Primary Rhesus Monkey Kidney Research Articles

Evaluation of Three Cell Culture Systems as Substrates for Influenza Virus Assay

The propagation of various influenza virus strains in primary rhesus monkey kidney (RMK), primary hamster kidney (HK), and the MDCK-USD canine kidney (CK) cell cultures was compared. Virus-infected cultures were examined for cytopathic effect (CPE) and by performing the hemadsorption (HAd) technique. The highest HAd titers were found most often in HK, followed by RMK and CK. However, the CK cell line provided the best substrate for detecting CPE. Although influenza B strains tended to grow to higher titers, these strains produced less CPE than did the A strains.

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.

Lipids of plasma membranes of monkey and hamster kidney cells and of parainfluenza virions grown in these cells

Plasma membranes have been isolated from primary rhesus monkey kidney (MK), baby hamster kidney (BHK21-F), and adult hamster kidney (HaK) cells, and detailed lipid analyses of the membranes, the whole cells, and parainfluenza virus SV5 grown in MK and BHK21-F cells have been carried out. The lipid compositions of the plasma membranes differ from those of the respective whole cells, and show characteristic features, including a high cholesterol and sphingomyelin content, and a cholesterol to phospholipid ratio which is higher than that of the whole cell. The lipid patterns of the membranes of the various cell types are significantly different from each other, especially with regard to phospholipids. In MK membranes, e.g., phos-phatidylethanolamine is the major phospholipid, and there is a relatively high content of phosphatidylserine, whereas in BHK21-F membranes, phosphatidylcholine is present in highest amount, and there is relatively little phosphatidylethanolamine and phosphatidylserine. Previous studies have shown that the plasma membranes of these cells exhibit significant biological differences, particularly in their interactions with SV5. SV5 virions grown in MK or BHK21-F cells have a lipid composition that is closely similar to that of the plasma membrane of the particular cell type in which the virus was grown. The data support the hypothesis that during the maturation of the virion by budding from the cell surface, the lipids of the plasma membrane are incorporated quantitatively into the viral envelope.

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.

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.

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.

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.

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.

On the role of the response of the cell membrane in determining virus virulence. Contrasting effects of the parainfluenza virus SV5 in two cell types.

The simian myxovirus SV5 multiplies in a continuous line of baby hamster kidney (BHK21-F) cells causing extensive cell fusion, followed by cell death. After inoculation of 15 PFU/cell, the latent period was 7 hr, the doubling time approximately 60 min, and the yield 7 PFU per cell. Giant cell formation began about 6 hr after infection and rapidly progressed to the formation by 14 to 18 hr of a single syncytium which disintegrated by 24 to 36 hr. In contrast, SV5 multiplies in primary rhesus monkey kidney cells for long periods of time producing high yields of virus with little cytopathic effect. High multiplicities of SV5 induced cell fusion in BHK21-F cells within 1 hr in the absence of virus multiplication but had no visible effect on monkey kidney cells. Time-lapse photomicrography has demonstrated that giant cells form by fusion of infected cells, and that some polykaryocytes divide. During aberrant division of polykaryocytes giant nuclei are formed from the nuclear material of several parent nuclei. The cytoplasmic development of viral antigens as demonstrated by immunofluorescence is similar in BHK21-F and monkey kidney cells. Synthesis of cellular DNA, RNA, and protein in monkey kidney cells is not shut off by SV5-infection, and in BHK21-F cells synthesis of these macromolecules is not inhibited until after extensive cell fusion has occurred 12 to 15 hr after infection. Persistently infected BHK21-F and monkey kidney cells have been serially carried through 11 and 28 cell passages, respectively. The results suggest that whether SV5 acts as a moderate virus, as in monkey kidney cells, or a virulent virus, as in BHK21-F cells, depends on the response of the cell membrane to the virus.

Milker's nodule virus infections in Dorset and their similarity to orf.

BETWEEN January 1964 and March 1965 seven farm workers on six Dorset farms developed lesions on the hand or forearm which were diagnosed as clinically typical orf infections. Material from each patient was examined by electron microscopy and tissue culture as previously described1,2. In each instance pox-virus particles were seen which in morphology and size were identical with orf virus3. In tissue culture four specimens produced the same cytopathic changes as orf virus and serial passage of three virus strains was obtained in primary human amnion and rhesus monkey kidney. No difference in behaviour was noted between these strains and orf virus strains previously grown from humans infected from sheep.

Differences in cytopathic effects of adenovirus in monkey kidney tissue culture.

Prototype strains for human adenovirus types 1 to 18 have been inoculated into primary Rhesus (Macacca mulatta) and African Green (Cercopithecus aethiops) monkey kidney cultures and give rise to two distinct types and a less obvious type of CPE. Rhesus kidney cultures give better resolution than do African Green. Types 1 to 18 are placed into 3 groups on the basis of the kind of CPE elicited and with a single exception these groups are identical to those suggested by Rosen(6) based on hemagglutination. Fourteen strains from type 12 were tested and all gave identical CPE, indicating that differences are probably type rather than strain oriented. Viruses from 2 different groups can be mixed with specific antiserum for one of the pair and the antiserum will selectively inhibit the CPE of only the homologous virus.

SUSCEPTIBILITY OF THE LLC-MK2 LINE OF MONKEY KIDNEY CELLS TO HUMAN ENTEROVIRUSES.

