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PubMed2004

Hepatitis C and steatosis.

Steatosis is a common finding in patients with chronic hepatitis C (CHC) due to a combination of the direct steatogenic effect of hepatitis C virus (HCV) and the prevalence of metabolic risk factors in the HCV population. Steatosis is now established as a risk factor for disease progression in CHC and significantly impacts therapeutic response. Research efforts should continue to focus on defining the complex viral and host interactions involved in the pathogenesis of HCV-related steatosis so that future therapeutic strategies may be accurately and appropriately targeted.

Antiviral Agents↗

PubMed2005

Steatosis and chronic hepatitis C virus infection: mechanisms and significance.

Chronic hepatitis C (CHC) and steatosis are common entities that have the potential to interact synergistically and result in significant morbidity. Steatosis is frequently observed in CHC and seems to have a significant impact on the natural history of the disease with respect to development of fibrosis and reducing the virologic response to current therapy. Research efforts should continue to focus on delineating the complex viral and host interactions involved in the pathogenesis of hepatitis C virus (HCV)-related steatosis. This may provide novel future therapeutic strategies that may help modulate disease progression in relation to steatosis in HCV infection.

Body Mass Index↗

PubMed2007

Separation of near full-length hepatitis C virus quasispecies variants from a complex population.

A long RT-PCR (LRP) protocol was developed recently for robust amplification of a near full-length HCV genomic sequence from clinical samples, followed by efficient cloning [Fan, X., Xu, Y., Di Biceglie, A.M., 2006. Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples. Biochem. Biophys. Res. Commun. 346, 1163-1172]. In the present study, the LRP protocol has been estimated for its error rate and the validation by sequencing fully the near full-length HCV inserts from six recombinant clones derived from a patient sample with complex viral diversity. These sequences were compared with the near full-length HCV sequence that was generated by direct sequencing of multiple overlapped PCR products from the same sample, referred to as the population sequence. Comparative analysis confirmed the artificial nature of the PCR-assembled population sequence and identified potential domains for linked viral mutations. The data also suggested that the hypervariable region 1 (HVR1) may be a biological marker for the phenotype at the quasispecies level. These observations emphasize the significance of the use of near full-length genomic sequences for HCV genetic studies and for reverse genetic analysis using authentic quasispecies variants.

Cloning, Molecular↗

PubMed1985

Interactions between hepatotropic viruses in patients with haemophilia.

Infections with the hepatotropic viruses non-A, non-B (NANB), hepatitis B (HBV) and delta agent (HDV) are described in two patients with haemophilia. The first patient illustrates the phenomenon of interference following a simultaneous exposure to NANB and HBV. The second patient, a carrier of hepatitis B surface antigen (HBsAg), acquired superinfection with HDV which resulted in acute hepatitis progressing to chronic hepatitis. Liver disease seen in multitransfused haemophiliacs may be significantly different to that seen in other patients. As a consequence of the infusion of blood clotting factor concentrates, these patients become the site of complex viral interactions.

Child↗

PubMedDate not supplied

The role of mRNA 5'-noncoding and 3'-end sequences on 40S ribosomal subunit recruitment, and how RNA viruses successfully compete with cellular mRNAs to ensure their own protein synthesis.

Since the elaboration of the scanning model to explain eukaryotic translation initiation, alternative hypotheses have gained support. Cap and 5' end-independent recruitment of the 40S ribosomal subunit conferred by the presence of an internal ribosome entry segment (IRES) in the 5'UTR of the mRNA is widely accepted, and has been formally and definitively proven for a picornavirus. However, the mechanism of IRES function remains essentially a black box. Using the complex viral IRESes as model systems, approaches taken to shed light on the mystery include systematic comparisons and molecular genetic analyses. The hypothesis that actively translated mRNAs are circular, rather than linear, molecules is based on rather indirect evidence. This model has invoked a revision of the image of 40S ribosomal subunit recruitment, to include recycling from the mRNA 3'- to the 5'-end in addition to true de novo 5'-end directed entry. Biochemical and genetic studies are used to define the network of interactions necessary for efficient ribosome recruitment. This has lent weight to the concept of mRNA 5'-3' cross-talk and clarified the mechanics of how this enhances translation efficiency. These refinements and revisions to the model of translation initiation form the core of this review, with current knowledge being considered from the perspective on how host-cell translation could yield to selective viral translation via the phenomenon of translational shut-off.

5' Untranslated Regions↗

PubMed1998

Sexually transmitted diseases. Present problems-future prospects.

The tools of molecular biology have brought us to a new level of understanding of the epidemiology and pathophysiology of sexually transmitted diseases, which has been augmented and concentrated by the challenge of HIV/AIDS. We enter the next millennium with an unprecedented knowledge of these infections, but lacking the resources and drive to apply this broadly, especially where most needed in the areas of socioeconomic deprivation. We may develop cures or vaccines for complex viral infections, but for whom? Eventually, the knowledge will flow and disseminate, but will practice change?

