Showing papers on "Virus published in 1997"

Hepatitis B virus infection.

Abstract: The hepatitis B virus (HBV), discovered in 1966, infects more than 350 million people worldwide.1 Hepatitis B is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma, accounting for 1 million deaths annually. Knowledge of the intricacies of viral infection and of the molecular biology of this fascinating virus has led to the successful development of a vaccine and to treatment sometimes capable of eradicating chronic infection. This review addresses many aspects of HBV infection, including the role of the immune system in determining the outcome of clinical infection, recent developments in molecular studies of the virus, and new . . .

Plasma Viral Load and CD4+ Lymphocytes as Prognostic Markers of HIV-1 Infection

Abstract: Background: The rate of disease progression among persons infected with human immunodeficiency virus type 1 (HIV-1) varies widely, and the relative prognostic value of markers of disease activity h...

Recovery of replication-competent HIV despite prolonged suppression of plasma viremia.

Abstract: In evaluating current combination drug regimens for treatment of human immunodeficiency virus (HIV) disease, it is important to determine the existence of viral reservoirs. After depletion of CD8 cells from the peripheral blood mononuclear cells (PBMCs) of both patients and normal donors, activation of patient CD4 lymphocytes with immobilized antibodies to CD3 and CD28 enabled the isolation of virus from PBMCs of six patients despite the suppression of their plasma HIV RNA to fewer than 50 copies per milliliter for up to 2 years. Partial sequencing of HIV pol revealed no new drug resistance mutations or discernible evolution, providing evidence for viral latency rather than drug failure.

Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection

Abstract: The capacity of HIV-1 to establish latent infection of CD4+ T cells may allow viral persistence despite immune responses and antiretroviral therapy. Measurements of infectious virus and viral RNA in plasma and of infectious virus, viral DNA and viral messenger RNA species in infected cells all suggest that HIV-1 replication continues throughout the course of infection. Uncertainty remains over what fraction of CD4+ T cells are infected and whether there are latent reservoirs for the virus. We show here that during the asymptomatic phase of infection there is an extremely low total body load of latently infected resting CD4+ T cells with replication-competent integrated provirus (<10(7) cells). The most prevalent form of HIV-1 DNA in resting and activated CD4+ T cells is a full-length, linear, unintegrated form that is not replication competent. The infection progresses even though at any given time in the lymphoid tissues integrated HIV-1 DNA is present in only a minute fraction of the susceptible populations, including resting and activated CD4+ T cells and macrophages.

Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy

Abstract: Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+ T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+ T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (<500 copies HIV RNA/ml) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4+ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4+ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.

RNA virus mutations and fitness for survival

Abstract: RNA viruses exploit all known mechanisms of genetic variation to ensure their survival. Distinctive features of RNA virus replication include high mutation rates, high yields, and short replication times. As a consequence, RNA viruses replicate as complex and dynamic mutant swarms, called viral quasispecies. Mutation rates at defined genomic sites are affected by the nucleotide sequence context on the template molecule as well as by environmental factors. In vitro hypermutation reactions offer a means to explore the functional sequence space of nucleic acids and proteins. The evolution of a viral quasispecies is extremely dependent on the population size of the virus that is involved in the infections. Repeated bottleneck events lead to average fitness losses, with viruses that harbor unusual, deleterious mutations. In contrast, large population passages result in rapid fitness gains, much larger than those so far scored for cellular organisms. Fitness gains in one environment often lead to fitness losses in an alternative environment. An important challenge in RNA virus evolution research is the assignment of phenotypic traits to specific mutations. Different constellations of mutations may be associated with a similar biological behavior. In addition, recent evidence suggests the existence of critical thresholds for the expression of phenotypic traits. Epidemiological as well as functional and structural studies suggest that RNA viruses can tolerate restricted types and numbers of mutations during any specific time point during their evolution. Viruses occupy only a tiny portion of their potential sequence space. Such limited tolerance to mutations may open new avenues for combating viral infections.

