Virological Assays Research Articles

Isolation from an Ant Myrmecia gulosa of Two Inducible O-Glycosylated Proline-rich Antibacterial Peptides

Reported here is the isolation and characterization of two antibacterial peptides synthesized in an ant Myrmecia gulosa in response to bacterial challenge. The peptides were purified by reversed-phase high performance liquid chromatography and characterized by peptide sequencing and mass spectrometry. Both peptides were formed from 16 amino acids, were rich in proline ( approximately 30%), and had N-acetylgalactosamine O-linked to a conserved threonine. The activity of a synthetic non-glycosylated isoform was markedly reduced demonstrating that glycosylation was necessary for maximum activity. The peptides were active only against growing Escherichia coli. They were inactive against stationary cells, Gram-positive bacteria, the yeast Candida albicans, two species of mammalian cells, and bovine pestivirus.

Biologie moléculaire et sécurité virale transfusionnelle

In this review we present four fields to which viral molecular biology has contributed: the discovery of blood-borne viruses and the knowledge of the natural history of infection by these viruses; the validation of the results of virological assays used in the biological screening of blood donations; the contribution of molecular biology in inquiries into viral transfusional contamination; the interest of molecular biology in viral transfusional epidemiology. We subsequently deal with the parameters of the discussion on the impact and the feasibility of a systematic screening of several viral genomes in blood donations.

An approach to the validation of markers for use in AIDS clinical trials.

Dr. Mildvan and coauthors have thoroughly reviewed and documented what is known about the validation of surrogate markers for use in clinical trials. They have proposed a classification system based on the usefulness of available immunologic and virological assays as measures of prognosis, drug activity, and therapeutic efficacy. The latter, a type II marker in the proposed classification, should estimate the proportion of treatment effect explained by change in the marker induced by therapy and, if complete, can substitute for clinical endpoints. HIV clinical trialists have had a long-standing interest in using surrogates for clinical endpoints to facilitate conduct of experimental protocols and to decrease the time and effort required to develop new treatment strategies. The approach outlined in this review by experienced clinicians, biostatisticians, and immunologists provides a framework to evaluate currently available and potential surrogate markers.

The role of PCR in the diagnosis and management of CMV in solid organ recipients: what is the predictive value for the development of disease and should PCR be used to guide antiviral therapy?

Cytomegalovirus remains a significant source of morbidity and mortality in immunocompromised hosts. The increased sensitivity of molecular diagnostic techniques (PCR, antigenemia) has resulted in our ability to detect viral replication earlier in the posttransplant period, before the onset of symptoms. With the advent of effective antiviral therapy, "preemptive therapy," guided by sensitive, early and specific predictors of CMV disease, has become a realistic objective. Although multiple studies have analyzed the sensitivity and specificity of these tests, their predictive value for the development of disease has not been defined. The purpose of this study was to evaluate the predictive value of a positive CMV PCR in the setting of solid abdominal organ transplantation. A total of 476 PCR assays were performed on 134 transplant recipients (102 kidney, 19 kidney/pancreas, 11 liver, 2 other) either as protocol serial samples or as dictated by clinical events. All samples were concomitantly analyzed using standard virological assays for CMV including culture, shell vial, and serology. Patients with any CMV seropositive donor/recipient (D/R) combination received ganciclovir prophylaxis in conjunction with antilymphocyte induction for 14 days. No subsequent CMV prophylaxis was used. The positive predictive value was 55% in all seropositive donor/recipient combinations. The highest risk group (seronegative recipient of seropositive donor) showed the highest positive predictive value, whereas seropositive recipients of either seropositive or seronegative donors showed positive predictive values of 45% and 25%, respectively. Negative predictive value was 100% for all groups. Early detection of CMV infection has important implications for patient management, including preemptive therapy, which can be guided by PCR, especially in high risk (D+/R-) patients.

Polymyositis, arthritis, and uveitis in a macaque experimentally infected with human T lymphotropic virus type I.

