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PubMed2006

Human rhinovirus induces robust IP-10 release by monocytic cells, which is independent of viral replication but linked to type I interferon receptor ligation and STAT1 activation.

Human rhinovirus (HRV)-induced respiratory infections are associated with elevated levels of IFN-gamma-inducible protein 10 (IP-10), which is an enhancer of T lymphocyte chemotaxis and correlates with symptom severity and T lymphocyte number. Increased IP-10 expression is exhibited by airway epithelial cells following ex vivo HRV challenge and requires intracellular viral replication; however, there are conflicting reports regarding the necessity of type I IFN receptor ligation for IP-10 expression. Furthermore, the involvement of resident airway immune cells, predominantly bronchoalveolar macrophages, in contributing to HRV-stimulated IP-10 elaboration remains unclear. In this regard, our findings demonstrate that ex vivo exposure of human peripheral blood monocytes and bronchoalveolar macrophages (monocytic cells) to native or replication-defective HRV serotype 16 (HRV16) resulted in similarly robust levels of IP-10 release, which occurred in a time- and dose-dependent manner. Furthermore, HRV16 induced a significant increase in type I IFN (IFN-alpha) release and STAT1 phosphorylation in monocytes. Neutralization of the type I IFN receptor and inhibition of JAK or p38 kinase activity strongly attenuated HRV16-stimulated STAT1 phosphorylation and IP-10 release. Thus, this work supports a model, wherein HRV16-induced IP-10 release by monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent JAK/STAT pathway activity. Our findings demonstrating robust activation of monocytic cells in response to native and/or replication-defective HRV16 challenge represent the first evidence indicating a mechanistic disparity in the activation of macrophages when compared with epithelial cells and suggest that macrophages likely contribute to cytokine elaboration following HRV challenge in vivo.

Cell Communication↗
PubMed2001

Hepatitis B virus harboring nucleotide deletions in the core promoter region and genotype B correlate with low viral replication activity in anti-HBe positive carriers.

BACKGROUND: Emergence of anti-HBe following seroconversion of HBe antigen indicates reduced hepatitis B virus (HBV) replication in the liver and low infectivity in the natural course of infection. However, some patients show continued replication or reactivation even in the presence of anti-HBe. OBJECTIVE: To clarify the cause of HBV replication, we investigated genotype differences and mutations in the core promoter and precore region in relation to virus titer. STUDY DESIGN: Using quantification of HBV DNA, nucleotide sequencing of the core promoter and precore region, and genotyping with the S gene by restriction fragment length polymorphism (RFLP), we analyzed sera of 26 anti-HBe positive carriers (28 serum samples). RESULTS: Various mutations were detected including C to T point mutation at nt 1653, A to T and G to A contiguous point mutations at nt 1762 and 1764 in the core promoter region, and G to A point mutation at nt 1896 in the precore region, but no common mutations were detected that were directly related to the virus titer from earlier reported mutations. In contrast, the mean titer of genotype B virus was 1.5 x 10(5) copies per ml and that of mutant HBV of genotype C having 8 base pairs (8-bp) deletion (nt 1768-1775) in the core promoter region was 7.9 x 10(4) copies per ml (mean titer). These titers showed commonly lower than that of genotype C virus without 8-bp deletion (median titer 5.0 x 10(6) copies per ml). Transition of genotype from C to B after viral reactivation and reduction of proportion of 8-bp deletion mutant at reactivation period was observed in a patient who demonstrated exacerbation of liver dysfunction due to immunosuppressive therapy and increased viral replication. CONCLUSIONS: These results confirm those of our earlier study describing low replication ability of 8-bp deletion mutant HBV in vitro, and also indicate that the presence of genotype B correlates with reduced titer of HBV.

Adult↗
PubMed1988

IgM anti-HBc in acute and chronic hepatitis B virus (HBV) infection: diagnostic value and correlation with viral replication and disease activity.

IgM antibody to hepatitis B core antigen (IgM anti-HBc) was determined in 348 subjects with hepatitis B virus infection, in order to evaluate, in an area of high hepatitis D virus (HDV) endemicity, its usefulness in discriminating acute HBV hepatitis from HDV superinfection of HBsAg carriers, and to see whether in chronic HBV infection IgM anti-HBc can be related to viral replication, disease activity, or risk of neoplastic transformation. The positive predictive value (PV) of IgM anti-HBc for acute hepatitis was low at the standard 2.1 cut-off (35.2%), but a cut-off of 7 raised the positive PV, retaining a negative PV of above 90% and a sensitivity of 59.5%. No significant relationship was found in chronic infection between IgM anti-HBc, the level of HBV replication or the type of liver disease. Patients with HDV infection were mostly IgM anti-HBc negative. Hepatocellular carcinoma was not associated with a raised prevalence of IgM anti-HBc.

