Plaque assay of Sendai virus in monolayers of a clonal line of porcine kidney cells.

The MN strain of Sendai virus formed distinct plaques in monolayers of PS-Y15 cells, an established porcine kidney cell line. The plaque-forming ability was neutralized by specific antibody to the virus. A linear relationship was found between the concentration of virus and the number of plaques. The sensitivity of this assay was about equal to that of the in ovo titration. When applied to the serum neutralization test, the end points obtained were comparable to those of the hemagglutination-inhibition and complement-fixation tests.     

Hyperpolarizing membrane potential changes in a cloned monkey kidney cell line

Electrophysiological properties of a cloned monkey kidney cell line, JTC-12, were studied. The mean resting potential and input resistance were –15.3 mV and 78 M, respectively. Spontaneous hyperpolarizations with increased membrane conductance were observed. Similar hyperpolarization could be elicited by mechanical and electrical stimulations. The mean reversal potential of these hyperpolarizations was –72.7 mV. Hyperpolarization could be also elicited in a chloride-free solution. These data indicate that: (1) JTC-12 cells exhibit spontaneous and induced hyperpolarizations, and (2) occurrence of hyperpolarization is related to an increase in membrane permeability to potassium ions.     

Plaque assay and improved yield of human coronaviruses in a human rhabdomyosarcoma cell line.

Propagation and plaque assay of human coronavirus prototypes were studied in two human cell lines: a diploid fetal tonsil (FT) and a heteroploid rhabdomyosarcoma (RD) cell lines. Plaques, observed within 2 to 3 days on FT cell monolayers with both 229E and OC43 viruses, appeared as colorless areas after staining with neutral red or crystal violet, whereas neutral red staining was required for visualization of plaques on RD cells. The plating efficiencies were approximately equal between the two cell lines, but virus assay by plaque formation was 15- to 30-fold more efficient than tube dilution assay with 50% endpoints. The discrepancy between 50% endpoint and plaque-forming unit values was striking and appeared to result from the fact that killing of cells (particularly RD cells) by coronaviruses was not accompanied by visible changes in the cells but killing was detected by the failure of infected cells to stain with a vital dye. The latent phase in one-step growth curves was 5 to 6 h for both viruses in either cell line, but the maximum yield of intracellular virus was reached in 18 to 20 h for FT cells and 24 to 28 h for RD cells. Virus release also differed between the two cell lines: in FT cells, the maximum yield of extracellular virus was reached 2 to 3 h later than that of intracellular virus, whereas in RD cells, the difference was 5 h for 229E virus and 10 h for OC43 virus. Although both cell lines appear equally useful for plaque assay, RD cells would be preferred for mass virus propagation because yields (5 X 10(8) plaque-forming units per ml) were 10-fold higher than in FT cells, a finding true for both virus prototypes.     

Morphogenesis of fowlpox virus in a baby hamster kidney cell line

Fowlpox virus (FWPV) recombinant vaccines are presently being tested as an antihuman immunodeficiency virus vaccine for humans. However, biosafety, as well as the morphogenesis of FWPV in mammalian cells, are not well understood. Currently, electron microscopy is the method of choice for analyzing virus morphogenesis in cell lines. In this study, four different electron microscopic techniques were used to study FWPV morphogenesis in the Syrian baby hamster kidney (BHK-21) cell line: direct negative stain electron microscopy, ultrathin section transmission electron microscopy, cryoimmunoelectron microscopy, and scanning electron microscopy. The study showed matured viruses, as well as other stages of fowlpox virus maturation, in BHK-21 cells that led to productive virus multiplication. A number of virus-containing vesicles and plasma membrane-associated mature viruses at an early stage in the budding process were observed. In addition, intracellular mature virus was observed in layers of the trans-Golgi network, a characteristic of intracellular mature virus wrapping that results in the formation of intracellular enveloped virus. The size and morphology of FWPV observed in this study are comparable with previously published data. This study presents the first morphological evidence for the release of FWPV by budding in BHK-21 cells.This work was presented in part as a late-breaking poster at the Microscopy and Microanalysis Congress in Quebec City, Canada, Aug. 4–8, 2002     

Plaque assay for avian metapneumovirus using a Japanese quail fibrosarcoma cell line (QT-35)

A plaque assay for detecting, isolating and titrating avian pneumoviruses using a Japanese quail fibrosarcoma cell line (QT-35) is described. Plaques are produced after application of either an agarose or carboxymethyl-cellulose (CMC) overlay onto cell monolayers infected with representative avian metapneumoviruses which belong to subgroup A, B or C. Virus plaques can be easily visualized by light microscopy or after staining. The parameters affecting plaque appearance include: cell seeding concentration, virus strain, overlay composition and incubation time following infection. Optimal conditions for plaquing involve seeding QT-35 cells at 40,000 cells per cm(2) when using a 1.5% CMC overlay or 100,000 cells per cm(2) when using a 1.0 or 0.8% agarose overlay. In both cases, cell monolayers are infected with virus 24 h post-seeding and clearly visible plaques develop in 6 days. Due to the robust nature of these cells, the incubation time can be extended to a maximum of 13 days after infection in order to produce larger plaques.     