To establish a simian model of human T lymphotropic virus type I (HTLV-I) infection and disease. Irradiated HTLV-I-producing cells were used to infect two 2-year-old rhesus macaque monkeys (Macaca mulatta). One monkey was also simultaneously inoculated with a cell-free suspension of simian immunodeficiency virus (SIV). Evidence of infection was monitored by serial clinical examinations and by serologic, molecular, and virologic assays. Both HTLV-I-inoculated monkeys became persistently infected following inoculation. Clinical disease was observed in the singly inoculated monkey, which developed arthritis (with synovial fluid positive for HTLV-I by culture and polymerase chain reaction), anterior chamber uveitis, and steroid-responsive polymyositis confirmed by electrophysiologic studies. The dually inoculated animal remained clinically healthy, despite high levels of SIV and HTLV-I virus expression and loss of HTLV-I-specific antibodies. These results indicate the utility of a nonhuman primate model for studying HTLV-I disease pathogenesis and the dynamics of SIV-1/HTLV-I retroviral coinfection.

Rates of p24 antigenemia and viral isolation in comparable white and black HIV-infected subjects

To determine the relative frequencies of HIV-1 p24 antigen and culture positivity in white and black patients. Volunteers in the US military's HIV natural history study were 46% white, 44% black, 7% Hispanic and 3% other. Focusing on the comparable groups of whites and blacks, a retrospective analysis was performed of the results of virologic assays collected over a 2-year period. p24 antigen was quantitated in sera with and without immune complex dissociation (ICD); viral isolation was performed by coculture of peripheral blood mononuclear cells. Results of the two virologic assays were very similar in the two racial groups, both overall and after stratification by CD4 cell count. As reported previously, the concentration of serum immunoglobulin G was found to be greater in black than white subjects. In contrast to results with ICD, sera tested without ICD resulted in differing (higher) rates of antigenemia in whites than blacks (P = 0.002). The frequencies of p24 antigen and culture positivity were found to be independent of race. Previously observed racial differences in antigen positivity were likely to be due to more extensive antibody binding in blacks than in whites.

Rates of p24 antigenemia and viral isolation in comparable white and black HIV-infected subjects

Objective: To determine the relative frequencies of HIV-1 p24 antigen and culture positivity in white and black patients. Design: Volunteers in the US military's HIV natural history study were 46% white, 44% black, 7% Hispanic and 3% other. Focusing on the comparable groups of whites and blacks, a retrospective analysis was performed of the results of virologic assays collected over a 2-year period. Methods: p24 antigen was quantitated in sera with and without immune complex dissociation (ICD); viral isolation was performed by coculture of peripheral blood mononuclear cells. Results: Results of the two virologic assays were very similar in the two racial groups, both overall and after stratification by CD4 cell count. As reported previously, the concentration of serum immunoglobulin G was found to be greater in black than white subjects. In contrast to results with ICD, sera tested without ICD resulted in differing (higher) rates of antigenemia in whites than blacks (P = 0.002). Conclusions: The frequencies of p24 antigen and culture positivity were found to be independent of race. Previously observed racial differences in antigen positivity were likely to be due to more extensive antibody binding in blacks than in whites. AIDS 1995, 9:325-328

Improved specificity of in vitro anti-HIV antibody production: implications for diagnosis and timing of transmission in infants born to HIV-seropositive mothers.