Acute Disease↗
PubMed1996

Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara.

The dynamics of plasma viremia were explored in a group of 12 simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) that had received prior immunization with either nonrecombinant or trivalent (gag-pol, env) SIV-recombinant vaccinia viruses. Three distinct patterns of viral replication observed during and following primary viremia accounted for significant differences in survival times. High-level primary plasma viremia with subsequently increasing viremia was associated with rapid progression to AIDS (n = 2). A high-level primary plasma virus load with a transient decline and subsequent progressive increase in viremia in the post-acute phase of infection was associated with progression to AIDS within a year (n = 6). Low levels of primary plasma viremia followed by sustained restriction of virus replication were associated with maintenance of normal lymphocyte subsets and intact lymphoid architecture (n = 4), reminiscent of the profile observed in human immunodeficiency virus type 1-infected long-term nonprogressors. Three of four macaques that showed this pattern had been immunized with an SIV recombinant derived from the attenuated vaccinia virus, modified vaccinia virus Ankara. These data link the dynamics and extent of virus replication to disease course and suggest that sustained suppression of virus promotes long-term, asymptomatic survival of SIV-infected macaques. These findings also suggest that vaccine modulation of host immunity may have profound beneficial effects on the subsequent disease course, even if sterilizing immunity is not achieved.

Animals↗
PubMed2005

Non-replicating viral vector-based AIDS vaccines: interplay between viral vectors and the immune system.

During the past 20 years, the development of HIV vaccines has come a long way. The focus has progressively changed from the traditional protein-based HIV vaccines that induce humoral immunity to the live recombinant viral vector-based HIV vaccines capable of eliciting both cellular and humoral immune responses. These new viral vector-based vaccines encoding multiple HIV antigens, delivered either alone or in heterologous prime-boost modalities elicited antigen-specific CTL responses in immunized hosts and protected animals from disease. The viral vector-based vaccines have proven to be potent vaccines in pre-clinical studies and foster the hope to put an end to the ever-increasing threat of the AIDS epidemic. Several unique features of viral vector-based HIV vaccines have contributed to their success, including their intrinsic immune-modulating properties, high transduction efficiency, and in vivo production of immunogens within the cell mimicking a natural infection without the associated health risks. In this review, we will discuss the characteristics of non-replicating viral vectors most commonly used for HIV vaccines with a particular focus on immune responses elicited by the vector particles alone and their effect on the potency of viral vector-based HIV vaccines.

AIDS Vaccines↗
PubMed1992

Study of viral replication in HIV-1-infected CEM T-cell subclones which are reduced in their ability to form syncytia.

The derivation of ethyl-methanesulfonate (EMS) mutagenized subclones from the CEM T-cell line has been described. These clones expressed CD4 and bound soluble gp120, however, two of the generated clones were markedly reduced in their ability to form syncytia after infection with either gp160-vaccinia vector or cell-free human immunodeficiency virus type 1 (HIV-1). Here, we asked at what stage(s) viral infection is blocked in these cells. Polymerase chain reaction (PCR) analysis revealed that at 6 and 72 h postinfection with HIV-1, cells of the syncytia-deficient clones expressed markedly reduced amounts of viral-specific DNA compared with cells of the parental line or the syncytia-positive clones. Long-term cultures revealed a marked delay in the appearance of reverse transcriptase (RT) activity in the supernatants of these subclones when compared with the parental line and viral replication did not lead to massive cell death. Syncytia formation in HIV-1-infected cultures of the syncytia-deficient subclones was enhanced by tumor necrosis factor alpha (TNF alpha) when added 24 h postinfection. In contrast, pretreatment with TNF alpha for 48 h followed by washing and infection of the cells with HIV-1 augmented syncytia formation of the syncytia-positive subclones, but not of the syncytia-negative subclones. Thus, the EMS-mutagenized subclones may provide a tool to study host cell factors required for the establishment of a productive HIV-1 infection and responsiveness to TNF alpha.

Clone Cells↗
PubMed1994

High levels of viral replication during acute hepatitis B infection predict progression to chronicity.