Enhancement of human rotavirus infectivity in a monkey kidney cell line by human expressed breast milk

Eight of forty (20%) human expressed breast milks enhanced the infectivity of human rotavirus (HRV) when assayed by immunofluorescence in a monkey kidney cell line (LLC-MK2). This enhancement was demonstrated with two HRV strains, one derived from a fecal specimen of a child with acute gastroenteritis, the other a tissue culture adapted strain. The phenomenon could have important implications in the pathogenesis of HRV infections and in future vaccine programs.     

Comparison of primary rhesus and cynomolgus monkey kidney cell cultures for viral isolation from clinical specimens.

Rhesus monkey kidney and cynomolgus monkey kidney cell cultures were compared for viral isolation by using clinical specimens that yielded 203 viral isolates. Cynomolgus and rhesus monkey kidney cells were comparable for the isolation of 22 adenoviruses, 12 coxsackieviruses, and one poliovirus. Four of 50 echoviruses and seven of ten herpesviruses were detected only in cynomolgus monkey kidney cells. Influenza virus was isolated in 84 instances, of which eight were detected only in rhesus and four only in cynomolgus monkey kidney cells. Rhesus monkey kidney cells yielded six more parainfluenza virus isolates. Except possibly for parainfluenza virus, cynomolgus monkey kidney cells appear to be as sensitive as rhesus monkey kidney cells for viral isolation from clinical specimens.     

Commercial simian virus antisera that inhibit virus replication in primary monkey kidney cell cultures.

The incorporation of hyperimmune serum into cell culture medium to control endogenous viral infections of primary cells can have a significant effect on the replication of other viruses. When commercial simian virus 5 or simian virus 40 antiserum was used with primary monkey kidney cell cultures, we found a significant inhibition (greater than 90%) of the replication of parainfluenza virus types 2 and 3 and reovirus type 1. In the viral diagnostic laboratory, the use of hyperimmune serum with primary monkey kidney cells may result in failure to isolate certain viruses if these cells are not first washed free of hyperimmune serum.     

Response of tissue-cultured cynomolgus monkey kidney cells to botulinum C2 toxin

C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two nonlinked protein components, designated components I and II. The toxin, a mixture of untrypsinized component I and trypsinized component II, induced marked morphological changes of tissue-cultured cynomolgus monkey kidney cells; the characteristic response of the cells to the toxin was rounding, which increased proportionally to log dose of the toxin. The components alone and a combination of untrypsinized components I and II showed little activity. The rounding of the cultured cells was not accompanied by inhibition of protein and nucleic acid syntheses of the cells, although the rounded cells ultimately lost viability. Immunofluorescence studies showed that component II, either trypsinized or untrypsinized, bound to the cell surface, whereas component I bound to the cells only in the presence of trypsinized component II. The present results support the previously proposed idea concerning the mode of action of C2T, that components I and II of C2T act together as a molecule with dual functions; component II as the recognizer of the receptor site on the cell surface membranes and component I as the effector in the cytoplasm by preferential inactivation of cytoskeletal actin, which results in alteration of cell morphology, and subsequently in cellular damage.     

Spontaneous contamination of primary green monkey kidney cell cultures by foamy virus

Examinations of 1653 batches of green monkey kidney cell cultures revealed contamination with foamy virus (FV) in 243 (16%) batches. From some of these cultures 65 strains of virus belonging to two serotypes were isolated. Tests on sera from 1122 monkeys revealed antibody to FV in 985 (87.8%) animals. No correlation between the presence of antibody in the serum and virus recovery from monkey kidney cell cultures was observed, however. This might be due either to the lack of the virus in the kidneys or to the failure in its isolation from primary cultures. Passages of cell cultures were shown to facilitate additional recovery of FV. These experimental results may be used for screening and selection of FV-free cultures and for control purposes in the course of vaccine manufacture.