Monkey Kidney Cell Cultures Research Articles

A rapid tissue culture assay for the detection of okadaic acid and related compounds in mussels

Okadaic acid, the main toxin of the diarrhoetic shellfish poisoning (DSP) group, shows high cytotoxicity to Buffalo green monkey (BGM) kidney cell cultures after different times according to concentration. A rapid method of detecting the presence of okadaic acid and related compounds in contaminated mussels by means of a cytotoxicity test is presented. The method is based on direct microscopic observation of toxin-induced morphological changes in BGM cell cultures. A high correlation ( r = 0.950; P < 0.001) was found between the official mouse bioassay and the cytotoxicity test conducted on naturally contaminated samples of Mytilus galloprovincialis.

Replication of hepatitis C virus in vitro; in monkey kidney or human fibroblast cell culture in serum-free medium for vaccine production : CNR World 9624 662; 15 August 1996

Replication of hepatitis C virus in vitro; in monkey kidney or human fibroblast cell culture in serum-free medium for vaccine production : CNR World 9624 662; 15 August 1996

Short-term in vitro culture and molecular analysis of the microsporidian, Enterocytozoon bieneusi.

The microsporidium, Enterocytozoon bieneusi, causes a severe, debilitating, chronic diarrhea in patients with the acquired immunodeficiency syndrome. Specific diagnosis of intestinal microsporidiosis, especially due to Enterocytozoon, is difficult and there is no known therapy that can completely eradicate this parasite. Preliminary studies indicate that a short term (about 6 months) in vitro culture of this parasite yielding low numbers of spores, may be established by inoculating human lung fibroblasts and/or monkey kidney cell cultures with duodenal aspirates and or biopsy from infected patients. The cultures may subsequently be used for the isolation and molecular analysis of parasite DNA.

The impact of interfacial interactions on poliovirus infectivity in detergent-treated cell monolayer cultures

Monolayer cultures of Buffalo green monkey (BGM) kidney cells were inoculated with different aqueous solutions of anionic detergents prior to infection with poliomyelitisvirus type 1 (PMV-1). Alteration of inoculated cell culture monolayers was assessed by polarized light microscopy. Based on their visual appearance, altered monolayers were arbitrarily assigned different rating categories (r.c.) ranging from 1 to 8. For each detergent concentration, the rate of monolayer destruction was recorded and graphically integrated into the plots of r.c. against detergent concentration. In addition to a direct detergentmediated alteration of the cellular monolayers, inoculation of detergent-conditioned BGM cultures with PMV-1 apparently affected virus-mediated alteration as well. The type of alteration as well as the initial rate of cytopathic destruction of the monolayer depended on several factors including the stereochemical properties of the detergent used, the final detergent concentration as well as the organic content and inoculation volume of the preconditioning solution, and the infectious titer of the viral inoculum. Moreover, infectivity kinetics revealed that cytopathic effects may be reactivated after a period of viral inhibition. In this way, at least two cycles of virus generation successively alternating with cell proliferation could be recorded simply by supplementing the system with energy for growth. From the presented results, it became apparent that modifications of cell surface activity contribute to virus infectivity mechanisms. Since treatment of aqueous PMV-1 suspensions has been previously described to largely affect viral infectious titers, viral uptake seems to be triggered by the phase behavior of viral particles upon contact with the plasma membrane. Therefore, aquatic samples that have been treated with anionic detergents in order to optimize virus isolation rates should be extensively washed prior to inoculation on cell culture.

Biological Activities of Pyrrolidinoindoline Alkaloids fromCalycodendron milnei

Certain genera of the tribe Psychotrieae, specifically Calycodendron and Psychotria, found on Pacific Islands, synthesize a series of Nb-methyltryptamine-derived alkaloids made by linking together 2 to 8 pyrrolidinoindoline units. Nine alkaloids of this class have been isolated from the aerial parts and stem bark of Calycodendron milnei, a species endemic to the Vaté Islands (New Hebrides), and examined for potential application as anti-cancer and anti-infective agents. All members of the series exhibited readily detected cytotoxic activity against proliferating and non-proliferating Vero African green monkey kidney cells in culture, with the most potent activity being exhibited by vatamine and quadrigemine C. Only hodgkinsine A exhibited substantial antiviral activity against a DNA virus, herpes simplex type 1, and an RNA virus, vesicular stomatitis virus. All members of the series showed readily detected anti-bacterial, anti-fungal, and anti-candidal activities using both tube dilution and disc diffusion assay methods. The most potent anti-microbial alkaloids were hodgkinsine A and quadrigemine C, which exhibited minimum inhibitory concentration (MIC) values as low as 5 micrograms/ml.

