Fetal Kidney Cells Research Articles

Anti-rabies treatment of dog-bite victims in Lagos, Nigeria: trial of suckling mouse brain and fetal bovine kidney cell rabies vaccines

Human rabies is common in Nigeria, a country known for rabies endemicity. Yet the supply of anti-rabies vaccines for human use (adult sheep brain origin, produced locally, and duck embryo and human diploid cell vaccines, imported) is grossly inadequate. This study involved controlled treatment of dog-bite victims with suckling mouse brain (SMBV) or fetal bovine kidney (FBKV) rabies vaccines in an effort to determine which type to recommend for production and use in Nigeria. Dog-bite victims treated were aged three to 74 years, with about equal numbers of males and females. Of the 136 patients, 116 (85.3%) completed at least the primo-series of vaccinations, and all have survived without any evidence of clinical rabies. Recipients of the SMBV showed local and generalized reactions in 11.1% and 2.5% of the cases, respectively. Recipients of the FBKV administered subcutaneously showed local and generalized reactions, in 12.5% and 9.4% of cases, respectively. There were no side effects attributable to the vaccine among patients who received the FBKV intramuscularly. By day 7, 26.7% of SMBV recipients and 28.6% of FBKV recipients showed antibody response, with titres of at least one Equivalent Unit ml −1. These percentages increased to 95.1 and 81.1, respectively, by day 14, and by day 20 (for SMBV recipients) or day 30 (FBKV recipients) the response was 100%. Titres dropped by day 90, but in no case to below 1 EU ml −1. We have concluded that both vaccines are equally efficacious and well tolerated. In view of the simple technology required, and the resultant lower cost, the SMBV is being recommended for production and use in Nigeria.

Further observations on the morphogenesis of human rotavirus in MA 104 cells.

Human rotavirus "KUN" strain was cultivated in a fetal rhesus monkey kidney cell line, MA 104 cells. Four types of virus particles in cells infected with KUN strain were clearly identified: nucleoid cores, single-shelled particles, double-shelled particles, and membrane band, "enveloped" particles. "Enveloped" particles were found only in the thin sections of infected cells. When first visible, the virus precursors appeared at the ribosome free membrane of rough endoplasmic reticulum (RER), increasing in size while simultaneously being coated with nucleocapsid, inner shell. Single-shelled particles were also synthesized within bundles of filaments of viroplasm in the cytoplasma. During subsequent virus maturation two types of "budding" processes were observed. Double-shelled particles arising at the RER membrane entered the cisternae of the RER through an exocytosis-like process. In contrast, the "enveloped" particles developed in the cisternae by being completely enclosed with RER membrane, and later during cytolysis released the single-shelled particles. These "enveloped" virus particles appeared to be the result of inefficient virus maturation at the last stage of outer capsid formation.

Étudede la réponse immunitaire humorale au «vaccin rabique purifié pasteur après traitement post-exposition: Study of the humoral immune response 023 to the «pasteur purified rabies vaccine in post-exposure treatment

Summary The purified inactivated Pasteur rabies vaccine was prepared on primary culture of foetal bovine kidney cells using the Pasteur fixed rabies virus (PV). A clinical trial in a post-exposure situation was carried out on a group of 130 volunteers. The vaccine was administered according to the WHO scheme for vaccines prepared on human diploid cells: 4 injections of 1.0 ml on days 0, 3, 7 and 14, followed by booster doses on days 30 and 90. This induced the production of neutralizing antibodies, detected from day 14. Neither local nor systemic adverse reactions of any importance were registered.

Characterisation of a polyomavirus in two foetal rhesus monkey kidney cell lines used for the growth of hepatitis A virus.

Electron microscopy and prolonged incubation of cell cultures have revealed the presence of a papovavirus in two foetal rhesus monkey kidney cell lines, FRhK-4 and FRhK-6, which are used to grow hepatitis A virus. The papovavirus, designated FRKV, was present in culture fluids from both cell lines and in thin sections of FRhK-4 cells. The size of the virus, 47 nm, places FRKV within the Polyomavirus genus. FRKV has been grown in primary human embryo kidney and calf kidney cell cultures. Haemagglutinin has not been demonstrated. Preliminary investigations indicate that FRKV is antigenically identical to or closely related to stump-tailed macaque virus and is not one of the primate polyomaviruses SV40, SA12, BK or JC. FRKV reacts with antibody that occurs in pools of foetal and newborn bovine sera suggesting that the virus might be of bovine origin.

