William F. Scherer

Preservation at Subzero Temperatures of Mouse Fibroblasts (Strain L) and Human Epithelial Cells (Strain HeLa).

Summary Mouse fibroblasts of strain L (Earle) and human epithelial cells of strain HeLa (Gey) have been preserved successfully at −60° to −70°C for 6 and 7 months, respectively. Glycerol in the suspending liquid greatly increased the percentage of surviving cells. Solutions of glycerol (5% for strain L and 20% or 30% for strain HeLa) in a solution for maintenance of animal cells (MS), in balanced salt solution, or in serum (horse serum for strain L cells, adult human serum for strain HeLa cells), were of approximately equal value for the periods of preservation studied. The cells survived storage poorly at −60° to −70°C when suspended in undiluted serum or in 5% dextrose in water. Strain L cells were preserved in slightly greater numbers after freezing over a period of 1–1.5 hours than after freezing in 3-5 minutes; strain HeLa cells survived both freezing procedures equally well. Better preservation of both cell types resulted from storage at −60° to −70°C than at −20°C, and from thawing in 1–2 min. rather than in 30–60 minutes. The value of low temperature preservation for handling and maintenance of strain L and strain HeLa cells and for preservation of strain stability is discussed.

The effect of host age, virus dose and route of inoculation on inapparent infection in mice with Japanese encephalitis virus.

Summary The occurrence of inapparent infection or death due to Japanese encephalitis virus has been related to the dose and route of inoculation of virus in young and old mice. The virus strain employed, which had been isolated from naturally infected mosquitoes and used in low passage, predictably produced death on intracranial inoculation at high dilutions with only a rare mouse surviving to develop antibody. In contrast, virus inoculated intraperitoneally, subcutaneously, intravenously, or intradermally produced only occasional deaths and usually at low virus dilutions; yet very small inocula provoked hem-agglutination-inhibiting antibody responses which correlated well with neutralizing and complement-fixing antibodies. Intravenous administration of 108 weanling mouse IC LD50 of virus consistently produced about 75% mortality; intranasal injection of virus killed mice, whereas duodenal inoculation (during laparotomy) or pharyngeal administration did not. A single previous inapparent infection of mice inoculated into the duodenum conferred immunity to intravenous virus challenge 46-54 days later. Six-month-old mice were more resistant than young mice to peripheral as well as intracranial inoculation although infectivity was about the same in both groups. The mechanisms involved in the determination of apparency of infection which can be correlated with host age, virus dose, and route of inoculation have yet to be learned.

Propagation of poliomyelitis virus in cultures of monkey and human testicular tissues.

Summary In experiments in which monkey or human testicular cells in tissue culture were infected with poliomyelitis virus, Lansing and Yale-SK strains, proof was obtained that the virus in 3 passage series had been propagated. At the time this report was written it had been established for these 3 passage series that the minimal dilution factor based on tissue replacements ranged from 1014.6 to 1018 and, when assessed by fluid replacement, from 1029 to 1036. The LD50 of each strain of virus was determined on successive transfers, and the identity of each strain of virus was established by neutralization tests and histopathological findings in monkeys dead from the injection of tissue culture virus. Control experiments and other tests made known that propagation of poliomyelitis virus did not occur in the absence of viable testicular cells and that an extraneous virus was not inadvertently acquired during the course of these studies. It is plain that the results of the present investigation substantiate the observations of other workers that poliomyelitis virus can propagate in extraneural tissues. From these findings it is apparent that the assumption of an obligate neurotropism for poliomyelitis virus no longer is tenable. This observation and the demonstration regularly of the natural occurrence in poliomyelitis of virus in the oro-intestinal tract emphasize the need for a renewal of investigative studies of the factors that make possible invasion of the central nervous system to result in the overt and residual manifestations of poliomyelitis.

THE APPLICATION OF MAMMALIAN CELLS IN CONTINUOUS CULTURE FOR ASSAYS IN VIROLOGY

Summary

Agglutination of a Pure Strain of Mammalian Cells (L Strain, Earle) by Suspensions of Vaccinia Virus.∗:

Summary Mouse “fibroblastic” cells from a pure strain of cells in vitro (L strain, Earle) were found to be agglutinated by suspensions of vaccinia virus, but not by the viruses of mumps and influenza A under the experimental conditions employed. The agglutination of L strain cells by suspensions of vaccinia virus was inhibited by vaccinial antibodies. The results of these experiments show that cellular agglutination by suspensions of vaccinia virus is not limited to erythrocytes.

Porcine Kidney Cell Cultures for Propagation and Assay of Japanese Encephalitis Virus.

Summary Cells cultured in vitro from porcine kidneys support growth of JE virus and are concomitantly destroyed. Each of 5 virus strains (from pig, bird, mosquito and man) was propagated serially through 5–28 passages. Virus harvests from porcine kidney cultures titrated 105–8 ID50/ml in mice and in PK cultures. Total destruction of cells by recently isolated viruses in low mouse passage occurred usually within 2–5 days after inoculation of cultures, and could be prevented by specific antiserum.

POLYVALENT GROUP B ARBOVIRUS ANTISERUM PRODUCED IN MONKEYS.

Summary Polyvalent group B arbovirus antiserum reactive by complement-fixation test was prepared in monkeys by sequential inoculation of Japanese encephalitis (JE), yellow fever, and TP 21 (Langat) viruses. Such serum should facilitate identification of newly isolated arboviruses as members of group B. Combinations of JE, dengue 1 and TP 21 or yellow fever viruses produced sera with lesser degrees of polyvalency, and use of only JE and yellow fever viruses yielded serum with only low CF titers to a few heterologous group B arboviruses. Examination of 27 monkeys for pre-existing arboviral neutralizing antibodies revealed most to be negative; however, one rhesus monkey from India was positive to Oriboca virus and another to Bwamba virus, suggesting that these or antigenically closely related arboviruses exist in India even though they are currently recognized to occur only in South and Central America or in Africa respectively.

Transportation of Human Cells Cultured in vitro.

Summary Living human epithelial cells, strain HeLa (Gey), readily survive transportation over periods of 1–3 days. Six factors in survival, i.e., 1) nutritional status of cells at the beginning of shipment, 2) availability of nutrients in relation to nutritional needs of cells during shipment, 3) mechanical trauma to cells, 4) duration of shipment, 5) temperature near cells and 6) the method for revival of cells following shipment, were studied to define satisfactory conditions for routine shipment of strain HeLa cells.