Acquired Immunodeficiency Syndrome↗

PubMed1996

Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein.

The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.

Amino Acid Sequence↗

PubMed1981

Friend disease in vitro.

In long-term marrow cultures, hemopoiesis can be maintained for several months, although erythropoiesis is normally suppressed at the most primitive level of development (the erythroid colony-forming cells). Infection of these cultures with a viral complex combining helper-independent murine leukemia virus (F-MuLV) and a spleen focus-forming virus (SFFVp) results in a productive infection of both the replication defective SFFVp and the F-MuLV. After infection, the cultures show a dramatic elevation in the numbers of late erythroid progenitor cells (CFU-E), many of which will grow in the absence of added erythropoietin, and a transient erythropoietin, independent erythropoiesis, including the production of mature, enucleated erythrocytes. Hemopoiesis eventually declines, with no evidence for the generation of Friend tumor cells. When erythropoiesis is induced in the long-term cultures by addition of anemic mouse serum before infection by polycythemia-inducing Friend virus, the generation of erythropoietin-independent CFU-E and erythrocyte formation is followed by the sustained production (greater than 40 wk) of primitive erythroid cells with low spontaneous levels (less than 5%) of hemoglobinization. Although these cells will produce spleen colonies in irradiated mice and can be cloned in soft-gel media, they do not produce autonomous, permanently growing cell lines in vitro, i.e., they retain a dependency upon the marrow-adherent layer for their continued growth. However, following a further passage on a "virgin" marrow environment, permanent cell lines can be established that are able to grow independently of environmental influences. Thus, this system is the first description of a complete in vitro system for the reproducible production and isolation of Friend virus-induced erythroid cell lines.

Anemia↗

PubMed1991

Firefly luciferase as a marker for herpesvirus (pseudorabies virus) replication in vitro and in vivo.

Insertion of reporter genes into complex viral genomes and monitoring virus replication by detecting the corresponding protein products is increasingly used in pathogenesis studies. We present here the isolation and characterization of a recombinant neurotropic alpha-herpesvirus, pseudorabies virus (PrV), which stably carries the gene encoding firefly luciferase. To express the enzyme the complete open reading frame for luciferase was fused to the promoter and first seven codons of the non-essential glycoprotein gX gene of PrV. A recombinant PrV carrying the luciferase gene inserted into the gX gene and exhibiting strong luciferase activity after infection of cultured cells was further characterized. Kinetic analyses showed that luciferase activity was detectable as early as 90 min after infection. Luciferase expression could be monitored in cell extracts in a luminometer. For facilitating plaque isolation of luciferase recombinant viruses it was also visualized in situ on sensitive film. Kinetic experiments in mice proved the suitability of luciferase as an excellent marker for following herpesvirus spread in the animal. By way of luciferase detection we show that PrV invasion of the central nervous system after intranasal infection of mice occurred independently of replication in non-neural tissues such as lung or thymus. Furthermore, comparison of isogenic luciferase recombinant PrV strains carrying intact or deleted glycoprotein gI genes showed differences in the organotropism between these two viruses.

Animals↗

PubMed2005

An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein and ribonucleoprotein.

Replication of the RNAs of influenza virus occurs in the nucleus of infected cells. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different nuclear localization signals (NLSs). Here, an import assay in digitonin-permeabilized cells was used to further define the contribution of these NLSs. Mutation of the unconventional NLS impaired the nuclear import of the NP. A peptide bearing the unconventional NLS could inhibit the nuclear import of the NP in this import assay and prevent the NP-karyopherin alpha interaction in a binding assay confirming the crucial role of this signal. Interestingly, a peptide containing the SV40 T antigen NLS was unable to inhibit the nuclear import of NP or the NP-karyopherin alpha interaction, suggesting that the NP and the SV40 T antigen do not share a common binding site on karyopherin alpha. We also investigated the question of which NLS(s) is/are necessary for the viral ribonucleoprotein complex to enter the nucleus. We found that the peptide containing the unconventional NLS efficiently inhibited the nuclear import of the ribonucleoprotein complexes. This finding suggests that the unconventional NLS is the major signal necessary not only for the nuclear transport of free NP but also for the import of the ribonucleoprotein complexes. Finally, viral replication could be specifically inhibited by a membrane-permeable peptide containing the unconventional NLS, confirming the crucial role of this signal during the replicative cycle of the virus.

Active Transport, Cell Nucleus↗

PubMed1999

Spi-1/PU.1 is a positive regulator of the Fli-1 gene involved in inhibition of erythroid differentiation in friend erythroleukemic cell lines.

