A global lab against influenza.
Explore the source record for details and available documents.
Environmental Monitoring↗
Biomedical subjects
S P Layne
Publications and source records attributed to S P Layne.
Diagnosis of influenza virus: coming to grips with the molecular era.
Influenza viruses continually circulate and cause yearly epidemics, which kill 20,000 people in an average year in the United States. Occasionally and unpredictably, pandemic influenza strains sweep the world, infecting 20% to 40% of the world's population in a single year. In 1918, the worst influenza pandemic on record caused 675,000 deaths in the United States and up to 40 million deaths worldwide. Despite the prevalence of this virus, molecular assays for influenza diagnosis, surveillance, vaccine strain selection, and research have lagged behind such assays for other common viral pathogens. The extreme genetic variability of influenza viruses makes the design of useful molecular-based assays challenging, but several different approaches have been successfully used. RT-PCR is effective for the initial diagnosis and has greater sensitivity than other available rapid assays. Molecular assays also can be used to subtype influenza isolates, and sequence analysis of hemagglutinin may assist greatly in surveillance studies and vaccine strain selection. RT-PCR for influenza also can be performed from tissue biopsy specimens for both retrospective diagnosis and research.
Animals↗
A practical method for simultaneously determining the effective burst sizes and cycle times of viruses.
We describe combined analytic and experimental methods for determining reproductive statistics from time-series data. Our computational methods derive four fundamental measures from laboratory experiments: (i) average number of viral daughters; (ii) mean viral cycle time; (iii) standard deviation of the viral cycling time; and (iv) viral doubling time. Taken together, these four reproductive statistics characterize "age-specific fertility," a quantity that provides complete information on the reproduction of the average viral particle. In this paper, we emphasize applications relating to HIV and experiments for assessing cellular tropism, viral phenotypes, antiviral drugs, humoral immunity, and cytotoxic cellular immunity. Nevertheless, our method is quite flexible and applicable to the evaluation of drugs against bacterial, fungal, and parasitic infections, antineoplastic agents against cancer cells, and perturbations involving pest and wildlife releases in ecosystems.
Anti-HIV Agents↗
Laboratory firepower for infectious disease research.
Explore the source record for details and available documents.
Automation↗
Factors underlying spontaneous inactivation and susceptibility to neutralization of human immunodeficiency virus.
To determine the factors governing inactivation and neutralization, physical, chemical, and biological assays were performed on a molecular clone of human immunodeficiency type 1 (HIV-1HXB3). This included quantitative electron microscopy, gp120 and p24 enzyme-linked immunosorbent assays, reverse, transcriptase assays, and quantitative infectivity assays. For freshly harvested stocks, the ratio of infectious to noninfectious viral particles ranged from 10(-4) to 10(-7) in viral stocks containing 10(9) to 10(10) physical particles per milliliter. There were relatively few gp120 knobs per HIV particle, mean approximately 10 when averaged over the total particle count. Each HIV particle contained a mean approximately 5 x 10(-17) g of p24 and approximately 2 x 10(-16) g of RNA polymerase, corresponding to about 1200 and 80 molecules, respectively. The spontaneous shedding of gp120 envelope proteins from virions was exponential, with a half-life approximately 30 hr. The loss of RNA polymerase activity in virons was also exponential, with a half-life approximately 40 hr. The physical breakup of virions and the dissolution of p24 core proteins were slow (half-life greater than 100 hr) compared to the gp120 shedding and polymerase loss rates. The decay of HIV-1 infectivity was found to obey superimposed single- and multihit kinetics. At short preincubation times, the loss of infectivity correlated with spontaneous shedding of gp120 from virions. At longer times, an accelerating decay rate indicated that HIV requires a minimal number of gp120 molecules for efficient infection of CD4+ cells. The blocking activity of recombinant soluble CD4 (sCD4) and phosphonoformate (foscarnet) varied with the number of gp120 molecules and number of active RNA polymerase molecules per virion, respectively. These results demonstrate that the physical state of virions greatly influences infectivity and neutralization. The knowledge gained from these findings will improve the reliability of in vitro assays, enhance the study of wild-type strains, and facilitate the evaluation of potential HIV therapeutics and vaccines.
CD4 Antigens↗
The auto-regulation model: a unified concept of how HIV regulates its infectivity, pathogenesis and persistence.
The life cycle of HIV can be divided into two distinct stages: intracellular and extracellular. The prevailing view is that the intracellular stage provides the only locus for regulating the virus in response to physiologic stimuli. Such regulation is accomplished by modulating the rates of transcription, translation and viral assembly. The extracellular stage consists of physical processes such as diffusion, adhesion and penetration of cells by viral particles. These latter processes are commonly thought to be "automatic" and not subject to regulation. For the past several years, we have developed means of more carefully measuring and characterizing the extracellular stage of HIV infection, and we have obtained evidence indicating that novel regulatory processes do, in fact, take place during this extracellular stage. We believe that this extracellular regulation permits HIV to adapt to a wide range of physiologic cell densities, to maintain persistent but slow growing infection, and to defeat the protective activity of humoral blockers. The overall purpose of this review is to consider our evidence for this hypothesis.
CD4 Antigens↗
Blocking of human immunodeficiency virus infection depends on cell density and viral stock age.
