Monkey Kidney Tissue Culture Research Articles

Effect of the purification of virus antigens on the production of specific complement-fixing antibodies.

The effect of viral purification procedures on the antibody response of guinea pigs to immunization with reovirus type 2 and echovirus type 19 was investigated. Three grades of antigens were employed: (i) infectious monkey kidney tissue culture fluid (TCF), (ii) virus sedimented in the ultracentrifuge and suspended in phosphate-buffered saline, and (iii) virus purified by centrifugation in CsCl density gradients. The antibody response of the guinea pigs was studied by the hemagglutination inhibition, complement fixation, and serum neutralization tests. Only sera produced from virus purified by CsCl density gradients reacted specifically with homologous antigen in the complement fixation test. Sera from animals receiving tissue culture fluid virus or sedimented virus cross-reacted with heterologous antigens such as tissue culture fluid from uninfected monkey kidney cells. All sera, however, reacted specifically in hemagglutination inhibition and serum neutralization tests. Sera from intranasally infected animals (reovirus type 2), even though reacting specifically in the complement fixation test, had much lower titers than sera from animals inoculated intramuscularly.

Serologic Surveillance for Adeno-Associated Satellite Virus Antibody in Military Recruits

Abstract A retrospective serologic survey for antibodies to adeno-associated satellite viruses was conducted on serum specimens collected from military recruits over a 7-year period. The results showed that 14% to 23% of the subjects possessed antibody titers of 16 or greater to any of the four satellite serotypes. Monotypic and heterotypic type 4 antibody seroconversions, which have been seldom seen in human but often in simian sera, occurred more frequently in men who received respiratory virus vaccines prepared in monkey kidney tissue cultures. It is speculated that such responses may have occurred from infections due to activation of latent defective viruses in these preparations or to antigenic components in the vaccines themselves. No definite relationship between these satellite viruses and disease was observed although there was a suggestion of an association between seroconversion to type 1 satellite virus in patients with the diagnosis of pneumonia. Sera from rubella patients showed significantly higher preexisting antibody titer to satellite virus types 1, 2 and 3 than did those of subjects with other illnesses. Only a few of the rubella cases showed seroconversion to the type 1 satellite virus.

Effect of the Purification of Virus Antigens on the Production of Specific Complement-Fixing Antibodies

The effect of viral purification procedures on the antibody response of guinea pigs to immunization with reovirus type 2 and echovirus type 19 was investigated. Three grades of antigens were employed: (i) infectious monkey kidney tissue culture fluid (TCF), (ii) virus sedimented in the ultracentrifuge and suspended in phosphate-buffered saline, and (iii) virus purified by centrifugation in CsCl density gradients. The antibody response of the guinea pigs was studied by the hemagglutination inhibition, complement fixation, and serum neutralization tests. Only sera produced from virus purified by CsCl density gradients reacted specifically with homologous antigen in the complement fixation test. Sera from animals receiving tissue culture fluid virus or sedimented virus cross-reacted with heterologous antigens such as tissue culture fluid from uninfected monkey kidney cells. All sera, however, reacted specifically in hemagglutination inhibition and serum neutralization tests. Sera from intranasally infected animals (reovirus type 2), even though reacting specifically in the complement fixation test, had much lower titers than sera from animals inoculated intramuscularly.

Human Diploid Cell Cultures in Diagnosis of Echovirus 30 Meningitis

Diagnosis of echovirus 30 meningitis was established by recovery of the virus from the cerebrospinal fluid (CSF) of four patients during a small outbreak. In three patients, viral isolation was easily achieved with human diploid tissue cultures (WI-38) whereas the same fluids failed to induce a cytopathic effect in monkey kidney tissue cultures. In one instance, isolation of the virus from CSF was successful in monkey kidney tissue but cultures could not be transmitted serially in these cells. There was no pleocytosis of the CSF in one patient, minimal pleocytosis in another, and more than 500 polymorphonuclear leukocytes per millimeter in another. The results show the value of human diploid cells for diagnosis in this type of infection and indicate variations that can occur in the cellular content of the CSF.

Human diploid cell cutures in diagnosis of echovirus 30 meningitis

Diagnosis of echovirus 30 meningitis was established by recovery of the virus from the cerebrospinal fluid (CSF) of four patients during a small outbreak. In three patients, viral isolation was easily achieved with human diploid tissue cultures (WI-38) whereas the same fluids failed to induce a cytopathic effect in monkey kidney tissue cultures. In one instance, isolation of the virus from CSF was successful in monkey kidney tissue but cultures could not be transmitted serially in these cells. There was no pleocytosis of the CSF in one patient, minimal pleocytosis in another, and more than 500 polymorphonuclear leukocytes per millimeter in another. The results show the value of human diploid cells for diagnosis in this type of infection and indicate variations that can occur in the cellular content of the CSF.