Change in Coreceptor Use Correlates with Disease Progression in HIV-1–Infected Individuals

Abstract: Recent studies have identified several coreceptors that are required for fusion and entry of Human Immunodeficiency Virus type 1 (HIV-1) into CD4+ cells. One of these receptors, CCR5, serves as a coreceptor for nonsyncytium inducing (NSI), macrophage-tropic strains of HIV-1, while another, fusin or CXCR-4, functions as a coreceptor for T cell line-adapted, syncytium-inducing (SI) strains. Using sequential primary isolates of HIV-1, we examined whether viruses using these coreceptors emerge in vivo and whether changes in coreceptor use are associated with disease progression. We found that isolates of HIV-1 from early in the course of infection predominantly used CCR5 for infection. However, in patients with disease progression, the virus expanded its coreceptor use to include CCR5, CCR3, CCR2b, and CXCR-4. Use of CXCR-4 as a coreceptor was only seen with primary viruses having an SI phenotype and was restricted by the env gene of the virus. The emergence of variants using this coreceptor was associated with a switch from NSI to SI phenotype, loss of sensitivity to chemokines, and decreasing CD4+ T cell counts. These results suggest that HIV-1 evolves during the course of infection to use an expanded range of coreceptors for infection, and that this adaptation is associated with progression to AIDS.

Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus.

Abstract: The HIV-1-specific cytotoxic T lymphocyte (CTL) response is temporally associated with the decline in viremia during primary HIV-1 infection, but definitive evidence that it is of importance in virus containment has been lacking. Here we show that in a patient whose early CTL response was focused on a highly immunodominant epitope in gp160, there was rapid elimination of the transmitted virus strain and selection for a virus population bearing amino acid changes at a single residue within this epitope, which conferred escape from recognition by epitope-specific CTL. The magnitude (> 100-fold), kinetics (30–72 days from onset of symptoms) and genetic pathways of virus escape from CTL pressure were comparable to virus escape from antiretroviral therapy, indicating the biological significance of the CTL response in vivo. One aim of HIV-1 vaccines should thus be to elicit strong CTL responses against multiple codominant viral epitopes.

Late escape from an immunodominant cytotoxic T-lymphocyte response associated with progression to AIDS.

Abstract: The precise role played by HIV-specific cytotoxic T lymphocytes (CTL) in HIV infection remains controversial Despite strong CTL responses being generated during the asymptomatic phase, the virus persists and AIDS ultimately develops It has been argued that the virus is so variable, and the virus turnover so great that escape from CTL recognition would occur continually, but so far there is limited evidence for CTL escape The opposing argument is that evidence for CTL escape is present but hard to find because multiple anti-HIV immune responses are acting simultaneously during the asymptomatic phase of infection We describe six donors who make a strong CTL response to an immunodominant HLA-B27-restricted epitope In the two donors who progressed to AIDS, CTL escape to fixation by the same mutation was observed, but only after 9-12 years of epitope stability CTL escape may play an important role in the pathogenesis of HIV infection

Transmission of Hepatitis C by Intrahepatic Inoculation with Transcribed RNA

Abstract: More than 1% of the world's population is chronically infected with hepatitis C virus (HCV). HCV infection can result in acute hepatitis, chronic hepatitis, and cirrhosis, which is strongly associated with development of hepatocellular carcinoma. Genetic studies of HCV replication have been hampered by lack of a bona fide infectious molecular clone. Full-length functional clones of HCV complementary DNA were constructed. RNA transcripts from the clones were found to be infectious and to cause disease in chimpanzees after direct intrahepatic inoculation. This work defines the structure of a functional HCV genome RNA and proves that HCV alone is sufficient to cause disease.

Expression cloning of new receptors used by simian and human immunodeficiency viruses

Abstract: Several members of the chemokine-receptor family serve, in conjunction with CD4, as receptors for the entry of human immunodeficiency virus type I (HIV-1) into cells1,2,3,4,5,6 The principal receptor for entry of macrophage-tropic (M-tropic) HIV-1 strains is CCR5, whereas that for T-cell-line-tropic (T-tropic) strains is CXCR4 Unlike HIV-1, infection with either M-tropic or T-tropic strains of simian immunodeficiency virus (SIV) can be mediated by CCR5, but not CXCR4 (refs 7, 8, 9, 10) SIV strains will also infect CD4+ cells that lack CCR5, which suggests that these strains use as yet unidentified receptors7,9,10 Here we use an expression-cloning strategy to identify SIV receptors and have isolated genes encoding two members of the seven-transmembrane G-protein-coupled receptor family that are used not only by SIVs, but also by strains of HIV-2 and M-tropic HIV-1 Both receptors are closely related to the chemokine-receptor family and are expressed in lymphoid tissues One of the receptors is also expressed in colon and may therefore be important in viral transmission Usage of these new receptors following experimental infection of non-human primates with SIV strains may provide important insight into viral transmission and the mechanisms of SIV- and HIV-induced acquired immune-deficiency syndrome

