Monkey Kidney Tissue Culture Research Articles

Concentrated Inactivated Measles-Virus Vaccine

Introduction In this report we shall describe the preparation and certain properties of concentrated, inactivated measles vaccines produced from monkey kidney tissue cultures. That they can stimulate the production of neutralizing antibodies in a significant proportion of measles-susceptible children if the concentration of killed antigen is adequate has been demonstrated recently, and the results appear in other reports of this Conference.1 Evidence is also at hand which suggests that as little as 1 or 2 doses of such vaccine may substantially reduce the severity of the natural illness in children.2 The demonstration that such vaccinated children, even with a minimal antibody response, are immune to a subsequent challenge with live, attenuated (Enders) vaccine 2 justifies description of the vaccine at this time. Methods Virus Strain. —The Edmonston strain of measles virus in human amnion cell culture, kindly provided by Dr. John Enders, was used throughout. It was further propagated in our

Morphology of Echo 4 virus grown in monkey kidney tissue culture

Morphology of Echo 4 virus grown in monkey kidney tissue culture

Formation of poliovirus in monkey kidney tissue culture cells

Cytochemical and fluorescent antibody techniques have been utilized to study the release of poliovirus. Electron microscopic studies of ultrathin sections of epoxyembedded monkey kidney cells infected with poliovirus (types 1 and 2 virulent and attenuated) have revealed cytoplasmic arrays of spherical particles each approximately 25 mμ in diameter. Similar particles were found occasionally in “blebs” at the plasma membrane. Particles were detected only when infected cultures were incubated at 30°C. At this temperature fully infective poliovirus is formed but its release is inhibited. Particles were not encountered in infected cultures incubated at 37° or in control preparations.

Characteristics of para-influenza 1 (HA-2) virus. III. Antigenic relationships, growth, interaction with erythrocytes, and physical properties.

Dick, Elliot C. (Tulane University School of Medicine, New Orleans, La.) and William J. Mogabgab. Characteristics of para-influenza 1 (HA-2) virus. III. Antigenic relationships, growth, interaction with erythrocytes, and physical properties. J. Bacteriol. 83:561-571. 1962-Hemagglutination-inhibition tests with rabbit and rooster immune sera showed HA-2-like strains of para-influenza 1 to be nearly as closely related antigenically to para-influenza 3 (HA-1) virus as to the Sendai strain of para-influenza 1; however, convalescent sera from humans infected with para influenza 1 virus exhibited four-fold or greater increases in antibody to HA-2 and Sendai viruses, but seldom to HA-1.Para-influenza 1 (HA-2) infectivity was destroyed within 2 hr at temperatures approaching 50 C; at 36 C, infective virus disappeared by 80 hr. At a temperature of 44 C or below, the hemagglutinin (HA) titer remained unchanged for 100 hr; but at temperatures above this, hemagglutinin was destroyed rapidly. Virus infectivity was found to be most stable at very slightly acid or neutral pH and to be increasingly labile at pH values more extreme than 5.7 and 8.7. Hemagglutinin was generally more resistant than infectivity to extremes of reaction; this was most notable in the alkaline range. In primary monkey-kidney tissue culture, virus was adsorbed in 5 hr, new infective virus was noted in 11 to 15 hr, and maximal amounts were detected at 60 hr and maintained through 132 hr. Production of hemagglutinin paralleled that of infectivity. The tcid(50)/HA ratio was approximately 4.5. With human type "O" erythrocytes, the HA titer of Copenhagen 222 virus suspensions was raised two- to fourfold by increasing the temperature of reaction and sedimentation from 4 C to room temperature. It could not be demonstrated that this thermal effect was due either to an increased total adsorption at an elevated temperature or to a greater rate of adsorption; the rate of hemagglutination was, however, increased by fourfold at the higher temperature.

Inactivated measles virus vaccine. II. Prevention of natural and experimental measles with the vaccine.

This paper reports experiences with a concentrated, alum-containing, formalininactivated measles virus vaccine. The Edmonston (Enders) strain, cultivated in monkey kidney tissue culture cells, was used. Three doses of 0.5 ml. of vaccine were administered intramuscularly to alternate children in 22 families. The other children received placebo injections. Reactions were minimal in both groups. Neutralizing antibodies appeared in 72% of the 18 children who received 3 doses. Those who acquired antibodies and were exposed to measles remained quite well while their placebo-treated siblings became quite ill. On subsequent challenge with live, attenuated measles virus vaccine, the immunized children remained well, even if their antibodies were no longer detectable, while the placebo group had some symptoms; both had excellent serological responses.

