Rhesus Monkey Kidney Cell Line Research Articles

POLUDANUM’S INFLUENCE ON REPRODUCTIVE AND RESPIRATORY SYNDROME, TRANSMISSIBLE GASTROENTERITIS AND AFRICAN SWINE FEVER VIRUSES IN PRIMARY AND CONTINUOUS CELL CULTURES

Today studies of the influence of interferon synthesis inducer on human and animal body is a topical issue. Interferon synthesis inducers are substances of natural or artificial origin stimulating production of body’s own interferon. One of the synthetic inducers of interferon is Poludanum. It is used for stimulation of cell immunity which is capable of preventing infection and disease development and has antiviral and immunomodulatory effect. Poludanum mainly stimulates induction of α-interferon and, to a lesser extent, β- and γ-interferon in cells and tissues which prevent virus reproduction in a cell. The paper presents results of poludanum’s influence on reproduction of some porcine viruses in primary and continuous cell cultures by induction of interferon synthesis and other cytokines lowering the level of cell infection. As a result of performed tests it was found out that there is a link between introduction of interferon inducer and change in the PRRS, TGE, ASF virus reproduction. There was detected a high level of Poludanum’s interferon inducing activity in pig testicular primary cell cultures and spleen compared to the continuous pig embryo kidney (SPEV) and rhesus monkey kidney (MARC-145) cell lines. Poludanum’s interferon inducing activity was moderate for MARC-145 cells and it was not determined for SPEV.

Quantification of Infectious Sendai Virus Using Plaque Assay.

Sendai virus (SeV) is an enveloped, single-stranded RNA virus of the family Paramyxoviridae. SeV is a useful tool to study its infectious pathomechanism in immunology and the pathomechanism of a murine model of IgA nephropathy. Virus quantification is essential not only to determine the original viral titers for an appropriate application, but also to measure the viral titers in samples from the harvests from experiments. There are mainly a couple of units/titers for Sendai viral quantification: plaque-forming units (PFU) and hemagglutination (HA) titer. Of these, we here describe a protocol for Sendai virus plaque assay to provide PFU using LLC-MK2 cells (a rhesus monkey kidney cell lines) and Guinea pig red blood cells. This traditional protocol enables us to determine Sendai virus PFU in viral stock as well as samples from your experiments.

The mRNA for GST Pi from FRHK rhesus monkey kidney cells codes for an enzyme with activity towards 1-chloro-2,4-dinitrobenzene in spite of an I68F mutation

A cDNA library was constructed from mRNA of the rhesus monkey kidney cell line, FRHK, and the cDNA sequence for an FRHK glutathione S-transferase (GST) Pi was determined using a RACE method. This represents the first full-length monkey GST Pi sequence to be cloned and determined. The similarity to the human GST Pi was found to be extensive (more than 97%), the deduced protein differing only in six amino acids (aa) positions. FRHK GST Pi was expressed in bacteria and a recombinant protein was purified which demonstrated significant activity towards the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3- para-nitrophenoxypropane. Western blots also showed significant amounts of protein, both in the FRHK cells and transformed bacteria. The FRHK GST Pi was found to contain a phenylalanine at aa position 68, a position which is otherwise invariably occupied by an isoleucine in the GST Pi, Alpha, Mu and Beta class enzymes investigated. An isoleucine in this position is thus not essential for activity in the FRHK enzyme, unlike the human GST π, where the exchange of Ile 68 to a tyrosine (Manoharan, T.H, Gulick, A.M., Puchalski, R.B., Servais, A.L., Fahl, W.E., 1992. J. Biol. Chem., 267, 18940–18945), resulted in total loss of activity. Phe 68 was mutated to Ile in the FRHK GST Pi enzyme to determine whether the wild type amino acid conferred an impaired catalytic site. The resulting mutant did not show any changes in activity towards CDNB, clearly demonstrating that isoleucine at position 68 is not essential. Thus, the first monkey GST Pi enzyme has been characterized, an enzyme with many similarities to the human forms although it differs in an otherwise conserved residue at aa position 68. This difference does not appear to affect the function of the FRHK GST Pi.

Protein C inhibitor is expressed in tubular cells of human kidney.

Protein C inhibitor (PCI) is a serpin that inhibits a number of proteases. PCI is found in urine and binds to kidney epithelial cells. To determine if kidney is a source of PCI, cDNA was produced from human kidney total RNA. Sequencing and restriction mapping showed identity between kidney and liver PCI cDNA sequences. Similar cDNAs were obtained from rhesus monkey kidney and liver RNAs. Conditioned medium from the rhesus monkey kidney cell line CCL7.1 was analyzed on immunoblots, showing a 57,000-D protein band that comigrated with human plasma PCI. Immunohistochemical staining and in situ hybridization of human kidney tissue sections showed that kidney PCI antigen and RNA were confined to tubular cells. The findings are consistent with the idea that PCI is synthesized and localized in kidney tissue where it may provide protease inhibitory activity and suggest that complexes of PCI with urokinase found in human urine may be produced locally in the kidney.