To assess the pattern of development of serologic markers during acute hepatitis B, levels of HBsAg, HBeAg, and hepatitis B virus (HBV) DNA were assayed in stored serum samples obtained sequentially from 12 subjects infected with HBV during experimental studies conducted in the 1950s. Six patients developed acute self-limited hepatitis, three developed chronic hepatitis, and three had an asymptomatic infection without HBsAg. HBsAg was the first serologic marker detected (mean = 52 days after exposure), followed by HBeAg (62 days) and HBV DNA (72 days). Peak HBsAg levels occurred before onset of symptoms and correlated with peak titers of HBeAg and HBV DNA. Patients who developed chronic hepatitis had higher peak levels of viral markers than those with self-limited disease: HBsAg (30 versus 5.4 micrograms/ml), HBeAg (1:2,000 versus 1:60 titer) and HBV DNA (3,192 versus 444 pg/ml). Thus, chronic HBV infection is characterized by high levels of viral replication appearing early during the acute phase of infection.

Acute Disease↗
PubMed2001

Passive transfer of antibodies protects immunocompetent and imunodeficient mice against lethal Ebola virus infection without complete inhibition of viral replication.

Ebola hemorrhagic fever is a severe, usually fatal illness caused by Ebola virus, a member of the filovirus family. The use of nonhomologous immune serum in animal studies and blood from survivors in two anecdotal reports of Ebola hemorrhagic fever in humans has shown promise, but the efficacy of these treatments has not been demonstrated definitively. We have evaluated the protective efficacy of polyclonal immune serum in a mouse model of Ebola virus infection. Our results demonstrate that mice infected subcutaneously with live Ebola virus survive infection and generate high levels of anti-Ebola virus immunoglobulin G (IgG). Passive transfer of immune serum from these mice before challenge protected upto 100% of naive mice against lethal Ebola virus infection. Protection correlated with the level of anti-Ebola virus IgG titers, and passive treatment with high-titer antiserum was associated with a delay in the peak of viral replication. Transfer of immune serum to SCID mice resulted in 100% survival after lethal challenge with Ebola virus, indicating that antibodies alone can protect from lethal disease. Thus antibodies suppress or delay viral growth, provide protection against lethal Ebola virus infection, and may not require participation of other immune components for protection.

Animals↗
PubMed1993

The feline immunodeficiency virus ORF-A gene facilitates efficient viral replication in established T-cell lines and peripheral blood lymphocytes.

Frameshift mutants corresponding to all of the identified open reading frames of feline immunodeficiency virus, including the ORF-A gene, which has an unknown function, were constructed in vitro. Upon transfection into cells, no significant difference between the phenotypes of ORF-A mutant clones and those of wild-type clones was demonstrated. Although only ORF-A mutant virus among all mutant viruses from transfected cells showed infectivity in established T-cell lines, the replication and propagation of the ORF-A mutant virus were efficiently reduced compared with those of the wild-type virus. Moreover, the loss of the function of the ORF-A gene resulted in a severe defect in productive infection in primary peripheral blood lymphocytes both in the amount of reverse transcriptase activity produced and in core protein expression. These findings demonstrate that the ORF-A gene of feline immunodeficiency virus is required for efficient viral replication and suggest that the ORF-A gene is likely to be important for natural infection.

Animals↗
PubMedDate not supplied

Insect virus: assays for viral replication and persistence in mammalian cells.

Viral pesticidal agents must be evaluated for potential health hazards prior to utilization. Assessment of the likelihood of replication in humans has included in vitro exposure of human cells to the potential pesticidal agent. Previous in vitro evaluation strategies have lacked positive controls. Thus, negative results, interpreted as no effect of the virus on human cells, could reflect basic deficiencies in the testing protocols. We designed a testing scheme for viral pesticides and used it to test the nuclear polyhedrosis virus of Autographa californica. Tests were aimed at evaluating potential replication or gene expression in primate cells. Parallel tests were run utilizing identical protocols with primate viruses known to produce the biological effect being evaluated. Thus protocols described were tested with positive viral controls.

Animals↗
PubMed1985

Latent and viral replicative transcription in vivo from the BamHI K fragment of Epstein-Barr virus DNA.

We mapped one latent and two replicative messages transcribed in vivo from the BamHI K fragment of the Epstein-Barr virus genome. The exon encoding Epstein-Barr nuclear antigen (EBNA), a major latent product, is 2,028 bases; the 3' end of this exon occurs 30 bases after the polyadenylation signal AATAAA, and the 5' end occurs within a splice acceptor site. The open reading frame which encodes the EBNA peptide is completely contained within this coding exon. The exon was faithfully transcribed after transfection of cloned BamHI-K into either COS-1 or TK- mouse L cells. In lymphocytes the abundance of the EBNA message is increased after cycloheximide treatment. The two viral replicative genes completely contained in BamHI-K were not transcribed in line X50-7, in which the genome is tightly latent. In contrast to the EBNA message, these mRNAs of 1.3 and 2.1 kilobases are inducible with phorbol ester and are unspliced. Their promoter regions are similar to those of each other and to replicative promoters mapped in other regions of the Epstein-Barr virus genome (P. J. Farrell, A. Bankier, C. Seguin, P. Deininger, and B. G. Barrell, EMBO J. 2:1331-1338, 1983). An unusual feature of these replicative genes is that the smaller mRNA begins within a long open reading frame of the larger mRNA. The identification of the structure of latent and replicative genes within one DNA fragment will facilitate analysis of regulation of expression for the two life cycles of the virus.

