Endogenous agents in primary cell cultures with special reference to latent viruses

Summary A survey study of primary cell cultures prepared from primate and nonprimate tissues has shown that viruses are commonly found as endogenous agents in these cultures. Cultures prepared from monkey tissues yielded the greatest variety of virus isolates. Almost all strain 2 guinea pig cultures contained a Herpes-like virus, and both strain 2 and Hartley animals contained C-type virus. Bovine embryo and rabbit kidney cell cultures were rarely infected with viruses. Toxoplasmas and microfilariae were also detected. Most of the endogenous viral agents were obtained by holding the cultures for long-term incubation while testing for cytopathic effect, hemadsorption, and staining with hematoxylin and eosin for virus-induced cellular inclusions. Fluorescent antibody staining and electron microscopy were found to be efficient for detection of certain types of viruses. The screening of animals by testing for the presence of neutralizing antibody was not an effective procedure in selecting virus-free animals for cell culture purposes. This study was supported in part by the Food and Drug Administration, Department of Health, Education and Welfare, Contract NIH-DBS-72-2105, and Research Grant AI 08648 from the Institute of Allergy and Infectious Diseases, National Institutes of Health. Presented at the Session in Depth on Endogenous Viruses in Cell Culture at the Twenty-fifth Annual Meeting of the Tissue Culture Association, June 1974.     

The production of nuclear polyhedrosis viruses in large-volume cell cultures

Methods were tested for growing cell lines from the fall armyworm, Spodoptera frugiperda, in roller bottle cultures. The effects of inoculum size, medium volume, and serum level were tested for effect on the cell yield. A protocol is described which gives yields of 3–5 × 10⁸ cells per bottle. Several protocols were then tested for producing the NPV of Autographa californica in this culture system and the results are described.     

Activation of tyrosinase in mouse melanoma cell cultures

Summary Tyrosinase activity increased in Cloudman S-91 mouse melanoma cell homogenates incubated at 37°C for a minimum of 8 h. Enzyme activity continued to increase for 48h at which time the maximal level of activation was observed. Activation did not occur at 4°C and did not occur in the cytosol fraction of the cell, suggesting that the response was localized to melanosomes. The activated enzyme was resistant to solubilization with the nonionic detergent, Triton X-100, and preparation of homogenates in this detergent did not inhibit the temperature-dependent activation of the melanosomal fraction of the cell. The activation process increased the V _Max of tyrosinase 10-fold and lowered the K _M by a factor of 2 as determined by the tyrosine hydroxylase assay. The increase in tyrosinase activity was detectable by three assay methods: tyrosine hydroxylation, melanin synthesis, and by tyrosine decarboxylation. The formation of melanin, however, was found to be 1/20 that of either tyrosine hydroxylation or decarboxylation, a finding which suggests that the melanin pathway may be blocked at 5,6-dihydroxyindole. The “self-activation” response could not be mimicked by incubating cell homogenates with cyclic AMP-dependent protein kinase. Activated tyrosinase could be inhibited by the addition of fresh cell extracts, a finding which suggests that tyrosinase inhibitors may be present in these cells. This investigation was supported by Public Health Service grants CA41425 and CA30393 awarded by the National Cancer Institute, Bethesda, MD and by a research grant from the Proctor and Gamble Company.     

Type C viruses of baboons: isolation from normal cell cultures

Four new type C viruses were isolated from putatively virus-negative baboon lung, kidney, and testicular cells by cocultivation with several permissive host cell lines. The baboon type C viruses are infectious for cells from various mammalian species, but do not replicate in any baboon cell lines so far tested. These viruses can be distinguished from other major classes of mammalian type C viruses, including previous isolates from primates, but are most closely related to endogenous feline viruses of the RD-114/CCC group. By immunologic criteria, viral host range, and nucleic acid hybridization studies, the baboon type C viruses are highly related to one another and represent a distinct new class of endogenous primate type C viruses.     

Insensitivity of stationary cultures of transformed mouse fibroblasts to agents stimulating DNA synthesis in normal cell cultures]

Stationary cultures of the mouse transformed cells L and S-40 sensitive to topoinhibition were found to be insensitive to the action of hyaluronidase, RNAase, and colcemid in doses known to stimulate multiplication of normal mouse fibroblasts. These cultures were still insensitive to the action of medium change and removal of a part of the monolayer.     

Why do human hepatitis viruses replicate so poorly in cell cultures?

The five viruses which classically cause hepatitis in man represent diverse families of viruses and share in common only a striking hepatotropism and substantial restrictions to replication in conventional cell cultures. Hepatitis A virus is unique among these viruses in that it is amenable to propagation in cell culture, but replication of this virus is much slower and less efficient than replication of other picornaviruses. This probably reflects less efficient cap-independent viral translation, as well as restrictions at other points in the replication cycle. We speculate that the significantly restricted replication of hepatitis viruses in cell culture reflects evolutionary forces controlling their transmission and propagation through human populations.     

Hypoxia enhances prostaglandin synthesis in renal mesangial cell cultures

In view of recent findings which suggest that renal prostaglandins mediate the effect of hypoxia on erythropoietin production, we have studied whether hypoxia is a stimulus for in vitro prostaglandin synthesis. Studies were carried out in rat renal mesangial cell cultures which produce erythropoietin in an oxygen-dependent manner. Production rates of PGE2 and in specified samples also of 6-keto-PGF1 alpha, as a measure of PGI2, and PGF2 alpha were determined by radioimmunoassay after incubation at either 20% O2 (normoxic) or 2% O2 (hypoxic) in gas permeable dishes for 24 hrs. Considerable variation in PGE2 production was noted among independent cell lines. PGE2 production appeared to be inversely correlated to the cellular density of the cultures. In addition, PGE2 production was enhanced in hypoxic cell cultures. The mean increase was 50 to 60%. PGF2 alpha and 6-keto-PGF1 alpha increased by about the same rate. These results indicate that hypoxia is a stimulus for in vitro prostaglandin production.