Isolation, characterization, and serial propagation of a bovine group C rotavirus in a monkey kidney cell line (MA104).

A virus (designated the Shintoku strain) which was morphologically indistinguishable from group A rotaviruses was detected in the feces of adult cows with diarrhea in Japan. The virus contained 11 segments of double-stranded RNA and had an electrophoretic migration pattern in polyacrylamide gels similar to that of other group C rotaviruses (4-3-2-2). Feces containing the bovine virus reacted with antiserum to porcine group C rotavirus (Cowden strain) but not group A or B rotaviruses in immunoelectron microscopy. The virus was adapted to serial propagation in roller tube cultures of a rhesus monkey kidney cell line (MA104) by using high concentrations of trypsin. Evidence for viral replication in MA104 cell cultures was demonstrated by immunoelectron microscopy and indirect immunofluorescence by using antiserum to porcine group C rotavirus and by electrophoretic analysis of extracted viral double-stranded RNA. A significant antibody response against the isolate was detected in convalescent-phase sera of cows which excreted the virus: no increased antibody response to bovine group A rotavirus was observed. To our knowledge, this is the first isolation of a group C rotavirus from cattle.

HT-29 cells: a new substrate for rotavirus growth

Susceptibilities of a continuous rhesus monkey kidney cell line (MA-104) and that of a human colon carcinoma cell line (HT-29) to infection by different human and animal rotavirus strains were compared. HT-29 cells appeared to be more sensitive to human rotavirus infection than MA-104 cells, whereas the latter cell line was more susceptible to animal rotavirus replication. The greater sensitivity to human rotavirus infection of HT-29 cells was confirmed by the successful, direct isolation of these viruses from faecal specimens. Human rotavirus infection of HT-29 cells was also followed by transmission electron microscopy. In ultra-thin sections, unenveloped particles of rotaviruses, representing infectious mature virions, were observed in large number. Moreover, many "double-shelled" particles were detected in negative-stained supernatants from infected cultures. Scanning electron microscopy of uninfected HT-29 cells showed that in the presence of Ca++, required for rotavirus growth, they are able to express some of the features of mature intestinal cells. In view of these results, HT-29 cells appear to be a useful in vitro model for the study of rotavirus infection.

Presumptive identification of enteroviruses with RD, HEp-2, and RMK cell lines

The limited supply of the Lim Benyesh-Melnick antiserum pools for the typing of enteroviruses has made this test inappropriate for routine use in most clinical laboratories. We studied the correlation between the enterovirus groups and the cell lines on which they displayed cytopathic effect in order to make identifications without using the neutralization test. This study indicated that a presumptive identification of the enterovirus group could be made on the basis of characteristic cytopathic effect displayed after passage into rhabdomyosarcoma (RD), HEp-2, and primary rhesus monkey kidney (RMK) cells. Echoviruses and group A coxsackieviruses could be isolated in RD and RMK cells but not HEp-2 cells. Group B coxsackievirus could be isolated in RMK and HEp-2 cells but not RD cells. Poliovirus could be isolated in all three cell lines. We recommend the use of these cell lines to make presumptive enterovirus group identifications for routine viral isolates.

Efficiency of isolation of human rotavirus in primary African Green monkey kidney cells

Out of 212 human rotavirus (HRV) containing fecal specimens, 173 (81.6%) yielded virus on first passage in primary African Green monkey kidney cells (AGMK), while additional 34 specimens, did not yield virus on first passage. However, following blind passages, 18 of the 34 yielded virus in passage levels 2–8, thus raising the overall isolation rate to 90.1%. The isolation rate of HRV strains obtained in embryonic Rhesus monkey kidney cell line (MA-104), was only 41.4%. ELISA tests performed on fluids from infected cell cultures proved to be an efficient tool to measure virus replication. No differences were encountered in the isolation rates between subgroup I and II strains, while viruses lacking the antigenic determinants of both subgroups did not grow at all. However, one of those unusual group A strains was isolated and grew well in AGMK cells. Primary AGMK and MA-104 cells supported the growth of tissue culture adapted virus most efficiently when compared with six human and primate cell types.

Isolation and Serial Propagation of Turkey Rotaviruses in a Fetal Rhesus Monkey Kidney (MA104) Cell Line

Turkey rotaviruses from the intestinal contents of poults were isolated and serially propagated in MA104 cell monolayers by a simple procedure. The initial virus isolation was done by low-speed centrifugation of the inoculum onto the monolayers, and subsequent passages were accomplished in roller-tube monolayers using trypsin-treated virus suspensions. Each of the turkey rotavirus isolates possessed the morphologic, antigenic, and genomic attributes characteristic of turkey group A rotaviruses. Attempts to isolate and serially propagate turkey rotavirus-like viruses in MA104 cell monolayers by this procedure were unsuccessful. Turkey reoviruses also did not serially propagate in MA104 cell monolayers by this procedure.