Analysis of small and large plaque variants of equine herpesvirus type 3 by restriction endonucleases

Six of 6 equine herpesvirus type 3 (EHV3) isolates, 5 of which were epidemiologically unrelated, produced a mixture of small and large plaque variants in equine foetal kidney cells under methylcellulose. In 4 of 4 instances the cleavage site(s) generating the Bam HI A fragment of large plaque variants was distinct from the site(s) for the same fragment of small plaque variants.

Translational order of bovine leukemia virus gag and env gene-coded proteins

The translational orders of bovine leukemia virus (BLV) gag proteins and env proteins have been determined by the technique of pactamycin mapping. BLV-producing fetal lamb kidney (FLK) cells were pulse-labeled with 14C amino acids and [ 3H]glucosamine in the presence or absence of pactamycin which inhibits initiation of protein synthesis but not elongation. Viruses were harvested from the cell culture medium and viral proteins were analyzed by SDS-polyacrylamide gel electrophoresis. For each protein the “pactamycin ratio” was determined. The lower this ratio was for a given protein, the closer the protein would be to the amino terminus of the precursor polyprotein. From these experiments the following translational orders were derived: gag, NH 2-p10-(p15 2·p15 1)-p24-p12-COOH; and env, NH 2-gp64-gp30-COOH. Investigation of isoelectric points of BLV proteins, a conserved property of retroviral proteins, supported the translational order of gag gene-coded proteins.

Induction of lymphosarcoma in sheep inoculated with bovine leukaemia virus

Five sheep were experimentally inoculated with BLV in order to study the humoral immune response in animals infected with bovine leukaemia virus (BLV). During experimental periods of 46 months, 2 sheep died with leukaemia and one sheep showed splenomegaly and proliferation of tumour cells. The other 2 sheep were clinically normal. All of the inoculated sheep developed antiviral antibodies 1 month after inoculation and BLV could be re-isolated in lymphocytes 2 to 3 months after inoculation. Antibody against glycoprotein antigen (gp51) of BLV appeared earlier than the antibody against protein antigen (p24) and antibody titres of the former were higher than those of the latter during the course of the experiment. The complement dependent antibody cytotoxicity test was performed for the detection of antibody against BLV-related cell membrane antigen with 2 different kinds of target cells; FLK cells which are foetal lamb kidney cells chronically infected with BLV and SF-28 cells which are sheep fibroblasts transformed with BLV in vitro. All 5 sheep developed cytotoxic antibodies against both types of cells. In sera from two leukaemic sheep, cytotoxic antibody titres against SF-28 cells gradually decreased 30 months after inoculation and finally became negative one to 3 months before they died of leukaemia. However, these leukaemic sheep persistently produced antibodies against gp51 and p24.

Suppression of natural cytotoxic activity of lymphocytes from cattle and sheep during the progress of bovine leukosis

Peripheral blood lymphocytes (PBL) from cattle and sheep exhibited natural cytotoxicity on a fetal lamb kidney (FLK) cell line that was persistently infected with bovine leukemia virus (BLV) by 20-h 51Cr release assay. This cytotoxic activity was tested using PBL from normal cattle and sheep or BLV-infected animals. Although cytotoxic activity was also found in PBL from normal animals and from BLV-infected, but clinically healthy animals, the activity in PBL from animals with persistent lymphocytosis or leukemic animals was markedly decreased. The cytotoxic activity of PBL from 3 sheep sequentially tested before and during the disease was found to decrease gradually with the progress of the disease, and finally no cytotoxic activity was found at the time of death due to leukemia. These results suggest that the natural cytotoxic activity in PBL may have a role in immunosurveillance for progress of the tumor.

Characterisation of a polyomavirus in two foetal rhesus monkey kidney cell lines used for the growth of hepatitis A virus

Characterisation of a polyomavirus in two foetal rhesus monkey kidney cell lines used for the growth of hepatitis A virus

A microcarrier cell culture system for large scale production of hepatitis a virus

Hepatitis A virus (HAV) was isolated from human faeces using a fetal rhesus monkey kidney cell line (Frhk-4). Infectious medium from passage 12 was used to inoculate a large (5000 cm 2) microcarrier cell culture maintained in suspension. The microcarriers used were swollen, collagen-coated dextran beads on which it was easy to propagate Frhk-4 cells. Intra- and extra-cellular virus levels were assayed and compared with conventional cultures in 25 cm 2 plastic flasks. The results show that virus production per cell was similar in both systems. The number of cells per area unit in confluent cultures was initially lower in the microcarrier culture but subsequently increased. Two to three weeks post inoculation the virus yield per area unit in the microcarrier system was half of that of the conventional culture. The lower cell density per area unit in the microcarrier system was compensated by the large growth area that could be maintained in a single vessel and the total production of virus was substantial. Weekly harvests of medium with HAV antigen titres around 10 −2 contained antigenic material sufficient for several thousands of anti-HAV IgM tests. Propagation of HAV in microcarrier cell cultures thus seems a safe and simple way to produce large amounts of HAV.

