Rhesus Monkey Kidney Cells Research Articles

Observations on reovirus type 3 in certain culicine mosquitoes.

Eighteen strains of reovirus type 3 were isolated from mice and tissue cultures inoculated with suspensions of mosquitoes caught in the southeastern part of the main island of the Fiji group between 17 April and 12 June 1967. Three strains were isolated originally from rhesus-monkey kidney cells and suckling mice and reisolated from human-embryo kidney cells and suckling mice. Seven further strains were reisolated from suckling mice. Strains of virus isolated from mosquitoes were inoculated into laboratory colonies of Aedes australis and Culex quinquefasciatus and into wild-caught Culiseta tonnoiri. Evidence was obtained of prolonged survival but not of multiplication in these species. C. quinquefasciatus fed on viremic suckling mice were able to transmit to further suckling mice for up to 4 days after the infective feed, but no longer. This is interpreted as mechanical transmission of a highly resistant virus that probably remained active while contaminating the mouthparts of the insects.

A Comparative Study of the Antitumor and Antiviral Activity of 1- -D-Arabinofuranosyl-5-fluorocytosine (FCA) and 1- -D-Arabinofuranosylcytosine (CA)

SummaryFCA was similar or slightly superior in activity to CA with regard to antitumor effects when tested against the following experimental tumors: ascitic leukemia L-1210, sarcoma-180, Ehrlich solid carcinoma and mammary adenocarcinoma EO 771. FCA was appreciably more active on a mg/kg basis than CA with regard to its effect against the solid forms of leukemia L-1210 in both semi-isologous BDF1 and homologous albino mice, as well as against Walker carcinosarcoma 256. Both substances were without effect against the Flexner-Jobling carcinoma in rats. FCA and CA exerted similar degrees of antiviral activity against herpes simplex keratitis in the rabbit and vaccinia virus in rhesus monkey kidney cell cultures. Both substances were inactive in vivo and in vitro against the following RNA-containing viruses: Columbia SK, Coxsackie virus B1 (GP), and influenza A (PR8).

Yellow fever virus. I. Development and evaluation of a plaque neutralization test.

Heretofore, the most reliable way of measuring yellow fever virus antibody was to use the mouse neutralization (MN) test employing either suckling or weanling mice. Certain disadvantages (e.g., expense both of animals and of maintaining a mouse colony, allergic reactions of many laboratory workers, and the relatively long time, 21 days, before end points are reached) are inherent in any program with mice or other laboratory animal species and have discouraged the use of the MN test by many laboratories. A previously reported plaque neutralization (PN) test with primary chick embryo cell cultures could not be consistently reproduced by later investigators. We have developed a convenient and reproducible PN test employing the MA-104 embryonic rhesus monkey kidney cell culture and a single agar-overlay procedure. When compared with MN tests with newborn (1 to 3 days old) and weanling (16 to 20 g, 24 to 28 days old) mice inoculated by the intracranial route, the PN test was the most sensitive for measuring neutralizing antibody; it was also less variable, less costly, and it achieved results in the shortest period of time. End points could be determined in 5 to 6 days for the PN test as compared to 21 days for the MN test.

Cytoplasmic membrane-bound vesicles in echovirus 12-infected cells

LLC-MK2 continuous line of rhesus monkey kidney cells infected with echovirus 12 were incubated with or without the inhibitor of viral RNA synthesis, 2-(α-hydroxybenzyl)-benzimidazole (HBB) and were examined by light and electron microscopy at intervals during a single cycle of viral replication. In the absence of HBB, cytoplasmic membrane-bound vesicles were first seen by electron microscopy in cells 4–5 hours after infection and increased in number with time after infection. Cytopathologic changes, observed by phase microscopy, developed 5–6 hours after infection. The addition of HBB to cultures at the time of infection of 1–2 hours later prevented the development of cytoplasmic membrane-bound vesicles and cytopathogenesis over a 7-hour period, but did not prevent the virus-induced disaggregation of cellular polyribosomes. However, if HBB was not added until 3 or 4 hours after infection, the development of membrane-bound vesicles was not prevented. No viral hemagglutinating particles were produced during a period of time equal to three cycles of viral replication when HBB was added to cells at 3 hours or less after infection and only 10% of maximal viral yields were produced when the compound was added 4 hours after infection. Since cytoplasmic membrane-bound vesicles (seen by electron microscopy) and viral cytopathogenesis (seen by light microscopy) (1) develop at closely parallel times after infection, (2) are both probably not direct consequences of virus-induced disaggregation of host-cell polyribosomes, and (3) both require for development the initiation of active viral biosynthesis, it is concluded that the formation of membrane-bound vesicles is a major ultrastructural reflection of viral cytopathogenesis.