Human herpesvirus KSHV encodes a constitutively active G-protein-coupled receptor linked to cell proliferation

Abstract: Kaposi's sarcoma-associated herpesvirus (KSHV, also known as human herpesvirus 8, or HHV 8) is a virus that is consistently present in Kaposi's sarcoma and in primary-effusion (body-cavity-based) lymphomas, malignancies that occur frequently, but not exclusively, in AIDS patients. KSHV is a gamma herpesvirus with homology to herpesvirus Saimiri and Epstein-Barr virus, both of which can transform lymphocytes. Cloning of a KSHV genome fragment revealed the presence of an open reading frame encoding a putative G-protein-coupled receptor that is homologous to a G-protein-coupled receptor encoded by herpesvirus Saimiri and to human interleukin-8 receptors. Here we show that the KSHV G-protein-coupled receptor is a bona fide signalling receptor which has constitutive (agonist-independent) activity in the phosphoinositide-inositoltrisphosphate-protein kinase C pathway. Furthermore, the KSHV G-protein-coupled receptor stimulates cellular proliferation, making it a candidate viral oncogene.

Regulation of apoptosis by viral gene products.

Abstract: Apoptosis is the process whereby individual cells of multicellular organisms undergo systematic self-destruction in response to a wide variety of stimuli. Apoptosis is a genetically controlled preprogrammed event which eliminates cells during development when they have become redundant or which functions as an emergency response after radiation damage, viral infection, or aberrant growth induced by the activation of oncogenes. In the case of virus-infected cells, the induction of early cell death would severely limit virus production and reduce or eliminate spread of progeny virus in the host. Thus, most animal viruses have evolved strategies to evade or delay early apoptosis to allow production of high yields of progeny virus. Over the past few years both the biochemical basis of apoptosis and its regulation by viral products have become clearer. For example, part of the apoptotic program includes the induction of cellular endonucleases which could target replicating viral DNA and prevent virus production at a very early stage. Thus, the problem facing the virus is to replicate and package large numbers of progeny genomes safely within newly synthesized viral capsids. Many viruses have evolved genes encoding proteins which effectively suppress or delay apoptosis long enough for the production of sufficient quantities of progeny. In addition, a growing number of viruses are now known to induce apoptosis actively at late stages of infection. This process may represent a final and important step in the spread of progeny to neighboring cells while also evading host immune inflammatory responses and protecting progeny virus from host enzymes and antibodies. Such virally induced apoptosis may also contribute to some clinical manifestations and cytotoxicity associated with several human diseases of viral origin. The purpose of this review is to summarize recent information on the induction and suppression of apoptosis by viral products as well as to propose how this knowledge may provide insights into basic cell biology and offer the potential of new therapeutic applications. A recent comprehensive review by Shen and Shenk (75) also deals with some aspects of the subjects covered below.

Kaposi's sarcoma-associated herpesvirus gene expression in endothelial (spindle) tumor cells.

Abstract: The recent discovery of DNA sequences of a new human herpesvirus in Kaposi's sarcoma (KS) has fueled speculation that this virus might cause KS. The mere presence, however, of a virus in a complex multicellular tumor like KS could just as well be construed as evidence of a passenger agent. We sought stronger evidence linking the KS-associated herpesvirus (KSHV) to tumor formation by using in situ hybridization to investigate the specificity, constancy, and timing of KSHV gene expression in KS tumor cells. Here we document expression of a 700-nucleotide viral RNA in every KS tumor examined, from the earliest histologically recognizable stage to advanced tumors in which the vast majority of identifiable spindle tumor cells contain this transcript. Two other KSHV RNAs were also detected in a smaller fraction of the tumor cells in all but the earliest lesion. These viral RNAs were expressed to relatively low levels in this subset; because one of these RNAs encodes a major viral capsid protein, these cells may be producing KSHV. We did not find these KSHV genes expressed in a variety of other tumors and proliferative processes, but we did detect viral gene expression in prostatic tissue, supporting a possible mechanism for sexual transmission of KSHV. The close relationship between KS and KSHV gene expression is consistent with the hypothesis that KSHV is directly involved in the etiology and pathogenesis of KS.

