Journal of human virology最新文献

Objectives: The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in adult Han:NMRI mice. The outbred model, in comparison with inbred models, represents better the natural variable susceptibility of the human population.

Study design/methods: We analyzed the replicating virus titer, the antibody response in the acute and chronic phase of disease, the histology of myocardial injury, and the persistence of viral RNA.

Results: NMRI mice infected with 5000 plaque-forming units (PFU) of the CVB3 variant "P"D, a lytic variant to human fibroblast lines, showed a peak of virus replication at day 14 and developed a severe acute myocarditis. The chronic myocarditis was characterized by progressive fibrosis, small foci of infiltrates, persistent viral RNA in the heart, and detectable anti-CVB3 IgG production and neutralizing antibody response up to day 98 postinfection.

Conclusions: CVB3"P"D is able to induce chronic myocarditis in NMRI mice. This model provides a method for examining and proving the mechanisms of myocardial pathogenesis and of developing therapeutic strategies.

Objectives: The biologic phenotype of HIV-1 primary isolates obtained from approximately 50% of patients who progress to AIDS switches from non-syncytium-inducing (NSI) to syncytium-inducing (SI). We evaluated possible associations between virus coreceptor usage, sensitivity to inhibition by beta-chemokines, and disease progression of patients who continue to yield NSI isolates after developing AIDS.

Study design/methods: Sequential virus isolates were analyzed for biologic phenotype using the MT-2 cell assay, for sensitivity to beta-chemokines using RANTES inhibition, and for coreceptor usage using U87.CD4 and GHOST.CD4 cells expressing different chemokine/orphan receptors or donor peripheral blood mononuclear cells (PBMC) defective in CCR5 expression. In addition, the env V3 region was sequenced and the length of the V2 region determined.

Results: All NSI isolates, regardless of patient status at time of isolation, were dependent on CCR5 expression for cell entry. Furthermore, there was no indication of broadened coreceptor usage of NSI isolates obtained from persons with late-stage AIDS. A majority of NSI isolates remained RANTES sensitive; however, virus variants with reduced sensitivity were observed. The V2 lengths and the V3 sequences exhibited no or minor changes at analysis of sequential NSI isolates.

Conclusions: Our data suggest that NSI isolates obtained from AIDS patients remain CCR5 dependent (ie, R5) and, in many cases, also remain sensitive to RANTES inhibition. However, virus variants with decreased sensitivity to RANTES inhibition may evolve during disease progression, not only as a result of a switch from NSI to SI but also in patients who develop AIDS while continuing to maintain R5 isolates.

Objectives: To investigate the subtype classification of the circulating virus strains among human immunodeficiency virus type 1 (HIV-1)-infected children in Greece.

Study design/methods: Since the beginning of the acquired immunodeficiency syndrome (AIDS) epidemic in Greece in 1982, 23 children have been reported to be vertically infected with HIV-1. Blood samples were available for 19 of these children, and the C2-C4 env region was successfully amplified by nested polymerase chain reaction (PCR) for 16 subjects. HIV-1 subtype was established by the heteroduplex mobility assay (HMA) in 16 subjects and confirmed by DNA sequencing and phylogenetic analysis in 8 subjects.

Results: Most subjects (9; 56%) fell into subtype B. However, a substantial proportion (44%) were classified as subtypes A (3; 19%), C (1; 6%), D (1; 6%), and I (2; 12%). According to epidemiologic information, 5 of 7 children infected with non-B HIV-1 subtypes were born to Greek parents.

Conclusion: These findings clearly suggest that non-B strains have been introduced into Greece, providing evidence that HIV epidemic in this country will probably change profile over time. In addition, subtype I was identified in 2 HIV-1-infected children, both of whom were born to Greek parents.

Objective: Clinical experience with HIV-1 protease inhibitors (PIs) in the treatment of AIDS frequently has shown that increases in CD4+ T-cell counts can be independent of HIV-1 inhibition by these drugs. This disconnection between viral load and CD4 counts led us to investigate how the PI ritonavir directly affects leukocyte activation in vitro, using peripheral blood mononuclear cell (PBMC) fractions derived from normal donors.

Methods and results: When uninfected PBMC cultures were treated for 72 hours with ritonavir at concentrations similar to or lower than that shown to be effective in vivo, an increase in cell viability was observed. The susceptibility of PBMCs to apoptosis was markedly decreased after ritonavir treatment and correlated with lower levels of caspase-1 expression, decreases in annexin V staining, and reduced caspase-3 activity. Induction in vitro of tumor necrosis factor (TNF) production by PBMCs and monocytes was inhibited by ritonavir in a time- and dose-dependent manner at nontoxic concentrations.

Conclusion: Based on our data, we conclude that the HIV-1 PI ritonavir is an immune modulator that may affect leukocyte activation and apoptosis as an important part of its therapeutic benefit.

Objective: Kaposi's sarcoma (KS) is an acquired immunodeficiency syndrome (AIDS)-defining neoplasm histologically characterized by proliferation of spindle cells, inflammatory cells, and abundant neovascularization. When the malignant cell line KSY-1 derived from an AIDS-KS tumor is transplanted subcutaneously into nude mice, prominent neovascular features develop. Using this mouse model of neoplastic KS, we set out to determine, using c-ets 1 markers specific for mouse or human tissues, whether vascular growth and inflammatory infiltrate induced by the transplanted KSY-1 cells is of host cell or transplant origin.