The relative susceptibility of the LLC-MK2 cell line and primary rhesus monkey kidney cultures to thirty-eight prototype strains of human enteroviruses is described; of these strains only ECHO types 16, 18, 21 and 23 failed to cause CPE in the continuous cell line. The efficiency of the two tissues for the isolation of enteroviruses from faecal extracts is compared. The results show that the LLC-MK2 cell line is very satisfactory for the isolation of Coxsackie B and polioviruses, but not so useful for the isolation of ECHO viruses. The identification of enteroviruses by neutralization tests in LLC-MK2 cells is successful.

Immunologic Characterization of Western Equine Encephalomyelitis Virus Strains

Summary Ten WEE strains and plaques derived from each were compared by the plaque reduction neutralization test with antisera prepared in guinea pigs. The analysis indicated that most of the strains were comprised of subpopulations of virus having antigenically distinct properties and that the quantitative distribution of common but distinct antigens determined in part the immunologic properties of each strain. Hyperimmune guinea pig sera prepared from plaque purified viruses were more specific in their cross-neutralizing properties than similar antisera prepared from unpurified strains. The antigenic variations of 100 WEE strains were studied using goat antiserum prepared from antigenically distinct plaque viruses derived from the McMillan strain of WEE. Three antigenic groups (phases) of the virus could be differentiated, and these were designated aphasic, phase I and phase II variants. There appeared to be a general pattern in the geographic distribution of these variants. The antigenic properties of phasic strains could be altered by serial intracerabral passage in infant mice or passage in primary rhesus monkey kidney cultures. No changes in antigenic properties were recognized when phasic strains were passaged in primary chick embryo cultures. Aphasic virus strains were antigenically stable when passaged in different host systems.

Production and Properties of an Excystment Inhibitor from Acanthamoeba sp.

A method for titrating excystment inhibitor produced by strain R of Acanthamnoeba sp., using young (1to 3-day-old) populations of R cysts, was developed. Much older cysts were not satisfactory because of their refractoriness to excystment inhibitor. The data indicate that this inhibitor is produced by the trophozoites and that the rate of production at any time is directly proportional to the number of trophozoites present; no production by cysts was detected. The excystment-inhibiting activity of the R culture supernatant was not affected by bromelin, chymotrypsin, papain, trypsin, deoxyribonuclease, or ribonuclease; however, bromelin did affect the cysts, increasing their resistance to excystment inhibitor and also their efficiency of plating in the absence of excystment inhibitor. Heating the culture supernatant at 60 C did not affect its capacity to inhibit excystment but at 100 C this capacity was greatly reduced. Treatment with HC1 or NaOH also greatly reduced the inhibitory activity. After dialysis, the material in the bag still exhibited high titer; but a small fraction of the inhibitory activity was found in the dialysate. NH4 was found to have some capacity to inhibit excystment; however, the dialyzable component of supernatant excystment inhibitor is probably not NHW+, since NH,1 gives a titration curve of a different shape. In the course of developing a method for cloning, titrating, and differentiating Acanthamoeba sp. by plating, we found that supernatants from cultures of these amebae had the capacity to block the production of colonies from plated cysts but not from plated trophozoites (Jensen and Dubes, 1961 and 1962).1 The results indicated that these amebae produce one or more substances which inhibit excystment. All three strains studied (R, S, and V) produced an excystment inhibitor (s); however, the cysts of one of the three (strain V) were refractory to inhibitor. In this report we describe a method for Received for publication 31 October 1963. * Part of these results were presented on 6 November 1963 at the meeting of the American Society of Parasitologists in Chicago (Jensen and Dubes, 1963). We have recently found that supernatants from cultures of three other acanthamebae also inhibit colony production by the corresponding cysts. These three were Acanthamoeba castellanii, kindly supplied by Dr. Max C. McCowen of Lilly Laboratories, and Acanthamoeba sp. strains D and N, which were isolated by us from primary rhesus monkey kidney tissue cultures in 1961. titrating excystment inhibitor and present some results from applications of this method to studies of the production and properties of this inhibitor. MATERIALS AND METHODS Acanthamoeba sp. strain R2 was used for the studies reported herein. Trophozoites and cysts of this strain were prepared by growth at 37 C in medium Z3 containing 0.05% Candida albicans strain FA cells. Previously described procedures (Jensen and Dubes, 1962) were used for washing the harvested amebae and for obtaining the supernatants from such cultures. Some cysts were stored at 4 to 8 C for stock purposes. R cultures initiated to provide amebae for testing for excystment inhibitor were observed daily. Trophozoite populations were harvested therefrom when the cultures were near the top of the growth curve but before noticeable encystment. Cyst populations were harvested at various times after the encystment of the bulk of the amebae and used on day of harvest. These cyst populations were assigned an age, which was the difference 2 This strain was originally isolated from a mixed culture of chick embryo and HeLa cells (Jensen and Dubes, 1962). This is a chemically defined medium containing inorganic salts, 13 amino acids, eight vitamins, and phenol red (Jensen and Dubes, 1962).

Species-specific hemagglutinogens in cultivated mammalian cells: II. The intracellular location of hemagglutinogens

Species-specific antigenicity as measured by inhibition of species-specific hemagglutinins is associated with particulate as well as soluble proteinaceous components throughout the cell including the nucleus. There is no indication for a particular, specially located cell constituent as carrier of species-specificity. In contrast, blood group B-specific antigen was not found in the nuclear cell fraction of primary rhesus monkey kidney cells, but in the particulate as well as soluble cytoplasmic fractions tested.