Microevolution of Type 3 Sabin Strain of Poliovirus in Cell Cultures and Its Implications for Oral Poliovirus Vaccine Quality Control

Screening for sequence heterogeneities in Sabin Type 3 strains of attenuated poliovirus demonstrated mutations that consistently accumulate to significant levels following 10 passages in cultures of primary African green monkey kidney (AGMK) cells or continuous cultures of Vero cells. Fourteen newly identified mutations were quantified by mutant analysis by PCR and restriction enzyme cleavage in passages and in batches of commercial vaccines made in AGMK and Vero cells from the Sabin original (SO) seed virus and from a seed virus rederived by RNA plaque purification (RSO or "Pfizer" seed). Nine of the 14 mutations were reproducibly observed in more than one series of passages. Although 5 other mutations were observed in only one set of passages each, their content gradually increased to a high percentage, suggesting that all the mutations that we found accumulated consistently, SO-derived samples accumulated more mutations than did RSO derived ones, and the number of mutations and the rates of their accumulation were higher in Vero than in AGMK cells. While the rates of accumulation of most mutations were higher when passaging was performed at 37°, a U → C transition at nucleotide 5832 occurred faster at 34°, the temperature used for vaccine production. Analysis of Type 3 oral poliovirus vaccine (OPV) monopools made by six manufacturers found only 5 of these newly identified mutations in vaccine batches (nucleotides 3956, 4935, 5357, 5786, and 5832). Some of the mutations were found in trace amounts (less than 0.1%) while others were present at up to 1.8% levels. The pattern of these mutations was characteristic for the type of seed virus and the cell substrate but demonstrated no correlation with results of the monkey neurovirulence test. Therefore the only mutation occurring in Type 3 OPV which contributed to neurovirulence in monkeys was the previously described reversion at nucleotide 472. Quantitation of reversion at nucleotide 472 can be utilized for assessment of acceptability of vaccine lots, while other mutations can be used for monitoring the consistency of vaccine production.

Study of Persistence of Enteric Viruses in Landfilled Disposable Diapers

Disposable diapers are one of many possible sources of infectious enteric viruses that are disposed of in landfills. A total of 218 disposable diapers were collected from 7 sites and 10 depths at three landfills. Of this total, 110 diapers were selected be processed based on fecal content using a 1.5% beef extract elution, organic flocculation-concentration method to recover viruses. The concentrated samples were assayed on Buffalo Green Monkey (BGM) Kidney cell cultures for the detection of enteroviruses and with cDNA probes specific for poliovirus, hepatitis A virus, and rotavirus. Enteroviruses were not detected in any sample assayed using cell culture techniques

The effect of serial passage on Sabin oral poliomyelitis vaccine virus in secondary monkey kidney versus Vero cells as measured by the monkey neurovirulence test

The three Sabin strains of poliomyelitis seed virus were serially passaged in either secondary monkey kidney or Vero cell cultures and the tenth passage of each virus harvest compared to non-passaged Sabin reference virus of the same type using the monkey neurovirulence test. All three types were further attenuated by passage in Vero cells, whereas only type 2 became further attenuated after passage in secondary monkey kidney cells. After passage in Vero cells, type 3 poliomyelitis virus became more heat stable, as measured by its replicative capacity at 40 degrees C.