Nuclear pores: the pore-annulus relationship in thin section

In situ determinations of the spatial relationship between the nuclear pore and its annulus were made in ultrathin sections of African green monkey kidney and mouse 3T3 tissue culture cells, embedded as monolayers. A variety of fixation procedures, enzyme digestions, serial sections, and rotational analysis of selected images were used to analyze the structure of the pore complex. The results indicated that the annulus of each pore consists of at least two types of subunits; that it extends both into the cytoplasm and into the nucleoplasm and fits within the margins of the pore at the level of the nuclear envelope. The pore complex is hourglass-shaped since the diameter of the cytoplasmic and nuclear extensions are greater than the nuclear pore. The octagonal nature of the nuclear pore itself was confirmed. In addition, the predominant number of subannular components was also found to be eight. These observations are discussed in the light of the findings of earlier investigators and serve to clarify the structural complexity of the pore complex. On the basis of our results, which have been confirmed by three-dimensional reconstruction of the pore-annulus relationship from serial sections, we suggest that the nuclear pore complex be included among the classical cellular organelles.

Biological Characteristics and Viral Spectrum of Serially Cultivated Fetal Rhesus Monkey Kidney Cells

SummaryA serially cultivated tissue, designated HL-8, has been developed from fetal rhesus monkey kidney. A limited supply, frozen in liquid nitrogen, is available (ECD).3 The tissue culture exhibits persistent aneuploidy with a mean diploid chromosome number. It displays contact inhibition, an epithelial-like appearance in sparse culture, and consistent morphology and viral susceptibility during an approximately 13-month lifespan in vitro. The viral spectrum is comparable to primary rhesus monkey kidney tissue culture. HL-8 was able to support the growth of enteroviruses as well as primary MK. Laboratory adenovirus and re-ovirus strains grew and could be passed on HL-8, as could vaccinia and rubella. Surprisingly, HL-8 was successful in the recovery of several myxoviruses including virulent and vaccine measles. As with other serially cultivated cells, HL-8 was significantly inferior for propagation of influenza A2 and parainfluenza 1.

Markers differentiating type 3 parainfluenza virus grown at 25 and 37 C.

Serial passage of the 64-2389 strain of type 3 parainfluenza virus in cercopithecus monkey kidney tissue cultures at low temperatures resulted in the selection of a variant which had a higher efficiency of plaque formation at 25 C than the parent line grown at 37 C. The cold variant, unlike the parent strain, plaqued readily at 25 C, and at 37 C it produced significantly larger plaques. Virus titers of the cold variant in hamster lungs were significantly lower and this was probably caused by the stimulation of interferon by the cold variant during the early phase of the infection. The cold variant, like the virus grown at 37 C, also induced the synthesis of interferon late in the infection. Hamsters responded to the intranasal inoculation of each virus line by the development of hemagglutinating-inhibiting antibodies in the sera.

Markers of Rubella Virus Strains in RK 13 Cell Culture

When tested on RK(13) cell cultures, strains of rubella virus could be differentiated by their ability to form small or large plaques. Large plaques were produced by the HPV-77 and Cendehill strains, and also by a laboratory stock strain (West Point), after only 14 passages in RK(13) culture. Five wild-type rubella viruses, isolated and passaged only a few times in African green monkey kidney tissue culture, grew well in RK(13) cell culture, but they were sensitive to agar inhibitors and, therefore, formed small plaques. On the other hand, RA27/3, an attenuated strain grown in WI-38 human fibroblast cells, developed low titers in RK(13) cells and also produced small plaques. We concluded that the morphological differences between small-plaque and large-plaque viruses depended on their sensitivity to agar inhibitors and on the pH of the medium during plaque formation.

Comparative Sensitivity of Tissue Cultures to Rubella Virus: Use of Guinea Pig Cells for Virus Titration

A series of 19 different primary and serial tissue cultures were investigated for their sensitivity to virulent or attenuated rubella virus (RV). Primary guinea pig tissues, a serial passage of baby hamster kidney, and primary human amnion were comparable to African green monkey kidney tissue cultures in their sensitivity. In general, primary human tissues were relatively insusceptible to the Gilchrist strain of RV. RV interfered with the growth of vesicular stomatitis virus. Based on this finding, it was possible to develop an assay method in guinea pig tissue cultures by using VSV as the challenge virus. This system appeared to be comparable in sensitivity to the use of primary monkey kidney tissue cultures for the detection of small amounts of RV and offers the advantages of economy, rapidity, and safety.

