequence in the hepatitis C virus 3' untranslated region inhibits translation of capped and polyadenylated mRNAs as a result of the hybridization.

Conclusions: The modest effect of the hepatitis C virus 3' untranslated region on translation suggests that it does not play a major role in mRNA translation. The inhibitory effect of the hepatitis C virus 3' untranslated region on translation of polyadenylated mRNAs supports the notion that translation of hepatitis C virus mRNAs occurs independently of a polyA tail.

Objective: The inter- and intrapatient genetic variation of GB virus C (GBV-C)/hepatitis G virus (HGV) was investigated to characterize the molecular epidemiologic profile of GBV-C/ HGV infection in China, an area endemic for viral hepatitis. The intrapatient variation of hepatitis C virus (HCV) from the same patients was compared to that of GBV-C/HGV.

Study design/methods: GB virus C/HGV RNA was amplified by polymerase chain reaction in 88 patients with hepatitis C, hepatitis B or presumed non-A-E hepatitis from three cities in China. Five clones of the GBV-C/HGV NS3 region were sequenced from each GBV-C/HGV RNA-positive patient. The corresponding region of HCV was also sequenced from patients co-infected with HCV. Representative sequences of the GBV-C/HGV NS3 region from each patient and those of isolates from other continents were subjected to phylogenetic analyses.

Results: GB virus C/HGV was detected in 22 (25.25%) of 88 patients: 9 (21.4%) of 42 patients with presumed non-A-E hepatitis, 10 (27.7%) of 36 patients with hepatitis C, 3 (30.0%) in 10 patients with hepatitis B and C, and in none of 60 volunteer blood donors. The extent of nucleotide variation was less between Chinese isolates (2.4-17%; median, 10.4%) than between Chinese isolates and seven isolates from outside China (10.5-19.5%; median, 15.3%). Intrapatient sequence variation ranged from 0 to 1.75%, with a mean of 0.57 +/- 0.51%. Phylogenetic analysis grouped most Chinese isolates into four geographically specific clusters with a divergence of 10% to 16% from each other. The ratio of nonsynonymous to synonymous substitutions of GBV-C/HGV (Ka/Ks 0.019) was much lower than for HCV (0.071) in the same patients.

Conclusion: Chinese isolates of GBV-C/HGV are genetically distinct. There are local strains as well as shared strains between different locales. The extent of amino acid sequence conservation suggests strong selection against nonsynonymous substitutions in the GBV-C/HGV genome.

Objective: To evaluate the mother-to-child transmission profile of human immunodeficiency virus (HIV) as well as human herpesvirus 6 (HHV-6) and to examine active replication of HHV-6 in the HIV-infected mothers and their newborns.

Study design/methods: This polymerase chain reaction (PCR)-based detection was done using DNA from peripheral blood mononuclear cells (PBMCs) and milk cells from the mothers, PBMC from the newborns, and DNA derived from plasma and cell-free milk fluid from mothers and plasma of the newborns. None of the mothers received antiretroviral treatment.

Results: HIV was transmitted to 50% newborns and, of 36 total mothers, 8 had actively replicating HHV-6 detectable in their plasma and 2 also had it in the lactosera. Among the neonates. HHV-6 was found in the PBMC DNA of seven and in the plasma fractions of five, the latter five newborns were all HIV-infected at birth.

Conclusion: Perinatally cotransmitted HHV-6 was always activated in the neonates who were born with HIV infection. Also, HHV-6 can be detected in the milk cells and the activated virus may be present in the lactosera of some of these HlV-infected mothers.

Background: The combination of didanosine (DDI) and lamivudine (3TC) has been not recommended in guidelines for the first-line treatment of human immunodeficiency virus (HIV) infection because two major considerations might reduce its efficacy. First, both drugs are non-thymidine nucleosides and might exert less antiviral activity on activated T cells. Second, the codon 184 mutation emerging under 3TC failure could confer cross-resistance to DDI. However, because the intracellular half-life of their active metabolites allows administration once daily, the resulting improvement in patient compliance should favor this combination over others.

Patients and methods: We analyzed the virologic and immunologic outcome at 6 months in 46 naive HIV-infected patients (41% were intravenous drug users) who began a treatment of a triple combination of DDI, 3TC, and indinavir (IDV) with the schedule optimized to improve adherence. Both DDI and 3TC were administered once daily (450 mg and 300 mg, respectively) and IDV was taken twice daily (1,200 mg). Patients chose schedules at their convenience, according to their job timings and preferences. All had a plasma viral load (PVL) of more than 500 HIV-RNA copies/mL at the time they entered the study, and overall the mean PVL value was 54,201 copies/mL.