Quantitative infectivity assays were used to study how the blocking activity of soluble CD4 (sCD4) is affected by sCD4 concentration, target cell density, and viral stock age. During incubation with 20 nM sCD4, human immunodeficiency virus type 1 (HIV-1) stocks underwent irreversible inactivation. In contrast, inactivation with 2 nM sCD4 was almost entirely reversible. At lower sCD4 concentrations (less than or equal to 2 nM) and target cell densities of 6.25 x 10(4) ml-1, sCD4 blocking activity for HIV-1 gave a gp120-sCD4 association constant (Kassoc) of 1.7 x 10(9) M-1, which agrees with chemical measurements. At the higher density of 1.6 x 10(7) cells ml-1, however, the blocking activity was 20-fold less. During incubation of HIV-1 stock optimized for infectivity by rapid harvest, sCD4 blocking activity increased 20-fold during a 3-h window. These results show that competitive blocking activity depends strongly on target cell density and virion age. Thus, unappreciated variations in HIV stocks and assay conditions may hinder comparisons of blockers from laboratory to laboratory, and the age of HIV challenge stocks may influence studies of drug and vaccine efficacy. The results also suggest that blocking of viral particles in lymphoid compartments will require very high competitive blocker concentrations, which may explain the refractory outcomes from sCD4-based drug trials in humans.
Acquired Immunodeficiency Syndrome↗
HIV requires multiple gp120 molecules for CD4-mediated infection.
Binding of glycoprotein gp120 to the T cell-surface receptor CD4 is a crucial step in CD4-dependent infection of a target cell by the human immunodeficiency virus (HIV). Blocking some or all gp120 molecules on the viral surface should therefore inhibit infection. Consequently, competitive receptor inhibitors, such as soluble synthetic CD4 (sCD4), synthetic CD4 peptides and immunoglobulins, have been investigated in vitro and in vivo, but little is known about the molecular mechanisms of these inhibitors. We have now quantitatively examined blocking by soluble CD4 in the hope of gaining insight into the complex process of viral binding, adsorption and penetration. At low sCD4 concentrations, the inhibition in three HIV strains is proportional to the binding of gp120. The biological association constant (gp120-sCD4 Kassoc) for HIV-2NIHZ is (8.5 +/- 0.5) x 10(7) M-1, whereas Kassoc for HIV-1HXB3 (1.4 +/- 0.2) and HIV-1MN (1.7 +/- 0.1) x 10(9) M-1 are 15-20-fold larger. For all three viral strains, the biological Kassoc from infectivity assays is comparable to the chemical Kassoc. The inhibitory action of sCD4 at high concentrations, however, is not fully explained by simple proportionality with the binding to gp120. Positive synergy in blocking of infection occurs after about half the viral gp120s molecules are occupied, and is identical for all three viral strains, despite the large differences in Kassoc. Our method of measuring the viral-cell receptor Kassoc directly from infectivity assays is applicable to immunoglobulins, to other viruses and to assays using primary or transformed cell lines.
CD4 Antigens↗
Analytic results for quantifying HIV infectivity.
In a separate paper, we developed a mathematical model describing HIV infection and used it to suggest experiments for quantifying characteristic viral parameters. In this paper we generalize the model to any well-mixed assay system. We also present complete and rigorous derivations of fundamental results needed for the design and analysis of HIV infectivity assays. The model is applicable to infectious agents with multiple receptors for their target cell (e.g. HIV, Epstein-Barr virus and Plasmodium), and to blockers (both reversible and irreversible), as long as blocker and target cells are the same diffusion compartment.
Acquired Immunodeficiency Syndrome↗
Quantifying the infectivity of human immunodeficiency virus.
We have developed a mathematical model that quantifies lymphocyte infection by human immunodeficiency virus (HIV) and lymphocyte protection by blocking agents such as soluble CD4. We use this model to suggest standardized parameters for quantifying viral infectivity and to suggest techniques for calculating these parameters from well-mixed infectivity assays. We discuss the implications of the model for our understanding of the infectious process and virulence of HIV in vivo.
Acquired Immunodeficiency Syndrome↗
Risk behavior-based model of the cubic growth of acquired immunodeficiency syndrome in the United States.
The cumulative number of cases of acquired immunodeficiency syndrome (AIDS) in the United States has grown as the cube of time rather than exponentially. We explain this by interactions involving partner choice and sexual frequency in a risk-behavior model with biased mixing. This leads to a saturation wave of infection moving from high- to low-risk groups. If this description is correct, then the decreasing growth rate of AIDS cases is not due to behavior changes; rather it is due to the intrinsic epidemiology of the disease.
Acquired Immunodeficiency Syndrome↗
The need for national HIV databases.
Researchers and public health officials involved in surveying and forecasting the course of the HIV epidemic require complete and unfiltered information from many sources. Governments should respond by establishing national HIV databases.
Acquired Immunodeficiency Syndrome↗
Dimensionality of the human electroencephalogram.
Explore the source record for details and available documents.
Anesthesia↗
Transient fluorescence in synchronously dividing Escherichia coli.
Using a spectrometer equipped with an optical multichannel analyzer as the detector, we observed the Stokes laser-Raman spectra of metabolically synchronous Escherichia coli from 100 to 2100 cm-1. After more than 400 separate recordings, at cell concentrations of 10(7)-10(8) per ml, no Raman lines attributable to the metabolic process nor to the cells themselves were found. However, we did find that synchronous E. coli cultures become more fluorescent during a limited phase of the division cycle. This transient increase in fluorescence may be ascribed to a variation in the redox state of a chemical species within the bacteria or to a variation of the intracellular optical field. The effect is reproducible in synchronous cultures and it is not seen in asynchronous ones. The results suggest that spectral features seen in previous laser-Raman spectra of synchronous bacteria (taken with scanning monochromators) are due to a time-dependent variation in bacterial fluorescence.
Cell Division↗