Spi-1/PU.1 and Fli-1 are two members of the ETS family of transcription factors whose expression is deregulated by proviral insertion in most erythroleukemic cell lines induced by the spleen focus-forming virus (SFFV) and Friend murine leukemia virus (F-MuLV) components of the Friend viral complex, respectively. In this study, we present evidence that transcription of the Fli-1 gene is positively regulated by Spi-1/PU.1 in SFFV-transformed cell lines: (i) all SFFV-transformed cell lines expressing Spi-1/PU.1 are characterized by a specific pattern of Fli-1 gene transcripts initiated in the -200 region instead of position -400 as reported for F-MuLV-transformed cell lines; (ii) these Fli-1 transcripts initiated in the -200 region are downregulated in parallel with that of Spi-1/PU.1 during hexamethylenebisacetamide (HMBA) induced differentiation; and (iii) Fli-1 transcription is upregulated in SFFV cells lines following stable transfection of a Spi-1/PU.1 expression vector. Furthermore, we found by transient transfection assays that the -270/-41 region of the Fli-1 gene displays promoter activity which is transactivated by Spi-1/PU.1. This promoter is strictly dependent on the integrity of two highly conserved ETS DNA binding sites that bind the Spi-1/PU.1 protein in vitro. Finally, we show that transfection of constitutive or inducible Fli-1 expression vectors in SFFV-transformed cells inhibits their erythroid differentiation induced by HMBA. Overall, these data indicate that Fli-1 is a target gene of the Spi-1/PU.1 transcription factor in SFFV-transformed cell lines. We further suggest that deregulated synthesis of Fli-1 may trigger a common mechanism contributing to erythroleukemia induced by either SFFV or F-MuLV.

Animals↗

PubMed1990

[Acute respiratory infections in children who have died and the role of the DIC syndrome in these diseases].

The results of several years study of the acute respiratory infections (ARI) (pneumonias) are summarized. Their high frequency in dying children is found (72% of autopsy cases in all Leningrad pediatric hospitals). Their etiology is usually complex: viral ARI were in 62.9%, bacterial--56.1%, mycoplasma--8.9%. Structural changes are described in some poorly known ARI (herpetic, cytomegalic, pneumocystic). The distribution of various ARI between hospitals and the etiology of children pneumonia in cases of the death at home (381 cases) or in hospitals (447 cases) are analysed when the autopsy was performed in the same pathology department with detail laboratory investigation. Pneumonias in these groups differed in the etiology and manifestations, first because of bacterial infections. In 114 children the incidence and manifestations of DIC syndrome were studied and this syndrome was shown to have a considerable importance in thanatogenesis particularly during first days of the disease and when the generalization of infections occurs.

Acute Disease↗

PubMed1989

Persistent suppression of virus-specific cytotoxic T cell responses after transient depletion of CD4+ T cells in vivo.

Co-administration of soluble Ag and anti-CD4 mAb has been successfully used to induce long term Ag-specific tolerance. The mechanisms underlying persistent immunologic unresponsiveness are unclear. We have now studied whether tolerance toward complex viral Ag expressed on Moloney sarcoma virus (MSV)-transformed tumor cells can be induced when given at the time of severe helper cell depletion. Although mice that had been injected with anti-CD4 mAb at the time of immunization regained the ability to recognize MSV Ag, their humoral and cytotoxic immunity to MSV were severely compromised. Ag-specific low responsiveness was maintained for more than 6 mo. To analyze the T cell repertoire of low responder mice we have estimated precursor frequencies of MSV-specific proliferative and cytotoxic T cells after the CD4+ T cell subset was fully reconstituted. There was no difference in the frequencies of control and low responder mice excluding clonal deletion as the mechanism maintaining low responsiveness. In co-culture experiments the defect in low responder mice could be localized to the regenerated CD4+ T cell subset, suggesting the induction of CD4+ suppressor-inducer cells. Alternatively, regenerated CD4+ cells in anti-CD4 conditioned mice had acquired a defect to provide help for MSV-specific responses. In spite of the potentials to induce low responsiveness to selected Ag by anti-CD4 conditioning, the risk to cause persistent virus-specific immunodeficiency might limit the clinical application of anti-CD4 therapy.

Animals↗

PubMed1982

Persistence of circulating HBsAg/IgM complexes in acute viral hepatitis, type B: an early marker of chronic evolution.

Serial serum samples from 110 patients with acute viral hepatitis type B were tested for HBsAg/IgM complexes by a newly developed solid-phase radioimmunoassay. In 102 patients the infection resolved and they recovered from the disease. In these patients, HBsAg/IgM complexes were either absent from the outset of disappeared from serum within four weeks of admission, long before HBsAg had cleared or serum alanine aminotransferase had returned to normal, 8 patients progressed to chronic HBsAg carrier state and chronic liver disease. In these patients, HBsAg/IgM complexes were detectable in the serum on admission, and never disappeared. These results indicate that persistence of circulating complexes containing HBsAg and IgM after the early phase of acute viral hepatitis type B is a predictor of disease chronicity. As early as the fifth week of illness those in whom chronic liver disease developed could be distinguished from those who recovered.