Toxoplasma gondii: Growth in ovine fetal kidney cell cultures

Serial, in vitro passage of Toxoplasma gondii (Rh strain) was successfully performed in a cell line derived from ovine fetal kidney cells. Invasion of this parasite into the kidney cells was easily discernible 1 hr after inoculation. The subsequent proliferation of the parasite was followed in the cytoplasm of the kidney cells. Very active endodyogeny and rosette formations, as many as 13 in a cell, were observed in the cytoplasm of the kidney cells 48 hr postinoculation. After 96 hr of incubation, the parasite population had increased about 132-fold. The virulence of T. gondii against mice was not attenuated after 2 years of in vitro growth which represented 100 serial passages through the kidney cell cultures. Although no “exotoxin” was produced by T. gondii grown in vitro, a Toxoplasma sp. agar gel immunodiffusion test antigen was isolated from the cell-free supernatant fluid of the kidney cell cultures which was identical to an antigen isolated from “toxogenic” organisms harvested from infected mice.

Isolation of a rotavirus from canine diarrheal feces.

Two samples of 60 canine diarrheal feces were positive by a reverse passive hemagglutination (R-PHA) test kit of human rotaviruses. An agent was isolated in a fetal rhesus monkey kidney (MA 104) cell culture from the sample with higher R-PHA titer, and it was identified as a rotavirus based on morphologic features by electron microscopy (EM) and common rotaviral antigen detected by immune EM and immunofluorescent test with an immune serum against a bovine rotavirus.

Evidence for a bovine origin of the polyomavirus detected in foetal rhesus monkey kidney cells, FRhK-4 and -6.

Rabbit antisera to the stump-tailed macaque polyomavirus (STMV) which had been shown by immunoelectron microscopy and indirect immunofluorescence to react with the polyomavirus found in FRhK-4 cells (FRKV), also gave precipitin lines in counter-immunoelectrophoresis (CIE) and double diffusion in gel (GD) when reacted with FRKV. The reactions in GD showed identity with that of a rabbit antiserum to FRKV. Naturally occurring antibody to FRKV (anti-FRKV) was found by CIE in 48 per cent of 353 cattle, 1/106 pigs and 1/20 goats but not in any of 13 other species including 45 rhesus monkeys and 97 humans. Each of 9 anti-FRKV positive samples from cattle, the goat serum, but not the pig serum gave a line of identity with the rabbit antiserum to FRKV in GD against FRKV. Detection of anti-FRKV in colostrum deprived newborn calves and in commercial foetal calf sera (FBS) indicates that intra-uterine infection of cattle with FRKV may occur. FRKV adapted readily to growth in secondary calf kidney cultures and grew more rapidly and to higher titres than in the FRhK-4 cultures. We conclude that FRKV is probably another strain of STMV and that the natural hosts of these viruses are cattle and not primates. Evidence of intra-uterine infection of cattle implies that infectious FRKV may be present in some FBS and may thus have gained entry into various susceptible cell lines, particularly primate kidney.

The role of calmodulin in human renal cell carcinoma

Calmodulin is a ubiquitous intracellular calcium binding protein which has been shown to be associated with cell cycling. Previous studies using animal tumor models have suggested a positive correlation between tumor calmodulin content and rate of tumor growth. We studied the role of calmodulin in renal cell carcinoma (RCC) cell lines and compared this with short term normal fetal kidney cell lines. The effects of calmodulin inhibition was determined using the calmodulin inhibitor W13 (Naphthalene-sulfonamide) and its less active partner W12. Cell size, calmodulin content and inhibition studies using W13 did not reveal any simple correlations for the RCC cell lines, although the RCC lines did have a higher content than the fetal kidney cell lines. Calmodulin content determination of RCC and normal adult kidney tissue failed to show any difference. We conclude that, contrary to previous reports using animal models, there is no simple relationship between tumor growth rates and calmodulin content for human RCC.