In vitro anti-HIV antibody production (IVAP), initially introduced as a method for diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in infants, has been limited in its application because of poor specificity and sensitivity early in life. The aims of this study were to improve the specificity of the IVAP assay and to evaluate its sensitivity in conjunction with assays of HIV culture, polymerase chain reaction (PCR), and p24 antigen. To prevent false-positive reactions resulting from maternal serum-derived cytophilic anti-HIV IgG, additional preculture and washing steps for peripheral blood mononuclear cells (PBMCs) were introduced that resulted in dramatic improvement in specificity of IVAP. The sensitivity of the revised IVAP at age < 3 months in 20 infected infants was, however, only 25%; of 15 infected infants initially negative in IVAP, 13 became positive at a mean estimated age of 4.4 +/- 1.8 months. When correlated with virological assays, a failure to respond in IVAP at age < 1 month was often associated with negative virological identification, whereas a positive IVAP response at age < 3 months was always associated with positive results in all virological assays. Moreover, conversion from negative IVAP to positive responses occurred subsequent to, and not concurrently with, a positive virological identification of infected infants. The revised IVAP methodology renders this assay potentially useful as an additional tool not only for the diagnosis of HIV infection, but for estimating timing of maternal-infant HIV transmission as well.

Dilution assay statistics

A parametric method of statistical analysis for dilution assays is developed in detail from first principles of probability and statistics. The method is based on a simple product binomial model for the experiment and produces an estimate for the concentration of target entities, a confidence interval for this concentration, and an indicator of the quality of the assay called the p value for goodness of fit. The procedure is illustrated with data from a virologic quantitative micrococulture assay used to quantify free human immunodeficiency virus in clinical trials. The merits of the procedure versus those of nonparametric methods of estimating the dilution inducing a 50% response rate are discussed. Advantages of the proposed approach include plausibility of the underlying assumptions, ability to assess plausibility of specific experimental outcomes through their likelihood, and plausibility of confidence intervals.

Use of virologic assays for detection of human immunodeficiency virus in clinical trials: recommendations of the AIDS Clinical Trials Group Virology Committee

Use of virologic assays for detection of human immunodeficiency virus in clinical trials: recommendations of the AIDS Clinical Trials Group Virology Committee

Oral Rehydration Therapy for Viral Gastroenteritis-Reply

In Reply. —We certainly concur that ORT is an effective, safe, well-recognized, proven, and economical method of treating acute GE in infants and young children. It was not the purpose of my presentation to discuss in detail various methods of therapy. Rather, the goal was to present highlights of our research program on viral GE and to give an overview regarding its etiology, epidemiology, clinical manifestations, treatment, and prevention. The case presented was drawn from one of 72 hospitalized infants and young children included in a 1977 publication that described the clinical features of rotavirus GE. 1 The case was not intended to set a standard for therapy of viral GE, but was presented as an example of a patient with acute GE associated with rotavirus infection as determined by virologic and serologic assays. In the Prophylaxis and Treatment section of this Grand Rounds, we described the treatment as follows:

Standardization of sensitive human immunodeficiency virus coculture procedures and establishment of a multicenter quality assurance program for the AIDS Clinical Trials Group. The NIH/NIAID/DAIDS/ACTG Virology Laboratories.

An independent quality assurance program has been established by the Division of AIDS, National Institute of Allergy and Infectious Diseases, for monitoring virologic assays performed by nearly 40 laboratories participating in multicenter clinical trials in the United States. Since virologic endpoints are important in evaluating the timing and efficacy of therapeutic interventions, it is imperative that virologic measurements be accurate and uniform. When the quality assurance program was initially created, fewer than 40% of the laboratories could consistently recover human immunodeficiency virus (HIV) from peripheral blood mononuclear cells (PBMCs) of HIV-infected patients. By comparing coculture procedures in the more competent laboratories with those in laboratories who were struggling to isolate virus, optimal conditions were established and nonessential reagents and practices were eliminated. Changes were rapidly introduced into a laboratory when experience dictated that such modifications would result in a favorable outcome. Isolation of HIV was enhanced by optimizing the numbers and ratios of patient and donor cells used in cultures, by standardizing PBMC separation procedures, by using fresh rather than frozen donor PBMCs, by processing whole blood within 24 h, and by using natural delectinated interleukin 2 instead of recombinant interleukin 2 products in existence at that time. Delays of more than 8 h in the addition of phytohemagglutinin-stimulated donor cells to freshly separated patient PBMCs reduced recovery. Phytohemagglutinin in cocultures and the addition of Polybrene and anti-human alpha interferon to media were not important in HIV isolation. The introduction of a consensus protocol based on this information brought most laboratories quickly into compliance. In addition, monthly monitoring has successfully maintained proficiency among the laboratories, a process that is critical for the scientific integrity of collaborative multicenter trials. Problems which might not be appreciated for months are now being resolved early, before data can be compromised unknowingly. This consensus protocol is recommended for any laboratory attempting to isolate HIV for the purpose of standardizing recovery and for accessing virologic endpoints in clinical trials.