Antigens, Viral↗
PubMed1991

Early viral replication in the brain of SIV-infected rhesus monkeys.

To investigate the mechanism of simian immunodeficiency virus (SIV) entry into the central nervous system (CNS) and the initial events leading to neuropathogenesis, SIV replication was studied by in situ hybridization in the CNS of 5 Rhesus macaques at 7 days, 1, 2, and 3 months after SIV intravenous inoculation. CNS infection was found to be a frequent and early event, as SIV was detected in the CNS of all the animals studied and as early as 7 days postinoculation. At the earliest stage, the infection localized mainly to perivascular cells. Using combined immunohistochemistry and in situ hybridization, infected cells were shown to express the CD68 marker, suggesting that infected mononuclear phagocytes crossing the blood-brain barrier represent the main source of virus in the CNS. Early viral replication coincided with neuropathologic changes, consisting in gliosis, perivascular infiltrates and rare glial nodules. Immunophenotyping of brain tissue showed that increased macrophage infiltration, microglial reactivity and MHC class II induction occurred within the first week of infection, indicating a possible immunopathologic mechanism in early CNS pathogenesis.

Animals↗
PubMed2001

Interferon resistance of cutaneous T-cell lymphoma-derived clonal T-helper 2 cells allows selective viral replication.

Cutaneous T-cell lymphomas (CTCL) comprise a heterogeneous group of lymphoproliferative disorders that are characterized by an accumulation of T-lymphocytes in the skin and occasionally in blood known as Sézary syndrome (SS). In most cases the dominant clone displays T-helper 2 cytokines. Because IFN-gamma is a natural inhibitor of T-helper 2 cells and IFN-alpha is frequently used in CTCL, the impact of IFNs on SS-derived purified clonal T-helper 2 cells was studied using anti-Vbeta antibodies. Moreover, IFNs are known to mediate virus resistance in normal cells. The isolated clonal CD4(+) cells, but not the nonclonal CD4(+) cells, appeared resistant to IFN-gamma and IFN-alpha stimulation in terms of human leukocyte antigen up-regulation and MxA induction caused in part by alterations in Stat-1 molecule mRNA and IFNgammaR1 mRNA transcription. The IFN resistance of the patient-derived clonal cells was then targeted by vesicular stomatitis virus infection after IFN-alpha priming, resulting in selective viral replication in clonal cells. In contrast, nonclonal cells of the same patient showed IFN-dependent MxA expression, which is a major mediator protein of viral protection. The IFN resistance of the dominant T-helper 2 cells might be important for lymphomagenesis. Interferon signaling deficiencies can be targeted for purging patients' cells in vitro. Furthermore, this approach may allow specific molecular interventions, resulting in the efficient treatment of CTCL and other IFN-resistant neoplasms such as lung cancer.

Clone Cells↗
PubMed2006

Impaired in-vitro growth of megakaryocytic colonies derived from CD34 cells of HIV-1-infected patients with active viral replication.

OBJECTIVE: To address the mechanisms of the thrombocytopoietic dysfunction that may follow HIV infection and to compare peripheral blood and bone marrow as sources of CD34 progenitor cells in HIV-infected patients. METHODS: The study used CD34 progenitor cells from 20 previously untreated HIV-infected individuals, 20 HIV-infected individuals treated with antiretroviral therapy and a control group of 20 HIV-uninfected healthy individuals to examine in-vitro megakaryocytopoiesis. There were no hematological abnormalities at baseline in the study groups. CD34 progenitor cells derived from peripheral blood and bone marrow were purified and cultured in medium containing thrombopoietin, interleukin-3, and interleukin-6. HIV-1 plasma viral load was determined by b-DNA technique. Expression of receptors for thrombopoietin, interleukin-3, and interleukin-6 was assessed on CD34 cells by flow cytometry, and numbers of receptors per single cell were calculated by Quanticalc software. RESULTS: Growth of megakaryocytopoietic colony-forming units (CFU-MK) were impaired in untreated HIV-infected individuals despite normal platelet counts. Viral load levels inversely correlate with CFU-MK growth and platelet counts. Antiretroviral drug-treated individuals showed normal megakaryocyte development. Similar results were obtained whether the CD34 progenitor cells derived from peripheral blood or bone marrow. CONCLUSIONS: These findings suggest that megakaryocyte differentiation is impaired before the onset of overt thrombocytopenia in HIV-infected patients and provide evidence for a direct link between viral replication and perturbed megakaryocytopoiesis, which appears to be prevented and/or restored by antiretroviral therapy. The results indicate that peripheral blood represents a suitable source of CD34 hematopoietic progenitors for studies of megakaryocytopoiesis in HIV disease.