Production of reference enteroviruses.

Forty-five human enterovirus reagents of certified purity and quality were prepared for use as seed viruses and as immunizing antigens. One of the reagents was ampouled as "untreated" seed virus, whereas 14 were ampouled as "MgCl(2)-stabilized" reagents. The remaining 30 reagents were ampouled as "untreated" seed viruses and as "MgCl(2)-stabilized" reagents. Thirty of the reagents were propagated on primary African green monkey kidney cells, 3 on primary baboon kidney cells, 3 on primary rhesus monkey kidney cells, and the remaining 9 on human amnion cells. Forty-two of the viral antigens were concentrated for use in the production of high-titered specific antisera in large animals.

Variation in neurovirulence of NWS influenza virus after repeated passages in different tissue culture systems. I. Derivation and neurotropic activity of NWS virus lines.

The NWS virus was repeatedly passaged in four different tissue cultures: cercopithecus monkey kidney cells, rhesus monkey kidney cells, human diploid cells (strain LEP) and chick embryo fibroblasts. After 10 or 11 passages virus stocks were prepared in chick embryos and tested in white mice for neurovirulence. Only one of the four virus lines, denoted as NWS-R, retained the high neurotropic activity of the original NWS virus, while two other lines proved almost without neurotropic activity.

Productive and abortive infections of simian and nonsimian cells with a simian adenovirus SV15: II. Viral biosynthesis and effects of chemical inhibitors

The viral biosynthesis of a simian adenovirus, SV15, was studied in both simian and nonsimian cells. In rhesus monkey kidney cells, viral complement-fixing (CF) antigen and infectious progeny virus were first detected 12 hours after virus inoculation. Synthesis of viral DNA was initiated at about 5–6 hours before the first appearance of complete virus; viral protein was first obtained 2–3 hours before the maturation of virus. In primary hamster kidney cells, SV15 induced an abortive infection. DNA synthesis was stimulated in SV15-infected hamster kidney cells as determined by the increased uptake of thymidine- 3H, but viral antigen could not be detected by CF test, immunofluorescence or agar gel double-diffusion methods. At no time could progeny infectious virus be detected by infectivity titrations. The use of polyoma virus or SV40 as a “helper” virus did not induce the production of SV15 infectious virus in hamster kidney cells. Fluorouracil and cytosine arabinoside were unable to inhibit cytopathic effect as well as the formation of eosinophilic inclusions in SV15-infected RhMK or HamK cells; these inhibitors did prevent the development of basophilic inclusions in infected RhMK cells. Actinomycin D and streptovitacin A inhibited cellular changes and eosinophilic inclusions induced by SV15 in both cell systems tested. In monkey cells, fluorouracil at concentrations of 12–50 μg/ml inhibited 90% or more of the infectious virus, whereas only a 50% reduction of complement-fixing antigen occurred at concentrations of 25–50 μg/ml. To obtain a greater than 90% reduction of CF antigen production, 100 μg/ml of FU was required. Cytosine arabinoside (10 μg/ml), actinomycin D (1 μg/ml), and streptovitacin A (3 μg/ml) completely inhibited viral antigen as well as infectious virus production.

