Monkey Kidney Tissue Culture Research Articles

Absence of Intestinal Viral Flora in Infants During Epidemic of Escherichia coli Gastroenteritis

SummarySystematic viral isolation attempts were made from an outbreak of infant gastroenteritis in which Escherichia coli 0–111:B4 was found. Treated stool samples collected from 25 infants were inoculated into monkey kidney and HeLa cell tissue cultures, embryonated eggs and a variety of young animals by various routes. No viral agent could be detected. It is concluded that this outbreak of gastroenteritis was caused by Escherichia coli 0–111:B4, uncomplicated by other microbial agents.

Immunochemical Studies of Poliovirus

Summary Through fractionation in the ultracentrifuge of partially purified concentrated poliovirus from monkey kidney tissue cultures, and by infectivity and serologic assay of the fractions so obtained, evidence has been obtained for the existence of four distinct viral particles. These have been designated ID+, NID+, NID- and NIC-, representing, respectively, infectious virus particles possessing immunologic D specificity, and a sedimentation coefficient of about 160 S, noninfectious particles of D specificity and 160 S, noninfectious particles of D specificity exhibiting a sedimentation coefficient of 80–90 S, and noninfectious particles of C specificity exhibiting a sedimentation coefficient of 80–90 S. The designation of C and D specificity refers to reactivity in complement fixation tests with suitable acute or convalescent phase sera, respectively.

EMPLOI D'UN MILIEU SYNTHÉTIQUE SIMPLIFIÉ POUR LA CULTURE IN VITRO DE CELLULES ANIMA LES ET DU VIRUS DE LA POLIOMYÉLITE

The nutritive medium SN-3 is composed of Hanks's balanced salt solution with ribose, glutamine, and cysteine added; the glucose concentration is doubled. This nutritive solution, without additional ingredients, can keep alive monkey or mouse kidney tissue cultures during a period of several weeks. When this medium is supplemented with serum, it stimulates the in vitro cellular proliferation of freshly trypsinized monkey kidney tissue and supports the growth of HeLa cells.We have used this simplified synthetic medium for the preparation of experimental lots of poliomyelitis vaccine. The virus concentration of the suspension of the three virus strains, Mahoney, MEF-1, and Saukett, produced with this new medium has been found as high as the concentration obtained with the more complex nutritive media usually used. We did not observe any important differences between the vaccine prepared with the SN-3 medium and that-prepared with the usual media.

Coxsackie B virus infection; presence of virus in blood, urine, and cerebrospinal fluid.

A 40-year-old male biochemist had an intermittent fever over a period of nine days. The only significant symptoms were malaise, chilliness, sweating, headache, myalgia, and minimal stiffness of the neck. The results of laboratory tests differed but little from the normal, except that the urinary excretion of phenolsulfonphthalein was consistently delayed. Since the patient had been working with viruses for four years, inoculations were made into monkey kidney tissue cultures and Hela tissue cultures, using specimens of blood, urine, and cerebrospinal fluid as well as swabs from throat and anus. Intraperitoneal and intracerebral inoculations were also carried out in mice. The specimens of urine were prepared for inoculation by a concentration technique. The organism was identified as a Coxsackie B virus, and it appeared in a urine specimen collected on the fourth day of illness. It was also found at various times in the blood, cerebrospinal fluid, and pharyngeal and anal swabbings. Its appearance in the urine suggests viral infection of the kidney as the probable cause of the retention of phenolsulfonphthalein.

Application of the “most probable number” method for estimating concentrations of animal viruses by the tissue culture technique

Justification for use of an MPN (most probable number) method for estimating concentration of Coxsackie B1 virus in monkey kidney tissue culture tubes was demonstrated by comparison of dosage-response data with a theoretical MPN curve based on one-particle infection and by variance analysis of 24 replicate MPN determinations. This method yields units comparable to plaque counts or plaque-forming units and affords unbiased estimates even when data do not bracket the 50% response dilution. It provides a means of testing for conformity to the one-particle infection theory and gives usable results from one dilution if both positive and negative tubes are present. For these reasons it is preferred to conventional LD 50 methods for virus quantitation where the dilution response data are known to follow the one-particle infection curve. A table of 5-tube, 4-fold dilution series and an approximation equation are presented for conversion of data to “most probable number of cytopathogenic units” (MPNCU).