Alteration of viral infectious behavior by surface active agents

Phosphate buffered saline (PBS) and wastewater retentate, which had been adjusted to the same level of pH and ionic strength by addition of a concentrated PBS solution, were experimentally seeded with polio- or parvovirus and treated with various concentrations of sodium dodecylsulfate (SDS) and dodecyltrimethylammoniumbromid (DTAB), respectively. Upon subsequent assessment for viral infectivity of the samples in Buffalo green monkey kidney cell cultures, infectivity modulating effects of DTAB in PBS and of SDS in retentate appeared to be largely affected by the electrical charge of the suspended virions. However, if PBS or retentate samples were treated with SDS or DTAB respectively, different isoelectric properties between polioand parvovirus particles were less likely to affect the detergent concentration required for optimal virus recovery. Moreover, in the presence of soluble organics, optimal virus recovery rates were obtained with much lower detergent concentrations if the samples had been treated with DTAB instead of SDS. Measurement of the effective critical micelle concentration as well as multiangle electrophoretic light scattering (MELS) seemed to provide a simple approach to monitoring colloidal stability of multicomponent viral particle (VP) suspensions upon the addition of ionic detergents. By measuring zeta potential distribution, MELS offers additional information about alterations to electrical viral surface properties. Since the behavior of VPs is well known to largely depend upon their electrical characteristics within the environment in which they exist, there is substantial evidence that MELS can provide valuable guidelines in studying optimal detergent-treatment conditions for virus recovery from aqueous suspensions.

Simian virus 40 prevents activation of M-phase-promoting factor during lytic infection.

Simian virus 40 (SV40) infection stimulates confluent cultures of monkey kidney cells into successive rounds of cellular DNA synthesis without intervening mitosis. As an initial step in defining the mechanisms responsible for viral inhibition of mitosis, M-phase-promoting factor (MPF) was examined in SV40-infected CV-1 cells passing from G2 phase into a second S phase. MPF is a serine-threonine protein kinase that is essential for mitosis in eukaryotic cells. In SV40-infected cells exiting G2 phase, there was a reduced amount of MPF-associated H1 kinase activity relative to that of uninfected cells passing through mitosis. Both subunits of MPF, cyclin B and the p34cdc2 catalytic subunit, were present and in a complex in infected cells. In uninfected cultures, passage through mitosis was associated with the dephosphorylation of the p34cdc2 subunit, which is characteristic of MPF activation. In contrast, the p34cdc2 subunit remained in the tyrosine-phosphorylated, inactive form in SV40-infected cells passing from G2 phase into a second S phase. These results suggest that although the MPF complex is assembled and modified normally, SV40 interferes with pathways leading to MPF activation.

Use of monoclonal antibody blends in the identification of enteroviral aseptic meningitis

Monoclonal antibody pools directed against group B coxsackievirus and echovirus antigens (Chemicon, Temecula, California) were evaluated as tools in the identification of enteroviral aseptic meningitis. Cerebrospinal fluid (CSF) and serial dilutions of stock coxsackievirus were inoculated into tube and shell vial Rhesus monkey kidney (RhMk) cell cultures. Positive cellular fluorescence was observed only in conjunction with cytopathic effect (CPE). The time from inoculation to CPE was similar with both tube and shell vial cultures.

Patent Evaluation : WO9301160-: Merck Sharp &amp; Dohme Ltd: Novel non-cyclic substance P antagonists

AbstractNovelty: Novel 3,3-diphenylpropan-1-ol-2-amine derivatives are claimed together with processes for their preparation. The compounds are non-peptide tachykinin antagonists. They are potentially useful for the treatment of a wide range of diseases in which substance P is implicated, particularly pain or inflammation, migraine and postherpetic neuralgia.Biology: Compounds were screened for their ability to displace [125I]-substance P from human neurokinin-1 receptors expressed in either monkey kidney or Chinese hamster ovarian cell cultures. A full table of results is disclosed. The specified compound has an IC50 value of 0.4 nM. No other biological tests are disclosed.Chemistry: Seventy-four compounds are exemplified by synthesis using standard methodology. 1-((375-Bis(trifluoromethyl)phenyl)methyloxy)-2-(2S)-(1-((carboxamido)ethylamino)-3,3-diphenylpropone is one of seventy-four specifically claimed compounds.

Rapid detection of respiratory viruses using mixtures of monoclonal antibodies on shell vial cultures.

