Journal of human virology最新文献

Objectives: Rabies virus variants associated with silver-haired bats (SHBRV) are responsible for most recent human rabies cases in the United States, which are not associated with a history of exposure. We compared their genotype and phenotype with those of dog rabies virus (DRV) variants, the classic cause of rabies in humans, to determine whether differences in these strains might have ramifications for therapeutic intervention, particularly vaccination.

Methods: Eleven silver-haired bat and 8 dog rabies virus isolates were characterized by sequencing the glycoprotein gene, by assessing their ability to replicate in neuronal versus nonneuronal cultures at optimal and suboptimal temperatures, by assessing their pathogenicity in mice, and by determining the resistance of these viruses to therapeutic immunization with commercial vaccines.

Results: SHBRV isolates were less genetically diverse, less neuronal cell specific, more temperature sensitive, but as pathogenic, on average, as DRV isolates. Immune protection was equivalent for SHBRV and DRV strains of similar pathogenicity.

Conclusions: SHBRV strains have unique characteristics that may explain their exceptional association with human rabies but have little bearing on their lethality in mice. The pathogenicity of a particular virus, rather than its antigenic makeup, determines the outcome of immunization.

An early step in the life cycle of human immunodeficiency virus type 1 is the reverse transcription of the viral RNA genome into double-stranded DNA, which is subsequently translocated to the cell nucleus. It is then integrated into host DNA and serves as a template for viral gene expression. Reverse transcription is catalyzed by the viral enzyme reverse transcriptase and is a complex process comprising a series of RNA-dependent DNA polymerization, DNA-dependent DNA polymerization, and RNase H reactions. Strand transfer reactions are required to complete the process. Reverse transcription is initiated when a molecule of host cell tRNA(lys3), which serves as a primer, is bound to the primer binding site of viral genomic RNA. The viral nucleocapsid protein is involved in each of the initiation of reverse transcription and in subsequent strand transfer or template-switching events. We review the interactions among reverse transcriptase, viral genomic RNA, the tRNA primer of reverse transcription, and viral nucleocapsid protein in the various steps of reverse transcription, including primer placement, initiation, and processive synthesis.

Objective: To evaluate human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) sequence diversity among distinct populations within India and to determine the prevalent subtype.

Study design/methods: Analysis of the 3'LTR was conducted from 28 HIV-1-positive samples: 1992-1993 (Pune, New Delhi) and 1995-1996 (Pune, Mumbai and Vellore). Genomic DNA was extracted from cocultivated peripheral blood mononuclear cells (PBMCs) and used for polymerase chain reaction (PCR) amplification and sequencing using dye terminator chemistry. Sequences were edited, aligned, and analyzed phylogenetically utilizing gap-stripped and bootstrapping parameters. Mobility shift assays were used to confirm binding activity.

Results: All nucleotide sequences were HWV-1 subtype C based on phylogenetic analysis. The isolates from Pune/Delhi formed subclusters when analyzed separately, irrespective of time or sample source. However, no significant subclustering was observed with isolates from Mumbai or Vellore or with the entire sample set when analyzed collectively. Subtype-specific enhancer analysis revealed an expected third NF-kappaB site but also revealed six isolates with insertions and deletions not previously described, one of which resembles an AP-1 binding site.

Conclusions: The results confirm the prevalence of HIV-1C and suggest increasingly complex phylogeny of HIV-1C within India, such that the previously observed subclustering may no longer adequately reflect the diversity of isolates currently circulating throughout India.

Background: The chemokine receptors CXCR4 and CCR5 have been identified as the major coreceptors for HIV-1 on CD4+ cells and macrophages. The natural ligands for these receptors are SDF-1 and the beta-chemokines (MIP-1alpha, MIP-1beta, RANTES), respectively, and are the products of a variety of immune cells, including CD8+ T lymphocytes.

Study design/methods: We hypothesized that the ability to stimulate the natural ligands for these receptors using an immune based therapy might influence in vivo chemokine receptor expression.

Results: In vivo CXCR4 expression remained stable after treatment with an HIV-1 Immunogen (REMUNE), whereas CCR5 expression on CD4+ T cells decreased (p < .05). Furthermore, HIV-1 antigen-specific production of beta-chemokines in vitro was also augmented (P < .05).

Conclusions: These preliminary results suggest that this HIV-1-specific immune-based therapy can stimulate antigen-specific beta-chemokine production in vitro and downregulate CCR5 receptor expression on CD4 cells in vivo.