Prognostic markers for AIDS.

Human immunodeficiency virus (HIV) infection causes a number of clinical syndromes and many laboratory abnormalities, often heralding the development of the life-threatening opportunistic infections or malignancies that are known as the acquired immunodeficiency syndrome (AIDS). Drawing heavily on the results of prospective cohort studies, particularly those that my colleagues at the National Cancer Institute and I have conducted, this paper reviews the relationship of AIDS to clinical signs and symptoms, immunologic measures, and viral assays. The risk of AIDS in the next 3 years is at least 25 to 50% for HIV-infected subjects who have oral candidiasis, unexplained fever, unexplained weight loss, a CD4+ lymphocyte count below 200 cells/microliter, or combinations of these. Elevated serum levels of beta 2 microglobulin and neopterin also appear to be strong predictive markers of AIDS, but further work is needed in diverse HIV-infected populations, such as intravenous drug users and persons in pattern II countries, such as Haiti and central Africa. Elevated levels of interferon or HIV-p24 antigen in the serum are insensitive but highly specific AIDS markers that may have predictive value independent of CD4 lymphocyte levels. Several potentially valuable immunologic (immunoglobulin levels, tumor necrosis factor, soluble interleukin 2) and virologic (HIV viremia) assays remain to be thoroughly evaluated or technically simplified. Data from prospective cohort studies have provided clinical and laboratory markers of AIDS risk that have proved essential for therapeutic trials and other clinical decisions. As effective treatments for HIV infection and its complications begin to emerge, these marker data will also prove invaluable for mathematic modeling of the scope, course, and public health response to the epidemic.

Expression of avian reticuloendotheliosis virus envelope confers host resistance

We constructed two reticuloendotheliosis virus (REV) envelope gene expression plasmids, one containing the REV-A envelope gene, the other the spleen necrosis virus (SNV) envelope gene. Cell lines were generated by transfecting each of the REV envelope plasmids into D17 cells, a canine cell line. The levels of REV envelope glycoprotein in the cell lines were assayed by immunoprecipitating the envelope glycoproteins from lysates of cells that were labeled with [ 35S]methionine. Virological challenge assays determined the degree of resistance of each of the cell lines to REV-A or SNV infection. The expression of either envelope gene protected the cells from infection by either REV-A or SNV virus. Several cell lines were significantly more resistant to REV infection than the parental D17 cells, and two lines were 25,000-fold more resistant, approaching the resistance of REV-infected D17 cells to reinfection. The resistant cell lines were not able to confer resistance to susceptible cells by cocultivation. The level of resistance was correlated with the uniformity of expression of the REV envelope glycoproteins by the individual cells in a cell line and not with the absolute level of expression by the population of cells.