Initial Genetic Characterization of the 1918 “Spanish” Influenza Virus

Abstract: The "Spanish" influenza pandemic killed at least 20 million people in 1918-1919, making it the worst infectious pandemic in history. Understanding the origins of the 1918 virus and the basis for its exceptional virulence may aid in the prediction of future influenza pandemics. RNA from a victim of the 1918 pandemic was isolated from a formalin-fixed, paraffin-embedded, lung tissue sample. Nine fragments of viral RNA were sequenced from the coding regions of hemagglutinin, neuraminidase, nucleoprotein, matrix protein 1, and matrix protein 2. The sequences are consistent with a novel H1N1 influenza A virus that belongs to the subgroup of strains that infect humans and swine, not the avian subgroup.

Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype.

Abstract: The biological phenotype of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to the severity of the HIV infection. Here we show that the two previously described groups of rapid/high, syncytium-inducing (SI) and slow/low, non-syncytium-inducing (NSI) isolates are distinguished by their ability to utilize different chemokine receptors for entry into target cells. Recent studies have identified the C-X-C chemokine receptor CXCR4 (also named fusin or Lestr) and the C-C chemokine receptor CCR5 as the principal entry cofactors for T-cell-line-tropic and non-T-cell-line-tropic HIV-1, respectively. Using U87.CD4 glioma cell lines, stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, we have tested chemokine receptor specificity for a panel of genetically diverse envelope glycoprotein genes cloned from primary HIV-1 isolates and have found that receptor usage was closely associated with the biological phenotype of the virus isolate but not the genetic subtype. We have also analyzed a panel of 36 well-characterized primary HIV-1 isolates for syncytium induction and replication in the same series of cell lines. Infection by slow/low viruses was restricted to cells expressing CCR5, whereas rapid/high viruses could use a variety of chemokine receptors. In addition to the regular use of CXCR4, many rapid/high viruses used CCR5 and some also used CCR3 and CCR2b. Progressive HIV-1 infection is characterized by the emergence of viruses resistant to inhibition by beta-chemokines, which corresponded to changes in coreceptor usage. The broadening of the host range may even enable the use of uncharacterized coreceptors, in that two isolates from immunodeficient patients infected the parental U87.CD4 cell line lacking any engineered coreceptor. Two primary isolates with multiple coreceptor usage were shown to consist of mixed populations, one with a narrow host range using CCR5 only and the other with a broad host range using CCR3, CCR5, or CXCR4, similar to the original population. The results show that all 36 primary HIV-1 isolates induce syncytia, provided that target cells carry the particular coreceptor required by the virus.

Kinetics of response in lymphoid tissues to antiretroviral therapy of HIV-1 infection

Abstract: In lymphoid tissue, where human immunodeficiency virus–type 1 (HIV-1) is produced and stored, three-drug treatment with viral protease and reverse transcriptase inhibitors markedly reduced viral burden. This was shown by in situ hybridization and computerized quantitative analysis of serial tonsil biopsies from previously untreated adults. The frequency of productive mononuclear cells (MNCs) initially diminished with a half-life of about 1 day. Surprisingly, the amount of HIV-1 RNA in virus trapped on follicular dendritic cells (FDCs) decreased almost as quickly. After 24 weeks, MNCs with very few copies of HIV-1 RNA per cell were still detectable, as was proviral DNA; however, the amount of FDC-associated virus decreased by ≥3.4 log units. Thus, 6 months of potent therapy controlled active replication and cleared >99.9 percent of virus from the secondary lymphoid tissue reservoir.

Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development.

Abstract: We have isolated and sequenced a novel 11-kDa virucidal protein, named cyanovirin-N (CV-N), from cultures of the cyanobacterium (blue-green alga) Nostoc ellipsosporum. We also have produced CV-N recombinantly by expression of a corresponding DNA sequence in Escherichia coli. Low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. Continuous, 2-day exposures of uninfected CEM-SS cells or peripheral blood lymphocytes to high concentrations (e.g., 9,000 nM) of CV-N were not lethal to these representative host cell types. The antiviral activity of CV-N is due, at least in part, to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. The biological activity of CV-N is highly resistant to physicochemical denaturation, further enhancing its potential as an anti-HIV microbicide.

Virus dynamics and drug therapy

Abstract: The recent development of potent antiviral drugs not only has raised hopes for effective treatment of infections with HIV or the hepatitis B virus, but also has led to important quantitative insights into viral dynamics in vivo. Interpretation of the experimental data depends upon mathematical models that describe the nonlinear interaction between virus and host cell populations. Here we discuss the emerging understanding of virus population dynamics, the role of the immune system in limiting virus abundance, the dynamics of viral drug resistance, and the question of whether virus infection can be eliminated from individual patients by drug treatment.