A comparison of complement-fixation titres in poliomyelitis-infected tissue-culture fluids and mouse brains.

Ultra-centrifugation of emulsions from suckling mouse brains infected with the MEF1 strain of poliomyelitis virus separates a non-infective antigen, or soluble antigen, from infective virus. This antigen is responsible for most of the complement fixation and explains the high titres obtained. On the other hand, the same virus, and also Mahoney, Type 1 poliomyelitis virus, grown in monkey kidney tissue culture, fail to produce this soluble antigen, and this is probably a factor in the low complement-fixing titres obtained in tests with these fluids.The author would like to express her appreciation of Miss M. Butchart's valuable technical assistance.

Typing of ECHO viruses by a complement fixation technic.

Typing of ECHO viruses(1) by standard neutralization tests is an inconvenient and laborious procedure because of large number of prototypes. Recent findings, that complement-fixing (CF) antigens for ECHO viruses could be prepared consistently from monkey kidney tissue cultures and that these antigens reacted type specifically with ECHO antisera prepared in guinea pigs and monkeys(2), suggested the possibility of typing ECHO virus isolates by means of a CF technic. This preliminary report describes simple methods which have been successful in typing 28 ECHO virus isolates by such a procedure.†Methods. CF antigens were prepared by inoculating each of 4 rhesus kidney tissue culture tubes containing 1 ml of the following: lactalbumin hydrolysate 0.5%, calf serum 2%, Earle's solution 97.5%, with 0.1 ml of undiluted fluid from the original or an early monkey kidney passage of the ECHO isolate. After complete degeneration of tissue had taken place, the tubes were frozen and thawed, and pooled tissue culture flui...

Poliovirus mutants with altered responses to cystine.

SUMMARY: Three strains of poliovirus (Akron, antigenic type 1; Brooks, type 2; Mabie, type 3), which require exogenously supplied cystine (or a cystine-substitute) for optimal cytopathogenic action on monkey kidney tissue cultures, were serially passaged ten times in cystine-deficient monkey kidney tissue cultures in tubes. From various passage levels in this system were isolated three mutants of Akron, four of Brooks and two of Mabie. These nine mutants are designated the cystine-response mutants and are distinguishable from their ancestral viruses used to initiate the passage series by being relatively cystine-non-requiring. Two criteria suffice to distinguish the mutants of any strain from each other: (1) the degree of loss of requirement for cystine, and (2) the degree of inhibition by relatively high concentrations of cystine. The compositions of the virus populations in most of the passage levels were determined, and from these determinations the patterns of emergence of the mutant viruses were delineated.

Preparation of ECHO Complement-Fixing Antigens in Monkey Kidney Tissue Culture and Their Purification by Fluorocarbon

The fluorocarbon purification technic was applied to purification of complement-fixing antigens prepared in monkey kidney cultures from ECHO prototype strains. The effect of serum in tissue culture medium on the effect of fluorocarbon was studied. Adding serum to synthetic tissue culture medium improved the titers of ECHO CF antigens. Lactalbumin hydrolysate media containing serum represented the best media of several tried for the preparation of ECHO CF antigens in monkey kidney culture. The untreated antigens, when tested with guinea pig sera prepared by immunizing animals with monkey kidney grown virus, gave strong monkey kidney tissue reactions. However, fluorocarbon treatment removed the nonspecific tissue reaction and “purified” antigens gave specific reactions in CF tests with ECHO antisera prepared in guinea pigs and in monkeys.

Hamster Kidney Cell Tissue Cultures for Propagation of Japanese B Encephalitis Virus.