Hemagglutination and Hemagglutination-Inhibition Studies with Echo Viruses

Summary A study of several factors which may affect hemagglutinin (HA) production revealed that a dilute inoculum combined with a relatively late harvest time favored maximum HA titers of ECHO 6 viruses grown in monkey kidney tissue culture. Infectivity to HA ratios of 6 to 7 logs were obtained with viruses which attain high infectivity titers (B phase), while the ratios obtained with the low titer viruses (S phase) were approximately 4 to 5 logs. A survey of ECHO 6 strains revealed that 9 of 24 tested produced HA. HA production could not be correlated with infectivity titers, phase, or number of passages in tissue culture. Cross hemagglutination-inhibition (HI) tests with hyperimmune rabbit antisera revealed a high degree of specificity among the serotypes tested. Differences were noted among several ECHO 6 HA antigens in their ability to detect HI antibody. Nonspecific inhibition of ECHO virus hemagglutination was demonstrated. Adsorption of sera with 0.1% bentonite resulted in almost complete removal of nonspecific inhibitors, whereas this procedure had no apparent effect on specific HI antibody.

On the oncogenic effect produced in rats by tissue culture preparations, subjected to the action of human mammary gland cancer tumor extracts

In administering to newborn rats preparations from the monkey kidney tissue cultures subjected to the action of the extracts from cancer tissues of the human mammary gland, sarcoma occurred in 17–18% of the experimental animals at the age of 11–20 months, as well as in control animals in direct contact with them. Much more rarely (in 3.7% of the cases) the tumors occurred in rats in an experiment in which the initial material was passaged both in vivo and in vitro (mixed passage). Tumors did not appear in another comparable group of rats inoculated at the same period with the preparation from tissue cultures of human origin. A question is discussed on the appearance of tumors in rats and a possible connection of this phenomenon with the material investigated or the monkey kidney tissue cultures.

Fluorescent Microscopic Study of Sendai Incomplete Virus Production in Tissue Culture Cells

Summary When monkey kidney cells were infected with 1 EID50 of Sendai virus per cell, infectious virus was produced and the cytoplasmic inclusions could be observed by the fluorescent antibody indirect method. Antigen was never seen in the nuclei. Diffuse fluorescence of the cytoplasm without cytoplasmic inclusions could be seen after inoculation with 100 EID50 per cell. Here, presumably incomplete virue production with a low EID50:HA ratio had taken place. Incomplete Sendai virus was also formed in monkey kidney tissue culture in the presence of proflavine. The cytopathic effect appeared earlier and was more intense than in controls. The fluorescent antibody indirect technique detected the cells as large, structureless brightly fluorescing globi with a high concentration of antigen in the cytoplasm as well as in the nuclei. Acridine-orange staining showed profound changes in the nucleus and the lack of RNA in newly-formed antigen. The possible role of the nucleus in incomplete virus production is supposed.

An outbreak of aseptic meningitis associated with a previously unrecognized virus.

In 1959, 69 cases of aseptic meningitis were admitted to various hospitals in Scotland—all apparently due to a Hitherto unrecognized virus. This agent had the characteristics of an ECHO virus but differed from the 28 ECHO viruses at present recognized. Seventy-five strains of the virus were isolated, and human thyroid and human amnion tissue cultures proved much superior to monkey kidney tissue cultures for its isolation.

Transfer of nongenetic thermostability from genetically thermostable to genetically thermolabile poliovirus.

Adding the genetically thermostablecr i mutant of the Akron strain of type 1 poliovirus to the genetically thermolabile Akronca 30 mutant before infection of monkey kidney tissue culture sheets resulted in an average 17-fold increase in the heat-surviving fraction of theca 30 virus progeny. Mixing the 2 viruses after infection did not result in any increase. The progenies of at least most of these thermostable transferee particles were thermolabile. By this criterion the transferred thermostability was at least mainly nongenetic.