Productive and abortive infections of simian and nonsimian cells with a simian adenovirus SV15: I. Microscopic observations

The uptake and development of simian adenovirus SV15 in rhesus monkey and hamster kidney cells was followed by light and electron microscopy. Uptake of virus particles was noted in both cell systems after virus inoculation. Sequential nuclear changes were observed only in SV15-infected monkey cells. These changes included the early appearance of eosinophilic inclusions, followed by the formation of basophilic inclusions, then nuclear vacuolation, and finally nuclear distortion. Progeny virus particles were seen in the infected nuclei of monkey cells as early as 14 hours postinfection. In hamster cells, on the other hand, an abortive growth cycle developed after infection by this virus. Only eosinophilic inclusions were observed in the infected hamster cells, Progeny virus particles could not be found even at 40 hours postinoculation, at which time complete cellular degeneration occurred. Formation of eosinophilic inclusions in hamster cells appeared to be induced by the infectious virus, but not by the noninfectious virus preparations.

Food-borne virus:detection in a model system.

The purpose of this study was to develop methods for detection and quantitation of food-borne virus. Samples (25 g) of cottage cheese, contaminated with various quantities of coxsackievirus type A9, comprised the model system. Two of the methods presented have at least a 50% probability of detecting virus at levels below 5 plaque-forming units/25-g sample. Noteworthy aspects of these methods include use of a glycine-NaOH buffer (pH 8.8) containing approximately 1 m MgCl(2) as the diluent in which the sample is slurried, treatment of the slurry with Freon TF and bentonite to facilitate centrifuge clarification, and concentration of the clarified sample extract by a two-stage process employing polyethylene glycol followed by ultracentrifugation. Virus in the final 0.5-ml sample concentrate was detected and quantitated by the plaque technique in rhesus monkey kidney cell cultures. Processing of the sample requires approximately 2 days, and the inoculated cultures may have to be observed for as long as 7 days thereafter. If these levels of sensitivity are desired, and if 12 samples per day are tested on a routine basis, the cost savings achieved by employing these methods rather than testing sample extracts without concentration may range from 75 to 90%.

Effect of pyronin Y on adenovirus.

At concentrations (10–15 μM) not markedly toxic for the eta line of rhesus monkey kidney cells, commercial pyronin Y, at low to moderate pH values, strongly inhibited the cytopathic effects and multiplication, of human type 1 adenovirus on these cells. The inhibition occurred equally in the dark and under ordinary light conditions, and was readily reversible simply by replacing the old medium with fresh medium without pyronin Y. The major colored component of the commercial pyronin Y, purified by paper chromatography, was found to possess nearly full activity. High concentrations (5–50 mM) of pyronin Y inactivated adenovirionsin vitro, especially at low pH, and equally in the dark as in ordinary room light; but the low concentrations which inhibit adeno-virus multiplication did not inactivate virions. Adenovirus synthesized in the presence of pyronin Y was stable to room light. Serial passages of adenovirus in the presence of pyronin Y did not result in the emergence of resistant mutants.

Yellow Fever Virus

Heretofore, the most reliable way of measuring yellow fever virus antibody was to use the mouse neutralization (MN) test employing either suckling or weanling mice. Certain disadvantages (e.g., expense both of animals and of maintaining a mouse colony, allergic reactions of many laboratory workers, and the relatively long time, 21 days, before end points are reached) are inherent in any program with mice or other laboratory animal species and have discouraged the use of the MN test by many laboratories. A previously reported plaque neutralization (PN) test with primary chick embryo cell cultures could not be consistently reproduced by later investigators. We have developed a convenient and reproducible PN test employing the MA-104 embryonic rhesus monkey kidney cell culture and a single agar-overlay procedure. When compared with MN tests with newborn (1 to 3 days old) and weanling (16 to 20 g, 24 to 28 days old) mice inoculated by the intracranial route, the PN test was the most sensitive for measuring neutralizing antibody; it was also less variable, less costly, and it achieved results in the shortest period of time. End points could be determined in 5 to 6 days for the PN test as compared to 21 days for the MN test.