Bluetongue virus propagation and plaque assay: Variation due to medium and serum supplement

Two base media, minimal essential medium (MEM) and RPMI 1640, were supplemented with a variety of serum extenders and/or substitutes for the purpose of defining a medium formulation capable of supporting good cell (VERO) growth and virologic assays. Bluetongue virus (BTV), the prototype Orbivirus in the Reoviridae, was used in all studies. In general, VERO cells grown in RPMI performed better than those grown in MEM relative to cell growth, virus production and plaque assay. RPMI was better for supporting cell growth when serum extenders (NuSerum or SerXtend) were employed as supplements. Relative to virologic techniques, cells grown in RPMI produced higher virus titers in both propagation studies and plaque assays. The interval from infection to >90% cytopathic effect (CPE) was consistently shorter with RPMI as the base medium. Cell cultures supported with RPMI base medium, supplemented with 3.5% FBS with SerXtend, provided the best overall performance relative to: (a) amount of virus produced by infected cell monolayers, (b) sensitivity to productive infection under overlay conditions (revealed the highest titer of a standard virus stock) and (c) plaque assay quality including cell quality, plaque size and plaque clarity.

An old nemesis in new clothing: viruses playing new tricks by causing cytopathology in the absence of cytolysis.

Peter Medawar amply summarized viruses when he described them as piece of bad news wrapped in a protein coat [1]. Viruses were first separated from other microbial agents in specimens by using the Pasteur-Chamberland filter. Viruses known or suspected to cause disease have been characterized first by clinical picture and histological profile and confirmed by virological, immunologic, or molecular biologic assays. This procedure, which was first applied by Ivanovski and Beijerinck to infection of tobacco plants with tobacco mosaic virus and by Frosch and Loeffler to infection of cattle with foot and mouth virus, continues to be used to the present day with diseases like the spongiform encephalopathies and acquired immunodeficiency syndrome. Thus, the sine qua non for suspecting viral involvement has been a histopathologic picture of cellular necrosis, usually, but not always, including inflammatory infiltrates. Indeed, the histological picture frequently suggests the virus involved, e.g., cytomegalo-like cells during infection with cytomegalovirus, injury of anterior horn neurons in poliomyelitis, and appearance of Negri or Lyssa bodies within certain neurons of the central nervous system in rabies. In other instances, such as encephalitis or myocarditis, the cellular necrosis and associated perivascular cuffing are not specific enough to suggest a single etiologic agent any of several agents could be the cause. The decisive factors in the course of virally induced injury are initially the tropism of that virus for specific cells and then either the viral lytic ability per se or the interaction between components of the host immune response and the in-

Effects of Chemically or Virus-Induced Immunodepression on Response of Chickens to Avian Leukosis Virus

The effects of chemically or virus-induced immunodepression on the infection profile (development of viremia and antibody) and shedding of avian leukosis virus (ALV) were studied in progeny chickens of experimental or commercial breeder flocks. Chickens were infected with ALV subgroup A by contact at hatching and by oral inoculation at 4-5 weeks of age. In the first experiment, chickens were inoculated with a virulent strain of infectious bursal disease virus (IBDV) at 1 day or 6 weeks of age. In the second experiment, chickens were neonatally treated with cyclophosphamide (CY), or were inoculated with strain T of reticuloendotheliosis virus (REV) at hatching, or were inoculated with strain JM of Marek's disease virus (MDV) at 2 weeks of age. The infection profile and cloacal shedding of ALV in chickens exposed to ALV and inoculated with immunodepressive viruses or CY were compared with those in hatchmates exposed only to ALV. In two of four chicken lines tested in the first experiment, shedding of ALV, as determined by virological assays of cloacal swabs at 22 weeks of age, was significantly higher in chickens infected with IBDV at 1 day of age than in uninfected hatchmates. The rate of shedding of ALV in one of these two lines was also significantly higher in chickens infected with IBDV at 6 weeks of age than in uninfected chickens. Further, the frequency of ALV-antibody detection at 22 weeks of age was significantly lower in chickens of these two lines infected with IBDV at 1 day of age than in uninfected chickens. In the second experiment, neonatal treatment with CY significantly increased the frequency of viremic chickens of both experimental and commercial flocks. The frequency of ALV-viremic chickens at 22 weeks of age was considerably higher in the REV- and MDV-inoculated groups (54% and 44%, respectively) than in control hatchmates (29%), but only in chickens of the commercial line. These findings suggest that chemically or virus-induced immunodepression may lead to an increase in rates of viremia and shedding of ALV in chickens infected with virus after hatching, especially in certain genetic lines.

