Biomedical subjects
J L Melnick
Publications and source records attributed to J L Melnick.
Extrachromosomal sequences of hepatitis B virus DNA in peripheral blood mononuclear cells of acquired immune deficiency syndrome patients.
The primary etiologic agent of the acquired immune deficiency syndrome (AIDS) is a human T-lymphotropic retrovirus (the AIDS virus). However, the pathogenesis of this virus suggests that other cofactors may contribute to the development of clinically overt disease. The hepatitis B virus (HBV) has been implicated as a potential cofactor because HBV and AIDS virus infections frequently coexist, striking similarities exist in their epidemiologic patterns, and recent data indicate that HBV is lymphotropic. To establish the prevalence of HBV infections in lymphoid cells from individuals with AIDS-related disorders, sera and peripheral blood mononuclear cells (PBMC) from 16 males with AIDS virus infections were examined for the presence of HBV DNA by DNA X DNA blot hybridization. Fifteen (94%) of these individuals had serologic evidence of a recent or prior HBV infection. HBV DNA was detected in the PBMC of all of these patients, regardless of existing HBV serology. Among the 36 control individuals without AIDS-related symptomatology, PBMC-associated HBV DNA was detected in 8 of 14 carriers of hepatitis B surface antigen (HBsAg) and in 3 of 10 individuals immune to HBV, but it was absent from the PBMC of 12 individuals without HBV infection. In all instances, the HBV DNA was extrachromosomal and existed as replicative intermediates or high molecular weight oligomers of the viral genome. Replicative intermediates and serum-associated HBV DNA were detected in all hepatitis B e antigen-positive carriers, regardless of their clinical status. In contrast, the high molecular weight oligomers of HBV DNA were detected in the PBMC of all of the AIDS virus-infected patients examined, but in only 33% of those in the control group who had evidence of HBV infection. This finding suggests that a unique and complex HBV-host-cell interaction exists in patients infected with the AIDS virus.
Hepatitis B virus DNA in mononuclear cells and analysis of cell subsets for the presence of replicative intermediates of viral DNA.
To determine whether peripheral blood mononuclear cells (PBMCs) contain replicating forms of hepatitis B virus (HBV) DNA and to define which cell subset may be permissive for viral replication, we analyzed the PBMC DNA from 14 carriers positive for hepatitis B surface antigen (HBsAg) by Southern blot hybridization. HBV-related DNA, which was present exclusively in an extrachromosomal state, was found in the PBMCs of all five hepatitis B e antigen (HBeAg)-positive and three of nine HBeAg-negative carriers. Serum-associated HBV DNA was detected only in those HBsAg carriers whose PBMCs contained HBV DNA forms resembling replicative intermediates (1.0-3.2 kilobase pairs in the EcoRI digests). Analysis of PBMC subsets revealed that replicating forms of the HBV genome were present primarily in monocytes. Low levels of hybridization also were detected in B cells, whereas the T cell fraction (which contained natural killer cells) appeared to be devoid of these replicating forms.
Antigenic characterization and ELISA detection of adult diarrhea rotaviruses.
Recently, severe epidemics of diarrhea among both adults and children in China have been associated with an agent called adult diarrhea rotavirus (ADRV). We have studied ADRV from two areas of China (Jinzhou and Guangxi) and compared them with prototype group A, B, and C rotaviruses. The viral genomes were compared by electrophoresis of the RNA genome segments in polyacrylamide gels, and the antigenic relatedness of these viruses was examined by immune electron microscopic studies using virus preparations of either double- or single-capsid particles. Hyperimmune antisera (guinea pig and rabbit) to one strain of ADRV reacted with high titers with other strains of ADRV and with single-shelled capsids of a porcine group B rotavirus. The antisera to ADRV did not react with either group A or group C rotaviruses. Antisera to either bovine or porcine group B rotaviruses reacted with ADRV but not with the group A or group C viruses. Antisera to group A or group C virus reacted only with their respective homologous virus. These results and those of immunofluorescence studies place the human ADRV rotaviruses from China among the group B rotaviruses. We also report the development of an ELISA to detect ADRV; this ELISA should be useful for defining the epidemiology of these recently described rotaviruses.
Development of a method for concentration of rotavirus and its application to recovery of rotaviruses from estuarine waters.
As part of our studies on the ecology of human enteric viruses, an improved method for detection of rotaviruses in water was developed, and their presence in Galveston Bay was monitored. Samples (378 liters) of estuarine water adjusted to pH 3.5 and a final AlCl3 molarity of 0.001 were filtered through 25-cm pleated cartridge-type filters (Filterite Corp., Timonium, Md.) of 3.0- and 0.45-micron porosity. Adsorbed virus was eluted with 1 liter of 10% tryptose phosphate broth, pH 9.5. Primary eluates were reconcentrated to a final volume of 10 to 20 ml by a simple and rapid magnetic iron oxide adsorption and elution procedure. Two percent casein at pH 8.5 effectively eluted rotavirus from iron oxide. A total of 21 of 72 samples of water, suspended solids, fluffy sediments, and compact sediments collected in different seasons in Galveston Bay yielded rotaviruses. Recovery of rotaviruses varied from 119 to 1,000 PFU/378 liters of water, 1,200 PFU/1,000 g of compact sediment, 800 to 3,800 PFU/378 liters of fluffy sediment, and 1,800 to 4,980 PFU from suspended solids derived from 378 liters of water based on immunofluorescent foci counts on cover slip cultures of fetal monkey kidney cells.
Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes.
The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.
Human viruses in sediments, sludges, and soils.
Recent studies have provided a greater understanding of the movement of viruses in the environment by their attachment to solids. These studies have focused on solids-associated viruses present in wastewater discharged into the ocean and on viruses in sludge and wastewater that may be retained in soil following their land disposal. Such ocean or land disposal of wastewater and sludge may result in a discharge of one or more of 120 human enteric virus pathogens including those causing poliomyelitis, viral hepatitis A and acute gastroenteritis.Solids-associated viruses in effluents discharged into coastal waters accumulate in bottom sediments, which may contain 10 to 10 000 more virus per unit volume than the overlying seawater. Solids-associated viruses resuspended by water turbulence may be transported from polluted to distant non-polluted recreational or shellfish-growing water. Transmission of viruses causing hepatitis or gastroenteritis may result from contact by bathers or swimmers with these viruses in recreational waters, or from ingestion of raw or improperly cooked shellfish in which the solids-associated virus had been bioaccumulated.The land disposal of sludge and wastewater has a potential of causing infections in farm workers, contamination of crops, pollution of raw potable water sources or infiltration of ground water. Viruses retained on soils can be released by rain water and may contaminate ground water through lateral and vertical movements.
Virus vaccines: 1986 update.
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Combined live and inactivated poliovirus vaccine to control poliomyelitis in a developing country--five years after.
The Gaza Strip is an area in transition which in the 1960's had a high prevalence of malnutrition and infectious diseases. The infant mortality was approximatively 140 per 1000 live births. Pediatric Services were almost non-existant. Trivalent oral poliovaccine (TOPV) has been used since 1967. Coverage however did not exceed 70%. From 1973 a network of comprehensive Child Health Centers was spread throughout the area, a set of laws was passed which made vaccination obligatory and the community became heavily involved in health education. These measures resulted in a vaccination coverage, from fixed centers, of over 90% of the susceptible infant population. Though infant mortality decreased rapidly, poliomyelitis was less affected and the mean annual incidence of the paralytic disease until 1977 continued to be 10 per 100,000 inhabitants. Two outbreaks caused by poliovirus Type 1 were registered in 1974 and 1976 with an incidence of 18 per 100,000 inhabitants. In these outbreaks 34% and 50% of the affected children, respectively, had received 3-4 doses of (TOPV). A new vaccination schedule was implemented in 1978 combining TOPV and inactivated polio vaccine in the form of an injectable quadruple vaccine. In the first three years following this change the annual incidence of the paralytic disease dropped from 10 to 2.2 per 100,000 inhabitants. In the following 5 years (1981-1985) only 4 cases of paralytic poliomyelitis were discovered, an annual incidence of 0.16 per 100,000 inhabitants. A serosurvey was done in 1980 on 117 immunized children age 6 months to three years.(ABSTRACT TRUNCATED AT 250 WORDS)
A prospective study of herpes simplex virus infection in a defined population in Houston, Texas.
A seroepidemiologic study was conducted to determine the incidence of antibodies to herpes simplex virus in a population of middle-class Caucasian women. In utero exposure to diethylstilbestrol did not influence the frequency of herpes simplex virus infection. The frequency of infection increased with age and number of sex partners and a 50% increase in incidence of herpes simplex virus type 2 infection was noted during the 4-year period of the study. Subclinical herpes simplex virus type 2 infections apparently occurred in some women without preexisting antibodies to herpes simplex virus type 1.
A prospective study of association of herpes simplex virus and human papillomavirus infection with cervical neoplasia in women exposed to diethylstilbestrol in utero.
One thousand one hundred thirty-four women, enrolled in a prospective study of the development of cervical neoplasia in women exposed to diethylstilbestrol (DES) in utero, were examined at least once a year. Twenty-three women developed cervical intraepithelial neoplasia (CIN) of different degrees during the 7-year follow-up period and were matched with women who did not develop CIN during the study period. Serum samples were obtained at the time of entry into the study and at the time of diagnosis of neoplasia. In the control group, a second serum sample was obtained close to the time when CIN was diagnosed in the other group of women. The sera were studied for antibodies to both types of herpes simplex virus (HSV) by microneutralization assay and by a radioimmunoassay using, as antigens, glycoproteins VP123 for HSV type 1 and VP119 for HSV type 2. Women who developed CIN had, in the enrollment specimen which preceded the neoplasia, a higher rate of antibodies to HSV-1 than did the matched controls (22%), while the two groups did not differ in frequency of antibodies to HSV-2 (9%). Similar differences were observed in the sample drawn at the time of diagnosis of CIN. Women who later developed CIN and had, at the time of entry into the study, cervical epithelial changes associated with DES exposure, were infected with HSV-1 at a higher rate than women who remained free of neoplasia or women with CIN but without DES-associated cervical epithelial changes. Morphologic evidence of associated papillomavirus (HPV) infection was observed in 74% of CIN cases and no such observation was made in biopsy specimens taken prior to development of CIN. Morphologic evidence of HPV infection was found in a women of the control group having squamous metaplasia. HPV structural antigens were found in 3 of 10 CIN I and CIN II cases, and none in the CIN III specimens. The possible role of the HSV and HPV infection in the pathogenesis of cervical neoplasia is discussed.