Paul Ehrlich, gifted with knowledge, vision, and the capacity to bring his discoveries to practice, shaped the destiny of many biomedical scientific disciplines, including immunology, chemotherapy, hematology, cytology, and cancer research. His perceptive concept of receptors and ligands binding together in highly specific reactions was introduced just over a century ago in 1898 and is both fundamental and central to present day biomedical research. His mother country, Germany, commemorates his seminal contributions to science and to human welfare with an annual prize for achievements in fields that are related to his work. The 1999 Paul Ehrlich and Ludwig Darmsteaedter Prize was awarded to Robert C. Gallo, M.D. for his achievements in the pursuit of cancer related viruses and the growth in culture of human T-cells which led to the discovery of the first human retroviruses and, as a direct consequence, the discovery of the Human Immunodeficiency Virus, the third known human retrovirus. Below, excerpted from his acceptance lecture, is a concise personal history of those discoveries.

Objectives: We attempted to define optimal conditions for amplification of low copy number HIV-1 RNA sequences in plasma samples, applying improved conditions for nucleic acid extraction and amplification.

Methods: Several methodologic parameters were evaluated, including methods of RNA extraction, volumes of plasma samples, proportion of extracted RNA used as a template for amplification, and reverse transcriptase-DNA polymerase enzyme combination employed in cDNA synthesis and polymerase chain reaction amplification.

Results: With this improved assay, we were able to obtain sufficient amounts of amplified material for direct sequencing in 97% of all plasma samples in our study, including 88% of samples with viral loads <80 copies/mL, 78% of samples with viral loads <50 copies/mL, and even 2 (67%) of 3 samples with <20 copies/mL.

Conclusions: This procedure could be useful for testing resistance mutations in patients undergoing highly active antiretroviral therapy, in which the viral load is commonly <400 copies/mL, and even if it is <20 RNA copies/mL.

Objectives: To deliver antiretroviral agents or other foreign proteins into progeny virions and evaluate their inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication.

Study design/methods: HIV-1 encodes proteins in addition to gag, pol, and env, some of which are packaged into virus particles. One essential retroviral enzyme is integrase (IN), which has been used as a target for developing agents that inhibit virus replication. In previous studies, we demonstrated that intracellular expression of single-chain variable antibody fragments (SFvs), which bind to IN, results in resistance to productive HIV-1 infection in T-lymphocytic cells. Because the highly conserved accessory HIV-1 Vpr protein can be packaged within virions in quantities similar to those of the major structural proteins, this primate lentiviral protein may be used as a fusion partner to deliver antiviral agents or other foreign proteins into progeny virions. In these studies, the fusion proteins Vpr-chloramphenicol acetyl transferase (CAT) and Vpr-SFv-IN have been developed. Stable transfectants expressing these fusion proteins were generated from PA317 cells and SupT1 T-lymphocytic cells and analyzed using immunofluorescence microscopy. After challenge of SupT1 cells with HIV-1, p24 antigen expression was evaluated. The incorporation of these fusion proteins were evaluated by immunoprecipitation of virions using a Vpr antibody.

Results: Expression of the fusion proteins was confirmed by immunofluorescent staining in PA317 cells transfected with the plasmids expressing Vpr-CAT and Vpr-SFv-IN proteins. Stable transfectants expressing these fusion proteins were generated from SupT1 T-lymphocytic cells. When challenged, HIV-1 replication, as measured by HIV-1 p24 antigen expression, was inhibited in cells expressing Vpr-SFv-IN. It was demonstrated that Vpr-chloramphenicol acetyl transferase (Vpr-CAT and Vpr-SFv-IN proteins can be efficiently packaged into the virions and that Vpr-SFv-IN also decreases the infectivity of virions into which it is encapsidated.

Conclusions: An anti-integrase single-chain variable fragment moiety can be delivered into HIV-1 virions by fusing it to Vpr. Vpr-SFv-IN decreases HIV-1 production in human T-lymphocytic cells. The benefits of "intravirion" gene therapy include immunization of target cells as well as decreasing infectivity of HIV-1 virions harboring the fusion construct. Thus, this approach to anti-HIV-1 molecular therapies has the potential to increase inhibitory effects against HIV-1 replication and virion spread.

Objective: Human adeno-associated virus (AAV) is ubiquitous and known to establish latency by chromosomal integration. We have constructed a wild-type plus AAV vector, ins96-0.9Neo, containing the neomycin resistance gene open reading frame (Neo ORF) of 960 bases in length at map unit 96 of the virus. Ins96-0.9Neo was constructed in an unconventional manner in that the Neo ORF lacked a dedicated heterologous promoter. In this study, this wild-type plus AAV vector was to used to test AAV's packaging capacity and ability for chromosome 19 AAVS1 integration. However, when it was discovered that ins96-0.9Neo also transduced cells to G418 resistance, we also investigated the mechanism of Neo ORF expression in this vector.