Primary isolation of cytopathic bovine rotaviruses on fetal rhesus monkey kidney cells

Primary isolation of bovine rotaviruses was successfully performed on rolling cultures of MA104 cells following trypsin treatment of fecal samples and cells. Fifty-one fecal samples were obtained from 22 herds affected with naturally-occurring acute diarrhea in calves during a period of over two years. Rotavirus particles were demonstrated in only 10 fecal samples by electron microscopy. Fourteen cytopathic bovine rotaviruses were isolated from positive samples and could be serially cultivated on MA104 cells. The presence of virus was identified by specific immunofluorescence in infected cells. These data indicated that approximately 30% of the herds affected with acute diarrhea in their calves were associated with rotavirus infection.

Isolation of human rotavirus subgroups 1 and 2 in cell culture.

One strain of human rotavirus subgroup 1 (KUN) and one strain of subgroup 2 (MO) were isolated with the MA104 cell line, a fetal rhesus monkey kidney cell line. Their subgroup specificities and RNA patterns were identical to those of rotaviruses present in stools before cultivation. Distinct cytopathic effects consisting of obscure cell boundaries, cell fusion, cell rounding, cell detachment, and lytic foci were recognized at passage 3 of MO and passage 6 of KUN. No differences in cytopathic changes were found between the two isolates.

Sensitivity of bovid herpesvirus 2 replication to temperatures found in the natural host.

Two isolates of bovid herpesvirus 2 replicated poorly in bovine testicular cells and fetal kidney cells at 39--40 degrees C, temperatures commonly observed in virus-infected cattle. High viral titers occurred in replicate cultures at 30--37 degrees C. Persistent viral infections were noted in cultures maintained at 40 degrees C. Interferon-like activity was not responsible for the high-temperature restriction since the level was not significantly different between virus-infected cultures incubated at 35 degrees or 40 degrees C. Spontaneous viral inactivation was only three times as rapid at 41 degrees as at 35 degrees C. Analysis of temperature shift experiments with respect to results of growth studies and electron microscopy indicated a temperature sensitive event late in replication. The sensitivity of bovid herpesvirus 2 replication to elevated temperature provides a possible explanation for the extensive viral growth and occasionally severe lesions which are limited exclusively to the skin of infected cattle.

Studies on the multiplication of a porcine parvovirus

A porcine parvovirus has been characterized with regard to its replication in foetal porcine kidney cells and certain biophysical properties. Electron microscopy of infected cells at selected times postinfection revealed that porcine parvovirus replication took place within or near a series of granular intranuclear inclusions which may be contiguous with cellular heterochromatin. Developing virions were observed to aggregate into a nuclear-like amorphous mass which gradually disrupted as cellular integrity was lost. Purified virions were found to have a buoyant density in CsCl of 1.38 g/ml, while ‘empty’ particles has a buoyant density of 1.29 g/ml. The particle diameter was calculated to be approximately 22 nm.

Hepatitis A virus in cell culture. II. Growth characteristics of hepatitis A virus in Frhk-4/R cells.

The propagation of hepatitis A virus (HAV) is shown in a rapidly growing fetal rhesus monkey kidney cell line (Frhk-4/R). In the course of ten passages through Frhk-4/R cells the HAV, which was released in the supernatants of the cell cultures, was adapted to these cells. In the tenth passage 6 days after infection, it was possible to detect low amounts of HAV in the supernatants by radioimmunoassay (RIA). With indirect immunofluorescence 3 days after infection, a specific granular fluorescence was demonstrated in the cytoplasm of cells infected with HAV from the tenth passage. A persistent infection of Frhk-4/R cells appeared with a main peak at a density of 1.32 g/cm(3) in CsCl density gradients. In the 1.32 g/cm(3) fractions, typical HAV particles are shown by electron microscopy. Furthermore the HAV produced in Frhk-4/R cells is shown to be a useful antigen for diagnostic tests.

Enzootic bovine leukosis: Preparation of an antigen for the agar gel immunodiffusion test and statistical comparison with other antigens

The preparation of an antigen for the agar gel immunodiffusion test prepared by ultrafiltration of cell culture supernatant (chronically infected fetal lamb kidney cells) is described. The antigen obtained was compared by the single radial immunodiffusion test with an antigen prepared by ammonium sulfate precipitation (30%) and the commercial antigens (Pitman Moore, Seromed). The positive serum was used either undiluted or diluted 1:16 to test for the internal (P 24) and the external antigens (Gp 60) respectively. The results obtained indicated that the antigen prepared by ultrafiltration appeared to be much superior to the others, and contained Gp 60 which detects the commonly found antibodies in bovine leukosis virus-infected cattle.