Retrovirus resembling HTLV in macrophages of patients with AIDS.
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Persistently infected cultures as a source of hepatitis A virus.
Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.
An instrument for the immediate quantification of bacteria in potable waters.
A new semiautomated instrument is described which quantifies the number of bacteria in potable waters within 3 min, providing a permanent colorimetric record of the results. The bacteria detection device can measure as few as 100 CFU/ml in potable waters. In brief, a 100- to 1,000-ml sample of tap water is drawn through a large surface, customized filter housed in the device, and bacteria, rust, and humic acid in the water are concentrated thereon. A reducing agent is used to remove the rust and humic acid from the filter. The filter is inverted and backflushed to elute the bacteria which are collected and reconcentrated onto a 7-mm-diameter filter surface. The reconcentrated bacteria are stained, and the filter fibers are preferentially decolorized without removing the dye from the bacteria. The color intensity of the filter surface is compared with a color guide to determine the amount of bacteria in the test water.
Method for detecting viruses in aerosols.
A simple method with poliovirus as the model was developed for recovering human enteric viruses from aerosols. Filterite filters (pore size, 0.45 micron; Filterite Corp., Timonium, Md.) moistened with glycine buffer (pH 3.5) were used for adsorbing the aerosolized virus. No virus passed the filter, even with air flow rates of 100 liters/min. Virus recovery from the filter was achieved by rapid elution with 800 ml of glycine buffer, pH 10. The virus in the primary eluate was reconcentrated by adjusting the pH to 3.5, adding AlCl3 to 0.0005 M, collecting the virus on a 0.25-micron-pore Filerite disk (diameter, 25 mm) and and eluting with 6 ml of buffer, pH 10. With this method, virus could be detected regularly in aerosols produced by flushing when 3 X 10(8) PFU of poliovirus were present in the toilet bowl. Poliovirus-containing fecal material from two of four infants who had recently received oral polio vaccine also yielded virus in the aerosols when feces containing 2.4 X 10(7) to 4.5 X 10(7) PFU of virus had been added to the toilet bowl. Persons infected with a variety of natural enteric viruses are known to excrete this amount of virus in their daily stools.
Rapid, colorimetric method for the detection of microorganisms in blood culture.
A semiautomated instrument (Septi-Screen) and method are described that semiquantify bacteria colorimetrically in blood-inoculated cultures in 2 to 8 h. If the blood culture is positive, concentrates (of viable bacteria) are obtained free of blood cells. In pediatric cases and in septicemia attributed to indwelling catheters where bacteremia is often manifested by high bacterial counts, the total time required to obtain bacterial concentrates is only 2 to 3 h. In brief, at various intervals after inoculation of 10 ml of blood into 100 ml of culture fluid, 25-ml blood culture samples are passed through the Septi-Filter. Erythrocytes and leukocytes pass into the filtrate and are discarded, but bacteria are electrostatically retained on the filter surface. The entrapped bacteria are backflushed and recovered as a concentrate that is processed through a second filter (Septi-Count) for colorimetric quantitation. The bacteria are stained, and the filter fibers are decolorized. The color intensity of the filter surface is proportional to the number of bacteria in the concentrate processed. The total Septi-Screen processing time is 3 to 5 min.
Isolation of Inoue-Melnick virus and detection of antibodies in coded cerebrospinal fluid specimens from patients with disorders of the central nervous system.
Inoue-Melnick virus was isolated from coded cerebrospinal fluid samples from 10 of 25 patients with chronic diseases of the central nervous system. Of 13 multiple sclerosis patients and 12 patients with a variety of other symptoms and signs 4 and 6, respectively, were positive. Replicate samples from the same patient yielded the same results, either positive or negative. Neutralizing antibody was present in cerebrospinal fluid at a dilution of 1:25 in the virus-positive patients. Only a single virus-negative patient was positive for antibody at this dilution.
Persistence of Inoue-Melnick virus and antibody in cerebrospinal fluid.
Serial cerebrospinal fluid samples were obtained from 14 multiple sclerosis patients over a period of several months and were tested under code for Inoue-Melnick virus. In five of the positive patients, virus was present in 28 of 32 specimens collected over a period of 2 to 5 months. Four patients, from whom a total of 34 specimens were taken, yielded only a single isolate each. Five patients contributed 28 specimens, all of which were negative. In six patients (three virus positive and three virus negative), neutralizing antibody was detected in serum and in cerebrospinal fluid.