Eleven hundred and thirty-three clinical specimens submitted to the laboratory for diagnosis of respiratory virus infections were tested by direct immunofluorescence (DIF) for respiratory syncytial virus (RSV), by shell vial culture, and by conventional cell culture. The shell vial cultures were stained with 8 different monoclonal antibodies both 1 day and 3-7 days after inoculation. In order to limit the cost and the workload, mixtures of monoclonal antibodies were used. Coverslips with HEp-2 cells were incubated with a mixture of FITC-labeled monoclonal antibody to RSV and nonlabeled monoclonal antibody to adenovirus. When no RSV positive IF staining was observed after the first incubation step, the same coverslip was incubated once more with FITC-labeled anti-mouse antibody. A positive reaction at this stage indicated the presence of adenovirus. Similarly, cultures of tertiary monkey kidney cells were investigated with a mixture of two FITC-labeled monoclonals to the influenza viruses A and B and three nonlabeled monoclonals to the parainfluenza viruses 1, 2 and 3. If influenza virus or parainfluenza virus was detected, the exact type was determined by staining different parts of a duplicate coverslip. Shell vial cultures for cytomegalovirus (CMV) were always performed separately on human embryonic lung fibroblasts. Using this approach, we detected RSV (n = 248), CMV (n = 42), parainfluenza virus (n = 31), influenza virus (n = 28), and adenovirus (n = 6), in most cases after only one day of culture. For RSV, the sensitivity of the shell vial method was too low (74%) to allow omission of DIF (sensitivity 95%).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of hemagglutination and hemadsorption tests for influenza detection

Hemagglutination (HA) testing of the supernatant of primary rhesus monkey kidney cell culture tubes inoculated with clinical specimens was compared with hemadsorption (HAd) of the same tube for identifying the presence of hemagglutinin activity due to influenza. A total of 476 respiratory specimens were screened for influenza by this method. The 127 influenza or parainfluenza positive cultures showed 100% concordance between the two tests. All negative cultures were both also negative by HA and HAd. In contrast to the HAd method, HA testing was performed with less tube manipulation, less hands-on time, and less potential for cell culture tube contamination.

Advantages of multiple cell culture systems for detection of mixed-virus infections

Simultaneous infections by two or more viruses occur frequently, especially in immunosuppressed patients. In order to detect more than one viral agent in a single specimen, multiple cell systems have been employed in our laboratory. Specimens are routinely inoculated into four different cell cultures, namely: MRC-5, a human diploid lung fibroblast cell strain; A549, a human continuous cell line; primary guinea pig embryo (GPE) cell culture, and primary rhesus monkey kidney (RhMK) cell culture. For rapid detection of cytomegalovirus (CMV) antigen, MRC-5 cells grown in shell vials containing coverslips are also inoculated with the same specimens followed by centrifugation. During 1989, nine cases of multiple-virus isolations were obtained in this laboratory. In all nine patients, CMV was detected in MRC-5 cells. Five of the nine cases were co-infected with HSV-1, three were co-infected with adenovirus, and one was co-infected with both HSV-1 and adenovirus. All four adenovirus isolates were obtained in A549 cells. Of the six HSV-1 isolates, one was detected in all three cell cultures, e.g. MRC-5, A549 and GPE; one was detected in both MRC-5 and A549 cells, and four were isolated in a single-cell type only. For nine CMV-positive cases, five were obtained by both conventional and centrifugation cultures, two each were detected by centrifugation or conventional culture only. Thus for a maximum detection of viruses present in a single specimen, it is suggested that multiple-cell-culture systems, together with more than one technique, should be employed.