Respiratory and Enteric Viruses in Man and Animals

Most of the current studies of respiratory diseases of man and animals are aimed at determining their virus etiology, and considerable progress has been made, particularly since the advent of tissue culture methods. The relatively new hemadsorption technique (1) is used extensively in work with many of the respiratory disease viruses. Essentially, this is a system in which red blood cells of various species, depending on the virus, are added to the infected tissue culture tubes. The red blood cells will adhere to the surface of t,he infected cells. Hemadsorption has been used for establishing the identity of isolates as well as isolating the virus from clinical specimens. Hemadsorption is a specific phenomenon which is dependent upon the hemagglutinating properties of the virus. It was first reported in 1957 by Vogel and Shelokov who worked first with the influenza viruses and later extended these studies to certain other members of the myxovirus group. This technique has been used to good advantage in our laboratories. The recovery of parainfluenza 1, 3 (2), and possibly 4 (3) viruses, from children was largely due to the use of hemadsorption because many of the strains of parainfluenza 3, particularly, produced no cell degeneration on original isolation from children, and the virus was detected only by the use of hemadsorption. The first suggestion that a virus recovered from cattle with shipping fever might be a myxovirus resulted from the use of hemadsorption. It was then relatively simple, by using type-specific human parainfluenza 3 antiserum, to demonstrate the relationship of this virus to the human virus (4). When this newly described hemadsorption technique was used in conjunction with monkey kidney tissue cultures, two new myxoviruses were recovered from children with respiratory illnesses (2). One of the viruses, the present parainfluenza 3, was significantly associated with febrile illness during an outbreak of respiratory disease in a nursery. About the same time the present parainfluenza 1 virus was also recovered from children. In a study of 1,738 children in three hospitals in the Washington, D.C., area, one or the other of these two hemadsorption viruses was recovered from 54 (6 percent) of 879 patients with respiratory disease. It appeared that the two viruses were capable of causing a wide variety of clinical manifestations including croup, pneumonia, bronchiolitis, pharyngitis, and mild febrile disease. Table 1 shows the recovery of parainfluenza 1 and 3 viruses from children with various respiratory disease syndromes. Evidence of year-round occurrence of hemadsorption virus infections has been obtained (5). Other recent evidence relative to reinfection of Dr. Abinanti is with the Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Public Health Service. This paper was presented at the Conference of Public Health Veterinarians, November 2, 1960.

Serologic response of incividuals infected with para influenza viruses.

THE PARA influenza viruses Types 1 (hemadsorption Type 2), 2 (CA), and 3 (hemadsorption Type 1) are newly recognized members of the myxovirus family which have been recovered from persons with various types of respiratory illness.' 2 These agents possess certain biologic and antigenic properties which separate them from the influenza viruses.3'4 The para influenza viruses appear to play an important role in childhood respiratory illness. On a yearround basis, para influenza 1 and 3 viruses have been isolated from 6 per cent of children with all types of respiratory illness, and during certain months the recovery rate from children with upper respiratory illness has been as high as 20 per cent.5'6 Para influenza 1 virus has been the most important single agent associated with acute laryngotracheobronchitis (croup) of infancy and early childhood; this agent has been recovered from 20 per cent of patients with this syndrome.5'7 Recently para influenza 1 virus has been recovered from adults with febrile upper respiratory illness.8'9 During the course of a study of the microbiologic experience of infants and children under three years of age in a welfare nursery, we had the opportunity to observe outbreaks of infection with para influenza 1 virus and with para influenza 3 virus. This report will describe an evaluation of the available serodiagnostic procedures employing specimens derived from the nursery outbreaks. Throat swab specimens were obtained from the nursery children at weekly intervals and at the onset of a febrile illness (temperatures of greater than 100.6 F rectally). These specimens were tested for the presence of virus in monkey kidney tissue culture employing the hemadsorption technic. Neutralization tests were performed with fourfold dilutions of serum which were incubated for one hr at room temperature with 10 TCD50 of para influenza 1 virus or 100 TCD5o of para influenza 3 virus.

Growth of laboratory and naturally occurring strains of Eaton agent in monkey kidney tissue culture.

SummaryAn egg adapted strain of Eaton agent multiplied in rhesus monkey kidney tissue culture. Antigen concentration within the cells was insufficient to permit visualization by immunofluorescence. An eclipse phase was demonstrated during replication in monkey kidney cells. In a simultaneous test monkey kidney culture was found to be as sensitive as embryonated eggs for recovery of naturally occurring strains of Eaton agent. Fourteen strains were recovered in tissue culture from 17 patients with pneumonia.

Growth of small free-living amoebae in various bacterial and in bacteria-free cultures.