Production of Reference Enteroviruses

Forty-five human enterovirus reagents of certified purity and quality were prepared for use as seed viruses and as immunizing antigens. One of the reagents was ampouled as "untreated" seed virus, whereas 14 were ampouled as "MgCl(2)-stabilized" reagents. The remaining 30 reagents were ampouled as "untreated" seed viruses and as "MgCl(2)-stabilized" reagents. Thirty of the reagents were propagated on primary African green monkey kidney cells, 3 on primary baboon kidney cells, 3 on primary rhesus monkey kidney cells, and the remaining 9 on human amnion cells. Forty-two of the viral antigens were concentrated for use in the production of high-titered specific antisera in large animals.

Food-borne Virus: Detection in a Model System

The purpose of this study was to develop methods for detection and quantitation of food-borne virus. Samples (25 g) of cottage cheese, contaminated with various quantities of coxsackievirus type A9, comprised the model system. Two of the methods presented have at least a 50% probability of detecting virus at levels below 5 plaque-forming units/25-g sample. Noteworthy aspects of these methods include use of a glycine-NaOH buffer (pH 8.8) containing approximately 1 m MgCl(2) as the diluent in which the sample is slurried, treatment of the slurry with Freon TF and bentonite to facilitate centrifuge clarification, and concentration of the clarified sample extract by a two-stage process employing polyethylene glycol followed by ultracentrifugation. Virus in the final 0.5-ml sample concentrate was detected and quantitated by the plaque technique in rhesus monkey kidney cell cultures. Processing of the sample requires approximately 2 days, and the inoculated cultures may have to be observed for as long as 7 days thereafter. If these levels of sensitivity are desired, and if 12 samples per day are tested on a routine basis, the cost savings achieved by employing these methods rather than testing sample extracts without concentration may range from 75 to 90%.

Replication of SV5 RNA and the effects of superinfection with poliovirus

SV5-induced RNA synthesis has been studied in actinomycin-treated primary cultures of rhesus monkey kidney cells. Virus-induced RNA synthesis is first detected about 3 hours after infection, and during the period of maximum virus replication, RNA synthesis precedes virus production by 1 1 2 –2 hours. At its maximum rate, SV5-induced RNA synthesis is <1% of total cellular RNA synthesis. Superinfection with poliovirus of cells already producing SV5 does not inhibit, but rather slightly enhances, the ongoing replication of infective SV5. In contrast, puromycin causes a rapid inhibition of ongoing SV5 replication. The inoculation of poliovirus, 325 PFU/cell, in the presence of guanidine causes inhibition of cellular RNA and protein synthesis; however, such inoculation of cells previously infected with SV5 causes a stimulation of actinomycin-resistant, virus-induced RNA synthesis, and a slight enhancement of infective SV5 production. The results indicate that poliovirus does not shut off SV5 RNA-dependent RNA synthesis although it does shut off cellular DNA-dependent RNA synthesis. Furthermore, poliovirus does not inhibit SV5 protein synthesis under conditions in which it does inhibit cellular protein synthesis, thus providing an example of a functional distinction between SV5 messenger and cellular messenger RNA.

New combination of antibiotics for treatment of contaminated specimens for viral studies.

A study of a new combination of antibiotics (sodium oxacillin, colistin sulfate, and amphotericin B) has shown that 30 mug/ml of each in tissue culture maintenance medium can inhibit a group of the commonest bacterial and fungal contaminants while causing no observable cytopathology in rhesus monkey kidney, HeLa, or human amnion tissue cells. In addition, there was no inhibition of the titers of poliovirus, echovirus, coxsackievirus, and adenovirus in the three cell lines. Ten duplicate fecal samples, which remained contaminated with fungal or Pseudomonas organisms after treatment with penicillin and streptomycin, were treated with the new antibiotic combination. Contaminants were controlled in each, and a variety of viruses was recovered.