Results: Overall, nine patients did not complete the study period: two died of non-acquired immunodeficiency syndrome (AIDS)-related causes, three described severe gastrointestinal symptoms, one discontinued therapy voluntarily, and three were lost to follow-up. Among the remaining 37 patients, a PVL value of less than 500 copies/mL was reached by 31 (83.7%) patients, and values below 40 copies/mL were recorded in 71% (22/31) of them. Overall, a clinically significant increase in the CD4 cell count (more than 60 cells/microL) was seen in 70.2% (26/37) of patients. Treatment adherence, assessed using both self-reporting and the pill count method, was considered good (more than 90% of pills prescribed were taken) in all but four patients; three of them were among those six who did not reach undetectable PVL values at the sixth month. Mutations at codons 184, 74, 65, and 151, which confer resistance to 3TC and DDI, could be examined in 12 of the 15 patients having PVL values above 40 copies/mL at the sixth month. The codon 184 mutation emerged in 25%, meanwhile the codon 74 mutation only appeared in one patient; none carried the codon 151 mutant genotype. Nine months after beginning treatment, 29 (93.5%) of the 31 patients who reached less than 500 copies/mL at the sixth month still sustained PVL values below this threshold, and 25 (80.6%) had less than 40 copies/mL.

Conclusion: The combination of DDI and 3TC both once daily plus IDV twice daily is well tolerated, seems to favor a good adherence, and shows significant antiviral activity.

Objectives: The aim of this study was to understand the effect of metal cations on the hepatitis C virus (HCV) NS5B in vitro RNA-dependent RNA polymerase (RdRp) activity and its susceptibility to various inhibitors.

Methods: A recombinant full-length HCV NS5B protein was expressed in insect cells and purified to homogeneity. RdRp activity was assessed using standard filtration or polyacrylamide gel-based assays.

Results: Efficient inhibition of the HCV NS5B RdRp activity by gliotoxin, as well as by various substrate analogs, occurs in the presence of Mn2+, but not of Mg2+. Assays performed in the presence of both cofactors suggest that, in vitro, the enzyme's affinity for Mn2+ is higher than that for Mg2+. In addition, the RdRp activity, displayed in the presence of heteropolymeric templates, is significantly increased when the metal cofactor consists of Mn2+. Finally, steady state kinetics showed that the velocity of the reaction, as well as the affinity of the enzyme for its substrate, could both be affected by the nature of the divalent metal cation used.

Objective: An in vitro blood-brain barrier (BBB) system was developed using primary cultures of human brain-derived microvascular endothelial cells (MVECs), macrophages, neuronal cells, and human fetal astrocytes. This BBB system simulates important morphologic and permeability characteristics of the BBB in vivo. This system could be used to study human neurologic/neurovirologic disorders.

Study design and methods: Microvascular endothelial cells were cultured to 100% confluency on the upper side of 0.45-microm polyethylene tetraphthalte (PET) membrane inserts coated with MVEC attachment factor. Expression of ZO-1 (Zona Occludens 1), a protein specifically associated with tight junctions and the intercellular sealing of adjacent MVECs, was analyzed by immunocytochemical methods. The integrity of the BBB formed on the insert membrane was also assessed by the measurement of electrical current passage through the membrane; and, after the formation of complete BBB, a two-compartment system was developed using the cell culture insert upper surface, essentially a confluent monolayer of brain-derived MVECs housed on a six-well chamber surface. In the six-well chamber surface, there are different central nervous system (CNS)-based cells, ie, human astrocytes, immature neurons, mature neurons, and MVEC. The cell culture inserts were in close juxtaposition to the surface of the chamber, making an intimate contact with the cells, separated by an insert membrane. The MVEC surface of the insert was exposed to human immunodeficiency virus type 1 (HIV-1) strains 89.6, NL4-3, and IIIB. After 72 hours, the cells were fixed and used for in situ polymerase chain reaction (IS-PCR), whereas the supernatant was subjected to HIV-1 p24 antigen determination.

Results: Primary human brain MVECs are capable of forming tight junctions, revealed by the expression of ZO-1, as well as elevated transendothelial electrical resistance. Based on these characteristics, these cells formed an in vitro BBB, which then was used to study the transfer of HIV-1 through this barrier. It was observed that HIV-1 can infect MVEC and can cross it in vitro and infect the cells growing on the opposite side of the membrane. Infection of various CNS-based cells was confirmed by IS-PCR, as well as by HIV-1 p24-antigen determination. It was observed that the dual-tropic strain, 89.6, had a greater potential to create a breach in the in vitro BBB, followed by NL4-3 and IIIB.

Conclusion: This model system is relevant for evaluating HIV-1 neuropathogenesis and therapeutics designed to alter HIV-1 expression in human CNS-based cells. As such, the effects of highly active antiretroviral therapy on HIV-1 infection of the human CNS, a possible drug sanctuary site, can be evaluated using this technology.