Antigens, CD34↗
PubMed2003

Conformational changes in the herpes simplex virus ICP8 DNA-binding protein coincident with assembly in viral replication structures.

The herpes simplex virus (HSV) single-stranded DNA-binding protein, ICP8, is required for viral DNA synthesis. Before viral DNA replication, ICP8 colocalizes with other replication proteins at small punctate foci called prereplicative sites. With the onset of viral genome amplification, these proteins become redistributed into large globular replication compartments. Here we present the results of immunocytochemical and biochemical analysis of ICP8 showing that various antibodies recognize distinct forms of ICP8. Using these ICP8-specific antibodies as probes for ICP8 structure, we detected a time-dependent appearance and disappearance of ICP8 epitopes in immunoprecipitation assays. Immunofluorescence staining of ICP8 in cells infected with different HSV mutant viruses as well as cells transfected with a limited number of viral genes demonstrated that these and other antigenic changes occur coincident with ICP8 assembly at intranuclear replication structures. Genetic analysis has revealed a correlation between the ability of various ICP8 mutant proteins to form the 39S epitope and their ability to bind to DNA. These results support the hypothesis that ICP8 undergoes a conformational change upon binding to other HSV proteins and/or to DNA coincident with assembly into viral DNA replication structures.

Cell Line↗
PubMed2001

Improving immune function and controlling viral replication in HIV-1-infected patients with immune-based therapies.

Restoring and preserving immune function is a key component to successfully managing HIV-1 disease. Phase II/III studies have evaluated the safety and immunologic effects of immune-based therapies, including granulocyte-macrophage colony-stimulating factor, interleukin-2, and an inactivated HIV-1 immunogen, as adjuncts to antiretroviral therapy. Addition of each of these immune-based therapies to a background antiretroviral regimen enhanced, to varying degrees, immunologic function and suppression of viral replication in HIV-1-infected patients, suggesting a potential role for immune-based therapies in the treatment of HIV-1 disease. Further studies are needed to better characterize specific immunologic and virologic effects in different patient populations and to determine their impact on clinical outcomes.

AIDS Vaccines↗
PubMed1996

Mechanism of RSV-induced IL-8 gene expression in A549 cells before viral replication.

Previous studies demonstrated that respiratory syncytial virus (RSV) infection of A549 cells induced interleukin (IL)-8 gene expression and protein release from the cells as early as 2 h after treatment [M. A. Fiedler, K. Wernke-Dollries, and J. M. Stark. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L865-L872, 1995; J. G. Mastronarde, M. M. Monick, and G. W. Hunninghake. Am. J. Respir. Cell Mol. Biol. 13: 237-244, 1995]. Furthermore, the effects of RSV at the 2-h time point were not dependent on viral replication. The studies reported here were designed to test the hypothesis that active and inactive RSV induce IL-8 gene expression in A549 cells at the 2-h time point by a mechanism dependent on the activation of the nuclear transcription factor NF-kappa B Northern blot analysis indicated that IL-8 gene expression occurred independent of protein synthesis 2 h after A549 cells were treated with RSV. Analysis of nuclear extracts from RSV-treated A549 cells by electrophoretic mobility shift assays demonstrated that NF-kappa B was activated as early as 15 min after RSV was added to the cells and remained activated for at least 90 min. In contrast, baseline levels of NF-IL-6 and activator protein-1 (AP-1) did not change over this period of time. Deoxyribonuclease footprint analysis of a portion of the 5'-flanking region of the IL-8 gene demonstrated two potential regions for transcription factor binding, which corresponded to the potential AP-1 binding site, and potential NF-IL-6 and NF-kappa B binding sites. Mutational analysis of the 200-bp 5'-untranslated region of the IL-8 gene demonstrated that activation of NF-kappa B and NF-IL-6 were required for RSV-induced transcriptional activation of the IL-8 gene.

DNA Mutational Analysis↗