Antigenic and genomic identity between simian herpesvirus aotus type 2 and bovine herpesvirus type 4

Herpesvirus aotus type 2 (HVA-2) was isolated from a culture of kidney cells from a healthy owl monkey (Aotus trivirgatus). Bovine herpesvirus type 4 (BHV-4) is frequently isolated from diseased and even healthy cattle and occasionally from sheep, wild ruminants and cats. The two viruses are related antigenically, as was revealed by an indirect fluorescent antibody test using polyclonal antisera from experimentally infected rabbits or monoclonal antibodies raised against six BHV-4 proteins, three of which were glycosylated. The genome structures of the two viruses consist of a unique central sequence flanked at both ends by G + C-rich tandem repeats. Restriction maps (produced using EcoRI, BamHI and HindIII) of these two viruses were nearly identical but the unique sequence of the HVA-2 genome possessed two additional BamHI sites. Four genomic regions of variable size were detected, two located in the unique part, one in the repetitive part and one in the left junction between the unique and the repeated part of the genome; these slight variations were similar to those observed between various BHV-4 isolates. These results suggest that HVA-2 and BHV-4 belong to the same virus species; HVA-2 could be either a BHV-4 contaminant of owl monkey kidney cell cultures or an isolate from an owl monkey accidentally infected with BHV-4.

Virus Removal by Sand Filtration of Septic Tank Effluent

Septic tank effluent (STE) is applied to stratified sand filter columns at a rate of 6.1 cm/day (1.5 gal./day/sq ft) and the ability of the sand filter to remove enteric viruses is demonstrated. The sites of virus retention are determined. Seven 10-cm (4 in.) diameter sand columns are constructed of 25 cm (10 in.) of dry, coarse sand, 10 cm (4 in.) of dry, fine sand, and 25 cm (10 in.) of glass sand, with the sand layers separated by 5 cm (2 in.) of 1.9-cm (3/8-in.) rounded gravel. Household septic tank effluent containing poliovirus vaccine (Type I, strain LSc) is applied to the filters, and influent and effluent samples are collected. Enterovirus analyses are performed using plaque assays in buffalo green monkey (BGM) kidney cell cultures. Following the filter runs, the columns are dissected into 2.5-cm (1-in.) discs and the discs were assayed for enteroviruses. The filter columns retain all viruses when the virus loading remains less than 33,000,000 PFU/1 (plaque-forming units per liter). Analyses of the filter discs show most viruses are retained in the top few centimeters of biologically active sand, and a mass balance reveals virus inactivation in the sand filter.

Human malignant melanoma cell line (HMV-II) for isolation of influenza C and parainfluenza viruses

HMV-II, a human malignant melanoma cell line, was compared with other cell lines (MDCK, Vero, and LLC-MK2) and primary cultures of monkey kidney (PMK) cells for the isolation and quantification of influenza and parainfluenza viruses. HMV-II cells were superior to MDCK and LLC-MK2 cells in quantification of the influenza C virus and were used successfully in the isolation of the virus from clinical specimens. The HMV-II cell line was also more sensitive for isolating parainfluenza viruses from clinical specimens than were Vero and PMK cells; there was, however, no significant difference in the quantification of the viruses among these cultures. As far as influenza A and B viruses were concerned, the HMV-II cell line was significantly less sensitive than MDCK cells, and no virus was isolated from clinical specimens with HMV-II cells. Thus, HMV-II cells are useful for the isolation of influenza C and parainfluenza viruses as an alternative to embryonated hen's eggs and PMK cells.

Occurrence of nonspecific reactions among stool specimens tested by the Abbott TestPack rotavirus enzyme immunoassay

Sixty-five stool specimens obtained from children suffering from gastroenteritis were tested for the presence of antigen to rotavirus by the Abbott TestPack Rotavirus (TestPack) enzyme immunoassay kit. The Kallestad Pathfinder enzyme immunoassay, polyacrylamide gel electrophoresis, immune electron microscopy, and virus isolation were utilized as reference assays. Fifty-four specimens were in accord by TestPack and Kallestad Pathfinder. Among 11 discordant specimens positive with TestPack but negative by Kallestad Pathfinder, rotavirus was not identified by polyacrylamide gel electrophoresis, immune electron microscopy, or isolation in primary African green monkey kidney cell cultures. TestPack displayed a performance specificity of 83%. The inordinately high number of stool specimens reported as false-positive by TestPack precludes the incorporation of this antigen detection kit into our routine regimen of diagnostic virologic testing.

Herpesvirus simiae Contamination of primary rhesus monkey kidney cell cultures

Herpesvirus simiae Contamination of primary rhesus monkey kidney cell cultures