Inactivation of Hepatitis a Virus by Heat and Formaldehyde

The GBM strain of hepatitis A virus adapted to a fetal rhesus monkey kidney cell line (Frhk-4/R) was treated and inactivated at several different temperatures and with several different formaldehyde concentrations. Heat inactivation showed that no inactivation occurred after treatment for 60 minutes at 37°C. Treatment for 60 minutes at 50°C reduced infectivity by 1 log 10 step ; treatment for 60 minutes at 60°C, by 4 log 10 steps. Treatment for 30 minutes at 70°C resulted in complete inactivation of HAV. Complete inactivation by formaldehyde treatment was achieved only with a 0.35% concentration. Treatment with lower concentrations resulted in incomplete HAV inactivation.

A variant form of the 1,25-dihydroxyvitamin D3 receptor with low apparent hormone affinity in cultured monkey kidney cells (LLC-MK2). A model for tissue resistance to vitamin D.

Chandler et al. (Chandler, J.S., Chandler, S.K., Pike, J.W., and Haussler, M.R. (1984) J. Biol. Chem. 259, 2214-2222) previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) caused the induction of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) in a rhesus monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity 1,25-(OH)2D3 receptor. We have re-examined this phenomenon and report here that 24-hydroxylase induction is mediated by a receptor variant in LLC-MK2 cells with low hormone affinity. Dose response analysis showed that in contrast to LLC-PK1 (a typical receptor-positive cell line), the LLC-MK2 line was less sensitive to 1,25-(OH)2D3 by 2 orders of magnitude. Employing optimal concentrations of 1,25-(OH)2D3 for 24-hydroxylase induction in each cell type, the early time courses of this bioresponse were identical in LLC-MK2 and LLC-PK1 and were consistent with a nuclear action of hormone-receptor complexes. Moreover, the rank order of potency of vitamin D3 congeners as inducers of 24-hydroxylase activity in LLC-MK2 cells agreed well with their relative affinity for the 1,25-(OH)2D3 receptor. An examination of 1,25-(OH)2D3 receptor content via DNA-cellulose chromatography in LLC-MK2 cells incubated at ligand concentrations 10-25-fold higher than the normal 2 nM revealed a minimum of 1600 receptor-like molecules/LLC-MK2 cell. These results show that LLC-MK2 cells possess a variant receptor form with apparent low affinity for 1,25-(OH)2D3. This system should serve as a model for clinical syndromes characterized by the requirement for massive doses of vitamin D to prevent rickets.

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.

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.

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.

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.

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.

Hepatitis A-virus in cell culture: I. propagation of different hepatitis A-virus isolates in a fetal rhesus monkey kidney cell line (Frhk-4).

A fetal rhesus monkey kidney cell line (Frhk-4) was infected with different hepatitis A-virus (HAV) isolates GBG, GBM, GJA. The time-dependent absorption of the HAV isolates of Fhrk-4 cells was measured. Replication of all three isolates in these cells could be demonstrated intracellularly 8--10 weeks after infection, and release of HAV into the supernatant some 10--15 weeks after infection could be shown. The specificity of the virus determination by RIA from supernatants of HAV-infected cells from passages 1, 2, and 3 in Frhk-4 cells was shown with sera that were collected from a chimpanzee infected with MS-1 both before infection as well as during convalescence. These results were subsequently compared with sera collected from human patients before the onset of hepatitis as well as during convalescence. With immunofluorescence microscopy a cytoplasmic fluorescence could be shown in HAV-infected Frhk-4 cells and finally the release of 27 nm HAV particles into the supernatant of HAV-infected Frhk-4 cells could be demonstrated by immune electron microscopy.

Plaque assay of neonatal calf diarrhea virus and the neutralizing antibody in human sera

Neonatal calf diarrhea virus (a bovine rotavirus) formed distinct plaques in monolayers of MA-104 cells, an established macacus rhesus monkey kidney cell line, when diethylaminoethyl dextran and trypsin were included in the overlay medium. By using this plaque assay method, titration of neutralizing antibody to neonatal calf diarrhea virus was made feasible. It was demonstrated that some human sera contained neutralizing antibody to this agent.

Characterization of a long-term suspension culture of the LLC-MK2 monkey kidney cell line

The adaptation of the rhesus monkey kidney cell line LLC-MK2 to growth in continuous suspension culture is described. These cells have been grown in suspension for a total of over 22 months. The adapted cells were characterized and results indicate that they may be useful for commercial production of a number of vaccines.