A system for functional analysis of Ebola virus glycoprotein

Abstract: Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green fluorescent protein gene instead of the receptor-binding G protein gene (VSVΔG*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSVΔG*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSVΔG* complemented with VSV G protein (VSVΔG*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSVΔG*-ResGP but not to VSVΔG*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and/or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured in vitro.

Persistent infection with Theiler's virus leads to CNS autoimmunity via epitope spreading

Abstract: Multiple sclerosis (MS) is a T cell-mediated autoimmune demyelinating disease1, which may be initiated by a virus infection2. Theiler's murine encephalomyelitis virus (TMEV), a natural mouse pathogen, is a picornavirus that induces a chronic, CD4+ T cell-mediated demyelinating disease with a clinical course and histopathology similar to that of chronic progressive MS (ref. 3). Demyelination in TMEV-infected mice is initiated by a mononu-clear inflammatory response mediated by virus-specific CD4+ T cells targeting virus, which chronically persists in the CNS (ref. 4–6). We show that beginning 3–4 weeks after disease onset, T-cell responses to multiple myelin autoepitopes arise in an ordered progression and may play a pathologic role in chronic disease. Kinetic and functional studies show that T-cell responses to the immunodominant myelin proteolipid protein epitope (PLP139–151) did not arise because of cross-reactivity between TMEV and self epitopes (that is, molecular mimicry)7, 8, but because of de novo priming of self-reactive T cells to sequestered autoantigens released secondary to virus-specific T cell-mediated demyelination (that is, epitope spreading)9, 10. Epitope spreading is an important alternate mechanism to explain the etiology of virus-induced organ-specific autoimmune diseases.

Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.

Abstract: We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication.

Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee

Abstract: We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5′ and 3′ termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase–PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6–28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.

Macrophages as a Source of HIV During Opportunistic Infections

Abstract: The source of increasing viremia that characterizes the latter stages of human immunodeficiency virus (HIV) disease has remained a paradox because it occurs at a time when lymphoid tissue is quantitatively and qualitatively impaired, and the patients' CD4 T lymphocytes are steadily declining. Here, macrophages, both infected and uninfected with common opportunistic pathogens of HIV disease such as Mycobacterium avium complex and Pneumocystis carinii, were identified as highly productive sources of HIV in coinfected lymph nodes. These observations indicate that tissue macrophages are not only infected with HIV, but that common pathogens of HIV disease can dramatically increase their production of virus. Thus, prevention or successful treatment of opportunistic coinfections, or both, potentially benefits the patient twofold by limiting the pathology caused by opportunistic infection and by controlling induction of HIV replication.

