Delay of Poliovirus Plaque Formation with Use of Fresh Bovine Serum in the Growth Medium of Host Cellsd

Christine E. Smull, E. H. Ludwig
1969 Applied microbiology  
The plaque formation of poliovirus in HeLa cell monolayers was decreased up to 95% when the host cells were subcultured several times in a growth medium containing fresh bovine serum prior to their inoculation with virus. Extended incubation of infected cell monolayers indicated that the inhibition was in the form of a delay in plaque formation rather than a permanent inhibition of it. The inhibitory factor, which was found in most bovine sera, was very unstable and disappeared after storage of
more » ... ed after storage of the serum at -20 C for a few weeks or at 36 C within a few days. In HeLa-cell monolayers, the fresh serum brought about a decrease in the plaque formation of all three poliovirus types as well as that of poliovirus ribonucleic acid. The plaque formation of poliovirus in monkey heart and HEp-2-cell monolayers was decreased irregularly by the use of fresh serum in the growth medium of these cells. Speculations were made as to the possible mode of action of the bovine serum inhibitor. Chang and Geyer (3) reported higher titers of poliovirus and adenovirus when small doses of these viruses were inoculated into cell cultures nourished in a medium containing horse albumin instead of horse serum. Verna and Eylar (15) observed a decrease in the plaque formation of fibroma virus with the use of calf serum in the growth medium of the host cells. Patty and Dougherty (11) reported an inverse relationship between the number of plaques formed by footand-mouth disease virus in cell cultures and the concentration of bovine serum in the medium in which the cells were grown. Some studies concerning the plaque formation of poliovirus in HeLa-cell monolayers have shown that the infectivity was reduced greatly in certain cases. Investigation revealed that the reduction in plaque formation was due to the use of host cells which had been grown several times in a medium containing fresh bovine serum, even though the medium was removed and the host cells were washed prior to application of virus. This paper describes an investigation of this phenomenon. MATERIALS AND METHODS Cell cultures, viruses, and viral ribonucleic acid (RNA). HeLa, monkey heart, and HEp-2 cells were used. Pools of a wild-type 1 poliovirus were prepared in monkey heart cell cultures, whereas Sabin 1, 2, and 3 polioviruses were grown in HeLa cell cultures. Unless stated otherwise, all plaque studies were made with HeLa-cell monolayers and the wild-type 1 poliovirus. Poliovirus RNA was prepared by a modification of the phenol method of Gierer and Schramm (4). The methods of cell culture, preparation and assay of the virus pools, and extraction and storage of the RNA were described previously (13). Bovine sera. Samples of fresh bovine blood were obtained from an abattoir, and the sera were removed promptly by centrifugation. All sera were routinely sterilized by Seitz filtration and then stored at -20 C until used. Test for presence of inhibitory factor in fresh bovine sera. A pool of HeLa cells was prepared by trypsinization of several monolayers grown in 250-ml bottles. Half of the cells were grown in monolayer form with an LAH medium (13) containing 10%0 fresh bovine serum (stored for 2 days or less). The other half of the cells was grown in a similar manner, except that the growth medium contained 10% of a bovine serum which had been stored at -20 C for 1 month or more (control serum). In some experiments, fresh serum which had been stored at 36 C for at least 1 day was used as control serum. The cells were subcultured several times in the described media. In most cases, the cells were subcultured every 2 or 3 days and thus could be grown three times in the medium containing fresh serum within 8 to 10 days after it had been obtained. In all cases, the two groups of 449 on May 7, 2020 by guest http://aem.asm.org/ Downloaded from
doi:10.1128/aem.17.3.449-453.1969 fatcat:krhshv4yw5et5mw5birakcgpgq