Hartmannella glebae, H. agricola, and Naegleria gruberi fed on a Flavobacterium sp. developed tolerance for the yellow pigment. Differences in the formation of plaques in the bacterial growth and in the growth cycle were observed.Various species of bacteria were equally effective as food for all three amoebae, and richness of the amoeba cultures paralleled the richness of the growth of edible bacteria. Failure of Pseudomonas pyocyanea to support growth of these amoebae was attributed to the toxicity of the pyocyanin pigment, and inability of the amoebae to develop tolerance to this pigment was attributed to the relatively high solubility of the pigment in water.These amoebae showed no growth on agar in the absence of living edible bacteria; however, bacteria-free H. agricola and N. gruberi, but not H. glebae, were successfully maintained in monkey kidney tissue cultures. The amoebae exhibited no toxic effect on the epithelial cells and fed on cell debris. Ingestion of very large numbers of cysts by young cells or any number by old cells resulted in degeneration of the cells with a release of live cysts. Epithelial cell degeneration hastened by enteroviruses facilitated rapid growth of the amoebae.

Studies of gastrointestinal viral flora of cholera patients.

Virus isolation studies on rectal swab specimens of cholera patients were carried out in tissue cultures during the Bangkok, Thailand cholera epidemic of 1958 and 1959. An adenovirus or enterovirus was isolated from 6 of 44 patients with proven cholera. It was concluded that viruses which can be isolated in monkey kidney and HeLa cell tissue cultures were not involved in the cholera syndrome.

A Fluorescent Antibody Study of the Simian Foamy Agent

Summary The fluorescent antibody technique has been used to observe antigen localization during the progressive development of foamy degeneration in naturally infected monkey kidney tissue cultures and in inoculated tissue cultures of several types. The indirect fluorescent antibody technique has been useful as an antibody screening procedure and has allowed the selection of monkeys free of foamy agent from the colony for use in the preparation of tissue cultures.

Phase variation in ECHO virus type 6

Strains of ECHO virus type 6 isolated during an epidemic of aseptic meningitis in 1955 were shown to react poorly in neutralization test with homologous or heterologous ECHO 6 antisera (virus in S phase). However, after passage in monkey kidney tissue culture, the strains reacted very well with such sera (virus in B phase). Alteration in other biologic characteristics accompanied this change in phase.

Relative resistance of slow-growing strains of Toxoplasma gondii to pyrimethamine (daraprim).

Both Eyles 1 and Summers, 2 working independently, discovered that pyrimethamine (Daraprim) cured acute experimental toxoplasmosis in mice. A subsequent study of the action of pyrimethamine and sulfadiazine 3 revealed that in mice infected with moderate inocula of RH-strain organisms (less than 20,000 parasites) the drugs acted synergistically and were toxoplasmicidal, when given early enough in the course of infection. When larger inocula were used, however, the same combination of pyrimethamine and sulfadiazine, at the highest doses tolerated, did not kill all the parasites, although the mice survived. Similarly, Cook and Jacobs 4 noted that, while pyrimethamine was generally toxoplasmicidal at a concentration of 0.3 mg. % when tested against RH-strain organisms in monkey-kidney tissue cultures, occasionally some organisms remained alive. They reported also that nonproliferating parasites, suspended in tissue culture medium without cells, were apparently unaffected by the drug. Cook then developed a strain of RH toxoplasmas resistant to pyrimethamine

Further experiments on the adaptation of Mahoney, type 1, poliomyelitis virus to suckling mice.

The Mahoney, Type 1, poliomyelitis virus had been through six to-and-fro monkey kidney-tissue culture and suckling mouse passages. Early paralysis was produced in groups of mice inoculated directly from infected tissue-culture fluid, but mouse-to-mouse passage failed to produce paralysis except in an occasional mouse. At the 7th tissue-culture mouse passage, the virus harvested from paralysed mice was inoculated into 100 mice. One mouse developed paralysis within 24 hr. From this mouse a variant of Mahoney virus was isolated which has now been through seventy-five mouse-to-mouse passages and produces paralysis in 90–100% of mice within 3 days.The authors would like to express their deep appreciation of Prof. A. Kipps' constant interest and encouragement.

Some Effects of Enzymatic Digestions of Fixed Monkey Kidney Tissue Cultures on their Staining Patterns.

SummaryA large decrease in cytoplasmic pyroninophilia and thioninophilia resulted from digestion of formalin-fixed monkey kidney tissue culture with either chymotrypsin or ribonuclease. This pair of enzymes decreased cytoplasmic pyroninophilia and thioninophilia more than either enzyme alone. Digestion of similar cultures with desoxyribonuclease gave a large decrease in nuclear pyroninophilia and thioninophilia. The effects of these 3 enzymes on pyroninophilia of these cells were more clearly shown when a purified pyronin was used.