Summary(1) Monolayer HKC tissue cultures can be prepared on a routine basis by methods essentially identical with those currently in use for preparation of monkey kidney tissue cultures. In screen tests with 13 arthropod-borne agents and 3 types of polio-virus. HKC proved markedly susceptible to the CPE of Japanese B encephalitis. West Nile, Uganda S. Ilheus. Bunyamwera, and Semliki Forest viruses, only moderately susceptible to effects of the St. Louis encephalitis agent and the New Guinea C, Hawaiian, and Trinidad strains of dengue virus. In contrast, the Ntaya, Zika and Bwamba agents and the 3 types of poliovirus failed to show CPE. (2) Evidence has been presented for serial propagation of JBE virus through 8 serial transfers in HKC tissue cultures. CPE was eventually complete. After 6 passages the virus yield was greater than the starting inoculum, although the dilution factors had carried beyond the extinction point of infectivity of the original inoculum. (3) Identity of the Japanese B encephalitis ...

The effect of anticellular sera on virus multiplication in tissue culture

Viruses causing a cytopathogenic effect (CPE) in tissue cultures can be nonspecifically inhibited by anticellular sera. Not only CPE but actual multiplication of viruses is suppressed even when antisera are applied late in the growth cycle of some viruses. Different viruses vary in their ability to be inhibited and for certain viruses greater inhibition can be demonstrated in a cell system homologous for the anticellular serum. However, there is species crossing when antisera to human cells are tested in monkey kidney tissue culture. This phenomenon is due to an effect of the antisera on the cell and is not antiviral. The inhibitory activity can be removed by adsorption with red blood cells or the proper tissue cells. Limited evidence suggests that the antiserum action takes place at the surface of the cell or in the cytoplasm. Cross reactions in neutralization tests employing standard typing sera may be caused by this phenomenon.

Viability of monkey kidney tissue cultures stored at 5 degrees C.

Viability of monkey kidney tissue cultures stored at 5 degrees C.

Investigation on the inoculation of biological materials from schizophrenic patients in chick embryos, in guinea pigs, in white mice and in monkey kidney tissue culture.

Investigation on the inoculation of biological materials from schizophrenic patients in chick embryos, in guinea pigs, in white mice and in monkey kidney tissue culture.

The Isolation in Tissue Culture, Chick Embryo and Suckling Mice of Filtrable Agents from Healthy Dairy Cattle

Summary The isolation of eight filtrable agents from apparently healthy dairy cattle in cattle kidney tissue culture has been described. Evidence has been presented that they are viral in nature, serologically similar, fairly widespread in the cattle population and probably of little importance in human disease. Studies with the partition cell in the ultracentrifuge indicate that they are relatively small particles with a sedimentation constant of 150 to 200 Sved. Their host range includes monkey kidney tissue culture, chick embryos and 1-day-old suckling mice. The appearance of melanin-like granules in the amniotic fluid of pigmented Barred Rock chick embryos is of interest. The isolation of many new viral agents by the techniques outlined should be productive.

The primary interaction of poliomyelitis virus with host cells of tissue culture origin

The inorganic salts CaCl 2 and MgCl 2 promoted the attachment of poliomyelitis virus to monkey kidney tissue culture cells in suspension. The rate of virus attachment was found to be dependent on the concentration of these salts. No attachment was demonstrable at 10 −1 M or higher and 10 −5 M or lower concentrations of the above salts. Optimal attachment rates occurred at 10 −3 M calcium or magnesium chloride. Temperature exerted no significant effect on the virus attachment rate within the range of 37° to 1°. A nonadsorbing system of virus and cells was converted into a rapidly reacting one by the addition of CaCl 2 to a final concentration of 10 −3 M. These data suggested that the initial binding of poliomyelitis virus to host cells is mediated through electrostatic bonding.

Free living amoebae as contaminants in monkey kidney tissue culture.

Two strains of apparently the same free living amoebae were found to be spontaneous contaminants in monkey kidney tissue culture. The TC technic proved to be a suitable method for cultivation of the amoebae in the absence of bacteria. Pure cultures of the amoeba were obtained in many cell lines. Both cysts and trophozoites were found in TC. In monkey kidney tissue culture, cysts were often found in the cytoplasm of the cells. One strain was subjected to 16 consecutive passages in monkey kidney tissue culture while the other had 12. The average CPE titer of the TC fluid was 104 to 105 per 1.0 ml. The cysts in the TC fluid survived at −50°C for eight months, although the titer was greatly reduced.

