Biomedical subjects
Publications and source records attributed to P C Loh.
Cytomegalovirus was isolated from cell cultures derived from 3 of 16 surgical specimens of adenocarcinomas of the colon. Virus identification was accomplished through electron microscopical, cytochemical, and immunofluorescent procedures.
The susceptibility of a variety of cell lines of different mammalian origin to cytotoxic (CT) induction by either ultraviolet light-irradiated reovirus type 2 (UVR2) or viable reovirus type 2 plus the protein synthesis inhibitor, cycloheximide, was examined. The following groups of cells were found to be susceptible to CT-induction: certain tumor cells and spontaneously transformed cell lines of human origin and certain virally and spontaneously transformed cell lines of murine origin. The following groups of cells were found to be resistant: normal human diploid cell lines, primary and continuous cell cultures of subhuman primates, primary mouse cells, normal rat kidney cells and baby hamster kidney cells. Susceptibility to CT-induction could not be related to the adsorption of virus to cells, nor to the capacity of the cell to support virus replication.
Some biological properties of the human syncytial virus have been examined. A 13-day plaque assay in whole human embryo fibroblasts (HEF) has been developed using a liquid (growth medium) overlay. The plaques were 0.7-2 mm in diameter and often showed a clear central zone with irregular edges. Pretreatment of HEF monolayers with the polycation DEAE-dextran for either 30 min or 1 h was found to enhance plaque formation by a factor of from 2- to 7-fold. The plaque assay procedure required cell cultures undergoing active cell division. Adsorption kinetics and growth cycle studies in HEF indicated a relatively long adsorption period (3 h) and a relatively prolonged latent period of 24 h. Even under optimal conditions, virus yields were low and did not exceed 1 PFU per infected cell. Like other animal syncytium-forming 'foamy' viruses, the human virus induced both intranuclear and cytoplasmic antigens detectable by immmunofluorescence and was also markedly labile to freezing and thawing.
The polycation DEAE-dextran treatment of HeLa cells was found to interfere with the production of reovirus, appearance of viral cytopathology, and the induction of cytotoxicity by UV-irradiated reovirus. The data obtained showed that while the polycation pre-treatment of cells enhanced virus adsorption to cells, its addition early during virus adsorption or 2 h after infection markedly interfered with virus production. The interference was decreasingly effective when the polycation was added during the later stages of the infectious cycle. Isopycnic CsCl buoyant density ultracentrifugation analyses of infected cytoplasmic extracts revealed that there were more subviral particles formed in untreated infected cells than there were in polycation-treated cells. In vitro uncoating studies with infected cytoplasmic extracts indicated that the polycation did not interfere with the removal of the outer capsid structure of the complete virus, and uncoating occurred only in the presence of the polycation. Electron microscopical examination revealed significantly fewer virus particles present in polycation-treated infected cells. The accumulated data strongly indicate that interference by DEAE-dextran is an early event involving viral penetration. Corollary studies using other polycations, such as polybrene (hexadimethrine bromide and poly-l-lysine, revealed that while they also enhanced virus adsorption, they did not interfere with reovirus production, appearance of viral cytopathology, and the induction of cytotoxicity by UV-irradiated reovirus.
Although IPN virus failed to multiply at 30 degrees, it replicated at 16 degrees and 22 degrees in SWT cells. At 22 degrees the viral eclipse period lasted nearly 6 hr with maximal virion titers attained by 24 hr, whereas replication at 16 degrees was much slower. The replication of the virion was inhibited by 0.05 mug/ml of AD which did not interfere with the production of reovirus. Biochemical studies revealed that cellular DNA synthesis was markedly reduced (greater than 50%) soon after infection whereas total RNA synthesis was enhanced. The period of rapid increase in RNA synthesis paralleled the exponential production of infectious virus. Viral inclusion bodies, revealed by acridine orange-staining of virus-infected cells (SWT and RGG-2) late in the infectious cycle, were found to contain single-stranded RNA on the basis of their staining characteristics and sensitivity to RNase.
A monolayer culture of fibroblast-like cells was initiated and established from a trypsin digest of Omaka fish larvae (Caranx mate). The cell culture has been subcultured 105 times over a period of 30 mo and appears to be contact-inhibited. The Omaka cells grew optimally at 27 degrees in Eagle's basal medium plus 10% fetal calf serum but failed to grow at 16 and 37 degrees. At the lower temperature the cells remained viable and began to divide when the temperature was raised to 27 degrees. Unlike certain marine fish cell lines the Omaka cell did not require a higher osmolarity medium for optimal growth. Karyological analysis at the 43rd passage revealed a chromosomal modal number of 50 consisting of 7 submetacentric pairs and 18 acrocentric pairs of chromosomes. Virological studies indicated that Omaka cells supported the replication of poikilothermic viruses such as, FV-3 and IPN viruses, but not the mammalian viruses examined. Attempts to induce interferon by the synthetic polynucleotide poly I:C were unsuccessful.