Use of aggregating brain cultures to study the replication of herpes simplex virus types 1 and 2 in central nervous system tissue

A novel tissue culture system consisting of reaggregated embryonic mouse brain cells was used to examine the replication of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) within central nervous system tissue. Brain aggregates cultured 30–40 days in vitro demonstrated progressive maturation and differentiation into cells recognizable as neurons, astrocytes and oligodendrocytes, with the latter cell type exhibiting myelin production. Mature aggregates were infected with HSV and sampled at timed intervals postinfection for morphological, virological, and biochemical assays. By electron microscopy mature nucleocapsids were observed in the nucleus of peripheral cells at 9 h and in all cell types by 33 h. Virus-specific antigens were observed, using the immunoperoxidase test, within peripheral cells at 12 h postinfection (p.i.). By 24 h p.i., antigen production had progressed throughout the infected aggregates. Growth curves of HSV-1 and HSV-2 for intracellular and extracellular infectious virus production correlated well with virus-induced morphological changes and antigen production. SDS-polyacrylamide slab gel electrophoresis of isotopically-labelled proteins and glycoproteins synthesized from 4 to 24 h p.i. in virus-infected aggregates revealed typical HSV-1 polypeptide profiles and HSV-1 and HSV-2 glycoprotein profiles. Our results suggest that aggregating brain cultures may provide a useful and more accurate in vitro model for the study of HSV-induced neurological disease.

Serological and virological analysis of NIH (NIH × AKR) mice: Evidence for three AKR murine leukemia virus loci

The segregation of murine leukemia virus (MuLV) loci in backcrosses of AKR mice with NIH Swiss, C57BL/6, and BALB/c mice was followed by serological and virological assays as well as by DNA hybridization. In the backcross, NIH × (NIH × AKR), three main phenotypes of virus expression were observed, including viremic mice without any detectable free antibody against MuLV, nonviremic mice with high titers of antibody, and nonviremic mice without any detectable antibody. Of the three phenotypes, only the nonviremic, no-antibody mice were segregants for the lack of MuLV proviral DNA by DNA hybridization, by AKR-MuLV type-specific antigen expression, and by second backcross analysis. These mice represented approximately 13% of the population, suggesting the presence of three AKR-MuLV loci in AKR mice. In backcrosses of AKR mice with C57BL/6 mice only the antibody-positive, nonviremic and no-antibody, nonviremic phenotypes were observed. The segregation of AKR-MuLV loci in these crosses, analyzed serologically, was also consistent with the presence of three AKR-MuLV loci in AKR mice.

Comparative Serial Virologic and Serologic Studies of Symptomatic and Subclinical Congenitally and Natally Acquired Cytomegalovirus Infections

Infants with congenitally (38) and natally (17) acquired cytomegalovirus infection were prospectively studied by means of virologic and multiple serologic assays. These infections were characterized by chronic viral excretion (measured in years). The quantity of virus excreted in the urine during early infancy was significantly greater in infants who acquired infection in utero, particularly amon those born with overt disease; thereafter, all three groups (congenital symptomatic, congenital asymptomatic, and natal) excreted similar amounts of virus. The patterns of antibody responses, particularly the fluorescent antibody response to the early antigen and the complement-fixing antibody response, further indicated that congenitally infected infants (especially symptomatic ones) bear a greater antigenic burden than do natally infected infants. From a diagnostic standpoint, the test for fluorescent antibody to the late antigen was the most sensitive assay, whereas the test for complement-fixing antibody proved to be the least useful, The indirect hemagglutination assay, although performed only in infants with natal infection, was only slightly less sensitive than the fluorescent antibody procedure; by the former technique, diagnostic rises were detected in all but one infant after the onset of viruria.