Markers Differentiating Type 3 Parainfluenza Virus Grown at 25 and 37 C

Serial passage of the 64-2389 strain of type 3 parainfluenza virus in cercopithecus monkey kidney tissue cultures at low temperatures resulted in the selection of a variant which had a higher efficiency of plaque formation at 25 C than the parent line grown at 37 C. The cold variant, unlike the parent strain, plaqued readily at 25 C, and at 37 C it produced significantly larger plaques. Virus titers of the cold variant in hamster lungs were significantly lower and this was probably caused by the stimulation of interferon by the cold variant during the early phase of the infection. The cold variant, like the virus grown at 37 C, also induced the synthesis of interferon late in the infection. Hamsters responded to the intranasal inoculation of each virus line by the development of hemagglutinating-inhibiting antibodies in the sera.

Detection of sensitizing tissue culture components in viral vaccines

Adsorbed measles and poliomyelitis vaccines of monkey kidney tissue culture origin sensitize guinea pigs to protein components of the tissue culture, as shown by the passive cutaneous anaphylactic test. Unadsorbed vaccines are not sensitizing, and there is a correlation between the production of anaphylactic antibodies in guinea pigs by different vaccines and their ability to sensitize infants to a subsequent injection of attenuated measles virus vaccine.

Tissue culture method for toxigenicity testing of Corynebacterium diphtheriae.

A method for toxigenicity testing of Corynebacterium diphtheriae in tissue cultures was developed. Results were obtained by comparing destruction of the monkey kidney or, preferably, rabbit kidney monolayer by 0.1 ml of the C. diphtheriae culture in Elek's broth containing 20% rabbit serum with the appearance after the addition of 0.2 ml of a mixture of the C. diphtheriae culture and diphtheria antitoxin. The mixture of C. diphtheriae broth culture and 10 antitoxin units per ml was incubated for 1 hr at room temperature before it was added to the tissue cultures which were then incubated as long as 5 days; most results, however, were read in 72 hr. Elek's broth medium was superior to heart infusion broth for toxin production by C. diphtheriae. Addition of 20% rabbit serum improved toxin production in either broth. Numerous toxigenic and atoxigenic C. diphtheriae cultures were tested for toxigenicity in primary rabbit and monkey kidney tissue cultures. If properly controlled, this in vitro method appeared to have an advantage over the in vitro agar gel method; its results were comparable with the rabbit intradermal test. With the wider use of tissue cultures in most laboratories, we believe that the tissue culture method for toxigenicity would be more economical and easier to perform than the animal intradermal method.

Reversible inhibition of the reproducton of DNA viruses by glycosides of 2-phenethyl alcohol and related compounds

Phenylethyl alcohol and related compounds are able to inhibit the reproduction of DNA viruses but not the reproduction of RNA viruses in tissue culture. Since phenylethyl alcohol and particularly the analogous compounds P-nitrophenylethyl alcohol and phenylpropyl alcohol have a high toxicity for tissue cultures, an attempt was made to attain a similar inhibition of virus synthesis but a lower level of toxicity through administration of the glycosides of these aromatic alcohols. For this purpose the following glycosides were synthesized: Phenylethyl galactoside; P-Nitrophenylethyl galactoside; Phenylpropyl galactoside; Phenylethyl glucoside; Phenylethyl glucuronide. The corresponding glycosidases, β-galactosidase, β-glucosidase and β-glucuronidase could be detected and their activity measured in monkey kidney and HeLa cell tissue cultures. In addition, it was possible to localize the enzyme activities histochemically by using glycosides of 4-methylumbelliferone. The effect of the synthesized glycosides on the reproduction of DNA viruses (Vaccinia virus, Herpes simplex virus, SV-40) and RNA viruses (Poliovirus type 1) was investigated. The efficacy of the glycosides is dependent upon the type of glycone. The galactosides have the greatest inhibitory effect while the glucuronide of phenylethyl alcohol shows no effect at all on the replication of Vaccinia virus. The glycosides investigated possess a lower toxicity than the free alcohol; HeLa cells are more sensitive towards the compounds than monkey kidney cells. The use of virus inhibiting compounds in the form of glycosides therefore makes it possible for the active substances to be brought into the cell in a non-toxic form and to be transformed into the active compound by the cell enzymes.

Clinical trial of live attenuated rubella virus vaccine, HPV-77 strain.