Study design/methods: We investigated the ability of ins96-0.9Neo to produce virus at high titers and to retain the Neo sequences by Southern blot analysis. The ability of ins96 0.9Neo virus to transduce the Neo gene into cells was analyzed by colony formation under G418 selection, and the ability of ins96-0.9Neo to latently infect cells, including the AAVS1 region of chromosome 19, was investigated by a series of polymerase chain reaction (PCR) amplifications. Finally, the RNA expression of the Neo gene at map unit 96 was investigated by reverse transcriptase primer extension (RTPE) analyses with two different primers and by S1 nuclease protection.

Results: High titers of the ins96-0.9Neo virus could be generated (10(9) infectious units [IU]/mL without concentration), the Neo gene was retained in the encapsidated viral genome, infection by this virus resulted in G418 resistance, and significant integration was taking place within the AAVS1 sequences of human chromosome 19 on transduction. Analysis of mRNA by RTPE using both primers and by the S1 nuclease protection assay mapped the 5' end of the Neo transcripts to approximately 700 bases upstream of the Neo ATG at map unit 81 (nt 3793-3813), thus identifying a new AAV promoter.

Conclusions: These data demonstrate that ins96-0.9Neo will be useful for studying wild-type AAV integration and suggest that such wild-type plus recombinant AAV vectors may be useful for human gene therapy. The advantages of using such wild-type plus AAV vectors over defective AAV vectors include the ease in production of recombinant virus and the ability for site-specific integration into chromosome 19. This study also uncovered a previously unknown AAV promoter, p81. This finding suggests that the as yet uncharacterized ORF (nt 3922-4388) located just downstream of this promoter is likely an expressed gene. Furthermore, these data support our earlier findings that the AAV virion can package >900 bases more than can the wild-type.

Objectives: This paper describes a unique JC virus (JCV) variant recovered from the Highlands of Papua New Guinea that contains an inframe 21-bp deletion in the agnoprotein gene. We characterize the mutation and suggest possible roles for the deletion in JCV evolution.

Study design/methods: JCV DNA was extracted from urine and polymerase chain reaction (PCR) amplified using whole genome primers. PCR products were cloned, and multiple clones were sequenced. The JCV agnogene was PCR amplified to verify the presence of the agnogene deletion.

Results: This mutation creates a 21-bp deletion near the 3' end, which alters the predicted secondary structure of the messenger RNA and changes local codon usage at the 3' end of the agnogene. Protein secondary structure predictions suggest the deleted portion of the agnoprotein may be a flexible surface feature.

Conclusions: We describe the first stable coding region deletion in JCV that presumably signifies a single evolutionary event that led to the split from other Highlands viral groups and occurred well after the human expansions that led to the peopling of the Southwest Pacific.

Background: Potent combination antiretroviral therapy can reduce HIV plasma viral load (VL) to levels below the detection limit for as long as 2 years or more. A VL <500 HIV RNA copies/mL was until recently considered a reasonable therapeutic goal. However, lower levels seem necessary if VL rebounds and development of drug resistance are to be avoided.

Patients and methods: The clinical and virologic outcome at 1 year were prospectively examined in a group of 100 patients who began a triple combination antiretroviral therapy regimen consisting of stavudine (d4T), lamivudine (3TC), and indinavir (IDV). A modified ultrasensitive VL test with a detection limit of 40 copies/mL and a point mutation nested polymerase chain reaction (PCR) assay for detecting the codon 184 mutation conferring 3TC resistance were used for testing samples collected longitudinally from these individuals.

Results: Overall, VL values <40 copies/mL were reached in 45% and 32% of patients at nadir and at 12 months, respectively. More than half (24 of 45 persons) who achieved a level <40 copies/mL at nadir remained with undetectable VL at 1 year, whereas this occurred in only one fourth (7 of 28 persons) of those having levels of 40 to 500 copies/mL (P < .05). However, rebounds in VL to >500 copies/mL at 1 year were seen at similar rates (26.6% and 25%, respectively) in persons achieving either complete (<40 copies/mL) or partial (40-500 copies/mL) VL suppression at nadir. In contrast, the codon 184 mutation emerged more frequently at 1 year in patients whose VL remained between 40 and 500 copies/mL at nadir than in those who reached a level <40 copies/mL (30.7% versus 0%; P < .05).

Conclusion: Plasma VL at nadir after beginning highly active antiretroviral therapy (HAART) predicts the 1-year outcome. The achievement of levels of viremia <40 copies/mL are desirable during antiretroviral therapy if prolonged benefit is to be obtained. Because more than two thirds of persons with residual viremia do not show drug resistance, intensification strategies should be investigated for those patients with a good virologic response but without complete suppression during the first 6 months on HAART.