Biologic and Serologic Characteristics of Echo Viruses from West Virginia

Summary The biologic and serologic characteristics of 30 echo viruses from Charleston, West Virginia, have been described. This group has been characterized as human enteric viruses which are not related to poliovirus, Coxsackie, herpes or adenovirus groups, which are not pathogenic for monkeys or newborn mice, but which produce cytopathic effects in tissue cultures of rhesus monkey kidney cells. Twenty of the 30 were typable as echo types 3, 6, 7, 8, 9, 11, 14, or a new type no. 15. A number of the remaining untypable viruses show cross neutralization reactions in one direction only, indicating the possibility of mixtures of viruses, antigenic deficiencies, or variations within a single type as already known to exist in the echo-6 virus group. The growth characteristics in monkey kidney, HeLa, and Maben cell tissue cultures have been described, and some strains have been shown to differ markedly in their ability to grow in these 3 cell types. Neutralization of these echo viruses by donor sera has been correlated with some of their growth characteristics in tissue culture. Those echo viruses which produced cytopathic effects rapidly and produced high virus titers in monkey kidney cell cultures were strongly neutralized. Those which produced cytopathic changes slowly and yielded only low virus titers were only weakly neutralized by sera from their human donors, as measured in tube cultures by the inhibition of the cytopathic response.

Virulence of polioviruses in relation to variant characteristics distinguishable on cells in vitro

Early terminal-dilution end point passages of the poliovirus strains Akron (antigenic type 1), Brooks (type 2), and Mabie (type 3) in monkey kidney tissue cultures resulted in the emergence of viruses of reduced virulence for monkeys. This reduction in virulence has not as yet been found to be associated with any change in the virus observable on cells in vitro. Through the use of specific selective environments, several variant viruses, which are characterizable on cells in vitro, were obtained from these viruses of reduced virulence. The distinguishing characteristics of these variant viruses are HeLa-adaptation, altered response to cystine, and cold-adaptation. Tests in monkeys showed that virulence is essentially independent of HeLa-adaptation and cystine-response. The cold-adapted viruses obtained by passage in monkey kidney tissue cultures at 30°, however, were found to be even less virulent than their only moderately virulent ancestors. Viruses further adapted to the cold were obtained by passage at 23° and were found not only to be more cold-adapted but also to be more avirulent than their progenitor 30°-adapted viruses. One of these 23°-adapted variants (Mabie) has shown no virulence for the monkey by the intraspinal route of inoculation. These cold adapted viruses are deadapted to monkey kidney tissue cultures at temperatures as high as 37° and the data indicate that the virulence of these viruses is negatively correlated with the degree of this deadaptation. The relative avirulence of a cold-adapted virus therefore appears to be due to its generally reduced capacity to propagate at 37° or above.

Adaptation of Mahoney, type I, poliomyelitis virus to suckling mice.

The Mahoney, Type I, poliomyelitis virus undergoes two ‘cycles’ of multiplication in the CNS of suckling mice—the first within 48 hr. after intracerebral inoculation, and the second approximately 10 days later. The former may not be associated with paralysis whilst the latter usually is.By to-and-fro passage in suckling mouse brain and monkey kidney-tissue culture there is a gradual increase in the amount of virus obtained during the early multiplication cycle in suckling mouse brains and associated with this is the occurrence of early paralysis in 80% or more of these mice. The virus capable of causing early paralysis in infant mice is gradually lost by direct mouse-to-mouse passage.The occurrence of two variants is postulated, one of these apparently nonparalytic and multiplying selectively in suckling brains.The author would like to thank the late Prof. M. van den Ende for his stimulating discussions and advice, Dr P. K. Olitsky of New York for his constant interest, and Mr T. C. Norcott for his able technical assistance.

Adsorption-hemagglutination test for influenza virus in monkey kidney tissue culture.

Adsorption-hemagglutination test for influenza virus in monkey kidney tissue culture.