Adolescent↗

PubMed2005

Preliminary X-ray diffraction analysis of crystals from the recombinantly expressed human major histocompatibility antigen HLA-B*2704 in complex with a viral peptide and with a self-peptide.

The product of the human leukocyte antigen (HLA) gene HLA-B*2704 differs from that of the prototypical subtype HLA-B*2705 by three amino acids at heavy-chain residues 77 (Ser instead of Asp), 152 (Glu instead of Val) and 211 (Gly instead of Ala). In contrast to the ubiquitous HLA-B*2705 subtype, HLA-B*2704 occurs only in orientals. Both subtypes are strongly associated with spondyloarthropathies and the peptides presented by these subtypes are suspected to play a role in disease pathogenesis. HLA-B*2704 was crystallized in complex with a viral peptide and with a self-peptide using the hanging-drop vapour-diffusion method with PEG as a precipitant. Both crystals belong to space group P2(1)2(1)2(1). Data sets were collected to 1.60 A (complex with the self-peptide pVIPR) or to 1.90 A (complex with the viral peptide pLMP2) resolution using synchrotron radiation. With HLA-B*2705 complexed with pVIPR as a search model, unambiguous molecular-replacement solutions were found for the complexes of HLA-B*2704 with both peptides.

Crystallization↗

PubMed1986

[Course control and significance of circulating HBsAg-specific immune complexes in acute viral hepatitis B].

The occurrence and behaviour of circulating HBsAg-specific immune complexes were investigated in 52 patients suffering from acute viral hepatitis B. In 6 patients the illness took a chronic course. The determination was carried out by precipitation with polyethylene glycol 6,000 and the increase of HBsAg concentration after antibody splitting from the complexes by trypsin treatment. The results allow following conclusions: The early presence of immune complexes suggests a specific antibody response also against HBsAg before development of clinical illness. The timing and strength of this humoral immune reaction is sex dependent; females show anti-HBs earlier and in higher titers. The course of the concentration of circulating immune complexes depends on the immunoglobulin class of antibody and the quickness of virus elimination. The determination of circulating unspecific or HBsAg-specific immune complexes does not allow to draw any prognostic conclusion to the further course of acute hepatitis B. Only in chronificating cases, the future activity of the chronic HBV infection may be expected by the early behaviour of HBsAg immune complexes. Also patients developing chronic course of the HBV infection demonstrated a "peak" of the immune reaction 6-8 weeks after the beginning of disease, which could offer the point of time for starting an immunomodulating therapy.

Adolescent↗

PubMed2004

Tumor efficacy and biodistribution of linear polyethylenimine-cholesterol/DNA complexes.

Non-viral polymer/pDNA complexes were formed using linear polyethylenimine (LPEI) Mw 25 k conjugated to cholesterol in a T-shaped geometry (LPC-T) and pDNA encoding murine interleukin-12 (pmIL-12e). These complexes were subsequently injected weekly into BALB/c mice intravenously and locally for the treatment of murine renal cell adenocarcinoma (Renca) induced pulmonary metastases and subcutaneous (SC) Renca tumors, respectively. At the cessation of the pulmonary metastases study, the number of pulmonary metastases was significantly less (p < 0.001) with systemic injections of LPC-T/pmIL-12e formulations than with pmIL-12e alone or pmIL-12e complexed with LPEI, branched polyethylenimine (BPEI) Mw 25 k, or an LPEI/pEGFP control. In addition, biodistribution studies showed increased pulmonary levels of both the LPC-T carrier and pmIL-12e vector up to 3 hr after systemic injection of the LPC-T/pmIL-12e complexes into mice carrying pulmonary metastases. Furthermore, mice systemically treated with LPC-T/pmIL-12e showed a near linear profile in weight gain in the course of the pulmonary metastases study that suggests increased biocompatibility. Finally, due to favorable characteristics in vitro, LPC-T was also used for local (peritumoral) injection of SC Renca tumors. Tumor stasis and slight tumor regression were seen only with the LPC-T/pmIL-12e treated mice compared to BPEI/pmIL-12e, LPEI/pmIL-12e, and naked pmIL-12e controls. Thus, it was concluded that LPC-T is an effective carrier for passive targeting of the pulmonary tissue, treatment of Renca-induced pulmonary metastases, and local administration of Renca cell SC tumors.

Animals↗

PubMed2003

Engineered retargeting of viral RNA replication complexes to an alternative intracellular membrane.

Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles. We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase. Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain. We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p. Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER. FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels. Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes. Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A. We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.

Amino Acid Sequence↗