Sulfated Polysaccharides Extracted from Sea Algae as Potential Antiviral Drugs

Abstract: The inhibitory effects of polyanionic substances on the replication of herpes simplex virus (HSV) and other viruses were reported almost four decades ago. However, these observations did not generate much interest, because the antiviral action of the compounds was considered to be largely nonspecific. Shortly after the identification of human immunodeficiency virus (HIV) as the causative agent of the acquired immune deficiency syndrome (AIDS) in 1984, heparin and other sulfated polysaccharides were found to be potent and selective inhibitors of HIV 1 replication in cell culture. Since 1988, the activity spectrum of the sulfated polysaccharides has been shown to extend to various enveloped viruses, including viruses that emerge as opportunistic pathogens (e.g., herpes simplex virus [HSV] and cytomegalovirus [CMV]) in immunosuppressed (e.g., AIDS) patients. As potential anti-HIV drug candidates, sulfated polysaccharides offer a number of promising features. They are able to block HIV replication in cell culture at concentrations as low as 0.1 to 0.01 mu g ml(-1) without toxicity to the host cells at concentrations up to 2.5 mg ml(-1). We noted that some polysulfates show a differential inhibitory activity against different HIV strains, suggesting that marked differences exist in the tar get molecules with which polysulfates interact, They not only inhibit the cytopathic effect of HIV, but also prevent HIV induced syncytium (giant cell) formation. Furthermore, experiments carried out with dextran sulfate samples of increasing molecular weight and with sulfated cyclodextrins of different degrees of sulfation have shown that antiviral activity increases with increasing molecular weight and degree of sulfation. A sugar backbone is not strictly needed for the anti-HIV activity of polysulfates because sulfated polymers composed of a carbon-carbon backbone have also proved to be highly efficient anti-HIV agents in vitro. Other, yet to be defined, structural features may also play an important role. Sulfated polysaccharides may act synergistically with other anti-HIV drugs (e.g., azidothymidine [AZT]). They are known to lead very slowly to virus drug resistance development and they show activity against HIV mutants that have become resistant to reverse transcriptase inhibitors, such as AZT, tetrahydro-imidazo [4,5,1-jk] [1,4] benzodiazepin 2 (1H) thione (TIBO) and others. From studies on their mechanism of action we concluded that polysulfates exert their anti-HIV activity by shielding off the positively charged sites in the V3 loop of the viral envelope glycoprotein (gp120). The V3 loop is necessary for virus attachment to cell surface heparan sulfate, a primary binding site, before more specific binding occurs to the CD4 receptor of CD4(+) cells. This general mechanism also explains the broad antiviral activity of polysulfates against enveloped viruses. Variations in the viral envelope glycoprotein region may result in differences in the susceptibility of different enveloped viruses to compounds that interact with their envelope glycoproteins. The efficacy of polysulfates in the therapy and/or prophylaxis of retroviral infections and opportunistic infections remains to be demonstrated both in animal models and humans. It is important to consider not only treatment of patients who are already infected with HIV, but also prophylaxis and protection from HIV and/or other virus infections. Because (i) sexual transmission is responsible for the large majority of HIV infections worldwide; (ii) this transmission is mostly mediated via mononuclear cells that infect epithelial cells of the genital tract; and because (iii) polysulfates effectively inhibit cell-cell adhesion, polysulfates may be considered as potentially effective in a vaginal formulation to protect against HIV infection. (C) 1997 Elsevier Science Inc.

An increased prevalence of Epstein-Barr virus infection in young patients suggests a possible etiology for systemic lupus erythematosus.

Abstract: An unknown environmental agent has been suspected to induce systemic lupus erythematosus (lupus) in man. Prompted by our recent immunochemical findings, we sought evidence for an association between Epstein-Barr virus infection and lupus. Because the vast majority of adults have been infected with Epstein-Barr virus, we chose to study children and young adults. Virtually all (116 of 117, or 99%) of these young patients had seroconverted against Epstein-Barr virus, as compared with only 70% (107 of 153) of their controls (odds ratio 49.9, 95% confidence interval 9.3-1025, P 10, 95% confidence interval 2.53-infinity, P < 0.002). When considered with other evidence supporting a relationship between Epstein-Barr virus and lupus, these data are consistent with, but do not in themselves establish, Epstein-Barr virus infection as an etiologic factor in lupus.

Stable gene transfer and expression of human blood coagulation factor ix after intramuscular injection of recombinant adeno-associated virus

Abstract: We sought to determine whether intramuscular injection of a recombinant adeno-associated virus (rAAV) vector expressing human factor IX (hF.IX) could direct expression of therapeutic levels of the transgene in experimental animals. High titer (1012–1013 vector genomes/ml) rAAV expressing hF.IX was prepared, purified, and injected into hindlimb muscles of C57BL/6 mice and Rag 1 mice. In the immunocompetent C57BL/6 mice, immunofluorescence staining of muscle harvested 3 months after injection demonstrated the presence of hF.IX protein, and PCR analysis of muscle DNA was positive for AAV DNA, but no hF.IX was detected in mouse plasma. Further studies showed that these mice had developed circulating antibodies to hF.IX. In follow-up experiments in Rag 1 mice, which carry a mutation in the recombinase activating gene-1 and thus lack functional B and T cells, similar results were seen on DNA analysis of muscle, but these mice also demonstrated therapeutic levels (200–350 ng/ml) of F.IX in the plasma. The time course of F.IX expression demonstrates that levels gradually increase over a period of several weeks before reaching a plateau that is stable 6 months after injection. In other experiments we demonstrate colocalization of hF.IX and collagen IV in intersitial spaces between muscle fibers. Collagen IV has recently been identified as a F.IX-binding protein; this finding explains the unusual pattern of immunofluorescent staining for F.IX shown in these experiments. Thus rAAV can be used to direct stable expression of therapeutic levels of F.IX after intramuscular injection and is a feasible strategy for treatment of patients with hemophilia B.