Hand-foot-and-mouth disease in a father and son

Hand-foot-and-mouth disease is a highly infectious condition characterized by maculopapular and vesicular exanthema of the extremities and diaper area, with “aphthous-like” lesions of the mouth located anterior to the fauces. Although usually it is a mild clinical entity of short duration, a Coxsackie A-16 infection may lead to serious consequences, including death in infants. Three Coxsackie Group A. Type 16, viruses were isolated from a father and son. The viruses were obtained from the stools of each patient and from the lesions on the foot of the father. All strains were isolated in suckling mice and cultures of primary rhesus monkey kidney cells. The two stool isolates were recovered also in primary cultures of Vervet kidney cells. In one patient studied, there was a diagnostic rise in titer of neutralizing antibody to the virus. This report may be the first fully documented case history of this disease in an English-language dental journal.

Patterns of antibodies to SV40 in children following the last booster with inactivated poliomyelitis vaccines.

SummaryThe antibody patterns to SV40 were studied in 190 sequential sera obtained from 40 vaccinees, following their last booster dose with formalin-inactivated poliomyelitis vaccines. None of 10 subjects, immunized with vaccines prepared in cultures of minced rhesus kidney cells, developed detectable neutralizing antibodies to SV40. Seventeen of 30 subjects who received vaccines prepared in monolayer cultures of rhesus kidney cells, developed significant levels of neutralizing antibodies to SV40. In 6 of these cases, the antibody levels to SV40, remained constant for at least 3 years, while the antibody titers to Type II poliovirus showed a progressive decline. None of the sera tested contained detectable antibodies to SV40 tumor antigens.

Isolation of an unclassified enterovirus from healthy children.

A viral agent was isolated in primary rhesus monkey kidney cell cultures from rectal swabs obtained from 2 healthy children resident in Western Pennsylvania. Although the agent had many of the characteristics of the human enterovirus group no serological relationship to any of the recognized pro types was demonstrated. Sera drawn from 2 normal adult population groups showed the presence of antibodies in a significant proportion (16-18%) against the virus which was successfully reisolated from the 2 original specimens. The evidence suggests that this as yet unidentified agent is a member of the human enterovirus group.

New Combination of Antibiotics for Treatment of Contaminated Specimens for Viral Studies

A study of a new combination of antibiotics (sodium oxacillin, colistin sulfate, and amphotericin B) has shown that 30 mug/ml of each in tissue culture maintenance medium can inhibit a group of the commonest bacterial and fungal contaminants while causing no observable cytopathology in rhesus monkey kidney, HeLa, or human amnion tissue cells. In addition, there was no inhibition of the titers of poliovirus, echovirus, coxsackievirus, and adenovirus in the three cell lines. Ten duplicate fecal samples, which remained contaminated with fungal or Pseudomonas organisms after treatment with penicillin and streptomycin, were treated with the new antibiotic combination. Contaminants were controlled in each, and a variety of viruses was recovered.

An electron microscopic study of moderate and virulent virus-cell interactions of the parainfluenza virus SV5

The multiplication of the parainfluenza virus SV5 was studied in primary rhesus monkey kidney (MK) cells and in a line of baby hamster kidney (BHK21-F) cells. Virus adsorbs to the cell surface and appears to penetrate by phagocytosis. Virus morphogenesis is similar in the two cells; the helical nucleocapsid forms in the cytoplasmic matrix and aligns under regions of cell membrane which acquire viral surface projections. Assembly and release of virus particles at the cell surface occurs by a budding process involving the incorporation into the viral envelope of a unit membrane which is continuous with and morphologically identical to that of the host cell. Both spherical and filamentous virus particles are formed; filaments frequently contain regularly coiled nucleocapsid throughout their length. In MK cells, which yield high titers of infective virus for many days but show little cytopathic effect, a balance appears to exist between synthesis of nucleocapsid and its continuous release within mature virus particles. In contrast, in BHK21-F cells, which produce little virus and disintegrate after extensive cell fusion, large aggregates of nucleocapsid accumulate in the cytoplasm, suggesting a block in maturation at the cell membrane. The present electron microscopic observations support previous studies which suggested that the virulence and yield of SV5 may depend, at least in part, on the response of the cell membrane to the virus.