Fetal Rhesus Monkey Kidney Cells Research Articles

Discovery of Novel Human and Animal Cells Infected by the Severe Acute Respiratory Syndrome Coronavirus by Replication-Specific Multiplex Reverse Transcription-PCR

The severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of the recent outbreak of severe acute respiratory syndrome. VeroE6 cells, fetal rhesus monkey kidney cells, and human peripheral blood mononuclear cells were the only cells known to be susceptible to SARS-CoV. We developed a multiplex reverse transcription-PCR assay to analyze the susceptibility of cells derived from a variety of tissues and species to SARS-CoV. Additionally, productive infection was determined by titration of cellular supernatants. Cells derived from three species of monkey were susceptible to SARS-CoV. However, the levels of SARS-CoV produced differed by 4 log(10). Mink lung epithelial cells (Mv1Lu) and R-Mix, a mixed monolayer of human lung-derived cells (A549) and mink lung-derived cells (Mv1Lu), are used by diagnostic laboratories to detect respiratory viruses (e.g., influenza virus); they were also infected with SARS-CoV, indicating that the practices of diagnostic laboratories should be examined to ensure appropriate biosafety precautions. Mv1Lu cells produce little SARS-CoV compared to that produced by VeroE6 cells, which indicates that they are a safer alternative for SARS-CoV diagnostics. Evaluation of cells permissive to other coronaviruses indicated that these cell types are not infected by SARS-CoV, providing additional evidence that SARS-CoV binds an alternative receptor. Analysis of human cells derived from lung, kidney, liver, and intestine led to the discovery that human cell lines were productively infected by SARS-CoV. This study identifies new cell lines that may be used for SARS-CoV diagnostics and/or basic research. Our data and other in vivo studies indicate that SARS-CoV has a wide host range, suggesting that the cellular receptor(s) utilized by SARS-CoV is highly conserved and is expressed by a variety of tissues.

Effect of ribavirin on hepatitis A virus replication in vitro.

The effect of ribavirin on fetal Rhesus monkey kidney cells (FRhK-4) acutely or chronically infected with hepatitis A virus was studied. The effect of ribavirin on hepatitis A virus yield as detected by radioimmunoassay in acutely infected FRhK-4 cells was dependent on hepatitis A virus inoculum dose. Treatment with 100 μg/mL ribavirin completely inhibited hepatitis A virus growth in cultures infected with 100 to 800 tissue culture infectious dose 50 (TCID(50)) hepatitis A virus, but inocula of 800 to 1600 TCID(50) resulted in limited production of virus. The effect was time dependent and required more than 96 h of treatment to inhibit the virus completely. Ribavirin was less effective in treating cells persistently infected with hepatitis A virus, although there was significant inhibition of hepatitis A virus (82%) in persistently infected cells as well. Ribavirin had some inhibitory effect on cell growth; treatment with 25, 50 or 100 μg/mL ribavirin reduced cell growth by approximately 0, 20 and 40%, respectively.

Adsorption, purification, and growth characteristics of hepatitis A virus strain HAS-15 propagated in fetal rhesus monkey kidney cells.

A human fecal isolate of hepatitis A virus strain HAS-15 was adapted to rapid growth in FRhK-4 cells by more than 20 7-day passages. A cell culture-derived inoculum of strain HAS-15 was used at a multiplicity of infection of 80 radioimmunofocus-forming units per cell, and a one-step growth curve was determined. Both intracellular production and supernatant release of infectious virions were evaluated. Detection of virus release into the medium directly corresponded to intracellular production of infectious virions. A classical eclipse period was not observed during the growth curve determinations; however, detectable infectious virion production was absent for approximately 20 h after infection. This 20-h period was immediately followed by a 4-day logarithmic phase of virus production. A maximum intracellular virus titer of 10(9) radioimmunofocus-forming units per ml was achieved, and this level remained essentially constant for up to 14 days after infection. The infectious virus and viral antigen produced during the growth cycle were ascertained by a radioimmunofocus assay and by a radioimmunoassay, respectively. Cell culture supernatants were negative for viral antigen as determined by the radioimmunoassay, even though as many as 10(8) hepatitis A virus radioimmunofocus-forming units per ml were found. An adsorption study was also performed with strain HAS-15 by using FRhK-4 cells. More than 99.9% of the infectious virus was adsorbed at 25 degrees C in less than 20 min.

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.

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.

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.

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.

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.

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.