Study design/methods: KS tumors were induced by subcutaneous inoculation of 5 x 10(6) KSY-1 cells/200 microL in immunodeficient mice, and species-specific mouse and human riboprobes of the c-ets 1 protooncogene were used for in situ hybridization to define cell of origin.

Results: Five different tumors were examined. Tissue sections from all cases were hybridized with radiolabeled riboprobes for the presence of both mouse and human c-ets 1 mRNA. Tumor cells were labeled with the human c-ets 1 probe, whereas neovascular and inflammatory tissues were of mouse origin.

Conclusions: The finding that vascular but not tumor cells are of host origin supports the model of tumor-induced vascularization via a mechanism of tumor cell-derived cytokine-medicated pathogenesis.

Objective: Select C-C and C-X-C chemokines can suppress HIV infection. This is because their receptors are the gateways for HIV-1 entry, determinants of viral tropism and sensitivity. C-C chemokines are most effective against macrophage-tropic viruses, and C-X-C chemokines are most effective against T-tropic viruses. The epitopes on the chemokine molecule responsible for virus inhibition and for chemokines' specificities are not known. The objective of this study was to map the functional domains of prototypic antiviral chemokine, namely, RANTES (regulated-on-activation normal T-expressed and secreted).

Study design: Optimal folding of the chemokine molecule is thought to be important for its biologic activity. Anticipating that it will provide a native milieu for folding, we expressed recombinant RANTES molecules in an HIV-2-derived lentivirus mammalian expression system. We focused on the structural landmarks of RANTES to determine their role in its life and function.

Results: We found that the flexible amino-terminal region of RANTES was not important for its structural integrity or antiviral activity, either positively or negatively. It was also not important for binding to the CCR5 receptor. Modification of all other domains was detrimental, implying a functional role. However, a more careful analysis revealed that these domains were crucial for controlling stability, transport, and secretion of the molecule. Although all recombinant clones contained signal sequence and were transcriptionally active, they presented three different phenotypes: normal synthesis and secretion, normal synthesis but blocked secretion, and presumed normal synthesis but rapid degradation. Structural considerations and preliminary experiments showing a lack of effect of proteasome inhibitors suggested that the signal recognition particle pathway of translocation and proteasomal pathway of destruction may not be the major determinant of the life of the chemokine.

Conclusions: The amino-terminal domain of RANTES was not essential for its antiviral activity or for its binding to the CCR5 receptor. Although the 1-domain of the core and carboxy-terminal domain may contribute to the antiviral activity of RANTES, they were more important for its intracellular life.

Objective: To determine whether a unique human T-lymphotropic virus type I (HTLV-I) transmission cohort containing multiple disease manifestations could be used to establish a relationship between tax gene sequence and HTLV disease expression.

Methods: DNA was extracted from the peripheral blood mononuclear cells (PBMC) of the HTLV-infected persons in the cohort. A 1.1-kb fragment of tax was amplified by polymerase chain reaction (PCR) and cloned. Six to 12 individual clones were sequenced per person.

Results: Comparison to a reference ATK strain showed numerous differences; however, consensus tax sequences from all persons within the transmission cohort were identical. Intraperson variation was 0.1% to 0.3%. Tax sequences from the index case did not differ from those obtained from a transfusion recipient who developed tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). Tax sequences from the same index case did not differ from sequences obtained from the asymptomatic or ATL phases of a second recipient.

Conclusions: In this cohort there did not appear to be tax genotypes associated with specific disease manifestations of HTLV infection.

Objectives: This study was undertaken to examine 6-bp insertions following codon 69 in the reverse transcriptase (RT) coding region of human immunodeficiency virus type 1 (HIV-1) mutations in terms of incidence, presence of additional RT mutations, phenotypic drug resistance, HIV-1 RNA levels, and antiretroviral treatment history.

Study design/methods: A retrospective study of 121 nucleoside reverse transcriptase inhibitor (NRTI)-experienced subjects infected with HIV-1 was performed. Methods included quantitation of HIV-1 RNA levels, genotypic analyses of the RT and protease coding regions, and determination of phenotypic drug resistance.

Results: A 6-bp insertion following RT codon 69 was observed in viral isolates from 4 subjects. Two subjects had a history of zidovudine (ZDV)-based therapy, and two subjects had a history of stavudine (D4T)-based therapy without prior exposure to ZDV. The T69S mutation and the 6-bp insertion following RT codon 69 were the only RT mutations observed in the 2 subjects with a history of D4T-based therapy.

Conclusions: Six-basepair insertions occurred in virus from 4 of 121 (3%) NRTI-experienced subjects, including those without prior ZDV treatment, and was observed in the absence of the T215Y mutation. There was no apparent correlation between insertion incidence and HIV-1 viremia.

Objective: To identify cellular factors in human liver that interact with hepatitis C virus (HCV) 5' and 3' untranslated regions (UTRs) and therefore possibly are involved in the regulation of HCV transcription or translation.

Methods: We prepared cytoplasmic extracts from human liver biopsy samples and fractionated cellular factors on ion exchange columns. The various fractions from ion exchange columns were subjected to ultraviolet (UV) cross-linking with radiolabeled HCV 5' and 3' UTR RNA probes.

Results: Two major 45- and 46-kd proteins that interacted specifically with the HCV 5' and 3' UTR were identified. Using Western blot and immunoprecipitation, these proteins were identified as the La antigen.

Conclusion: Our results demonstrate that the La protein from human liver biopsy cells interacts specifically with the U-rich region in the positive strand of the HCV 3' UTR.