IN 1966, Parkman, Meyer, and their colleagues described preliminary vaccine trials with a strain of rubella virus which had been attenuated by 77 passages in primary African green monkey kidney (AGMK) tissue culture. 1,2 This strain, designated HPV-77, exhibited desirable qualities of immunogenicity and obvious attenuation in man. The present report describes our experience with HPV-77 virus in a series of vaccine trials which involved 48 children. Materials and Methods Vaccine. —The details of the preparation and safety testing of HPV-77 vaccine have been described by Meyer et al. 1,2 The vaccine was supplied in single-dose vials which were stored at −65 C until use. Each vaccinated child received rubella vaccine (HPV-77), 0.5 ml subcutaneously. This dose represented 500 tissue culture interfering doses50 (TCID 50 ), based on a titration in primary AGMK tissue culture using echovirus type 11 challenge. 3 Study Population and Experimental Design.—The 48 children were admitted

Problems in the detection of rubella virus in African green monkey kidney tissue culture.

SummaryThe sensitivity to rubella virus of 96 lots of AGMK tissue culture was evaluated using the enterovirus interference technique. Wide variation in sensitivity was found leading to the classification of 32 of the lots tested as resistant to rubella virus. The use of these resistant lots of tissue culture for the isolation or titration of rubella virus would lead to invalid test results. Appropriate controls are needed to assure the validity of studies in which rubella virus is detected with African green monkey kidney tissue culture.

Hybrid of monkey and human adenoviruses.

Following joint replication of monkey SA7 adenovirus (C8 strain) and human adenovirus type 2 in green monkey kidney tissue culture, a virus possessing the properties of a hybrid was obtained. It was designated Ad2C8. Ad2C8 preparations contained two types of viral particles: human adenovirus type 2, and hybrid particles. The hybrid virions multiplied in green monkey kidney cells in the presence of human adenovirus types 1, 2, and 3, but not 3 and 7, and acquired the capsid of the helper adenovirus. The hybrid can serve as a helper for human adenoviruses. It can apparently induce T antigen of the C8 virus but, in contrast to the latter, does not induce tumors in hamsters.

Tissue Culture Method for Toxigenicity Testing of Corynebacterium diphtheriae

A method for toxigenicity testing of Corynebacterium diphtheriae in tissue cultures was developed. Results were obtained by comparing destruction of the monkey kidney or, preferably, rabbit kidney monolayer by 0.1 ml of the C. diphtheriae culture in Elek's broth containing 20% rabbit serum with the appearance after the addition of 0.2 ml of a mixture of the C. diphtheriae culture and diphtheria antitoxin. The mixture of C. diphtheriae broth culture and 10 antitoxin units per ml was incubated for 1 hr at room temperature before it was added to the tissue cultures which were then incubated as long as 5 days; most results, however, were read in 72 hr. Elek's broth medium was superior to heart infusion broth for toxin production by C. diphtheriae. Addition of 20% rabbit serum improved toxin production in either broth. Numerous toxigenic and atoxigenic C. diphtheriae cultures were tested for toxigenicity in primary rabbit and monkey kidney tissue cultures. If properly controlled, this in vitro method appeared to have an advantage over the in vitro agar gel method; its results were comparable with the rabbit intradermal test. With the wider use of tissue cultures in most laboratories, we believe that the tissue culture method for toxigenicity would be more economical and easier to perform than the animal intradermal method.

Rubella Virus: Role of Interferon during Infection of African Green Monkey Kidney Tissue Cultures

Summary African green monkey kidney cell cultures infected with low or high multiplicities of rubella virus were found to produce small or large amounts of interferon respectively. Following high multiplicity infection of these cell cultures with rubella virus there was continuous production of virus and interferon over a period of 38 days. Infected cell cultures also developed cellular resistance which was characterized as interferonlike by its broad antiviral effect against many viruses and by its inhibition by dactinomycin (actinomycin D). The sensitivity of rubella virus to monkey interferon was substantially greater than that of vesicular stomatitis virus. Rubella virus yield decreased when lower virus inputs were used. This appears to have been due to interferon production during early virus growth cycles. These facts suggest that much of the observed inhibition of homologous and heterologous virus could be accounted for by the amount of interferon produced, the degree of resistance induced, and the high sensitivity of rubella virus to the interferon system. The findings also suggest that persistent infection of African green monkey kidney cultures by rubella virus is associated with persistent activity of the interferon system.

Growth and cytopathic effect of H type rhinoviruses in monkey kidney tissue culture.

SummaryGrowth and production of CPE in MKTC has been demonstrated for 5 rhino-virus strains, NIH 1734, 353, 11757, 1059, and 33342, previously considered to be H strains.