Schwann cells from neonatal rat sciatic nerve can be maintained and grown in culture in the absence of neurons. We are interested in substantially expanding such cultures for use in the study of Schwann cells, their growth responses, and their interactions with neurons. However, it was important to determine if expanded cell populations retained their distinguishing biological properties and their ability to differentiate when recombined with neurons. Therefore, we have compared the functional properties of extensively expanded populations of sciatic nerve Schwann cells to those of embryonic dorsal root ganglion (DRG) Schwann cells that had been briefly expanded in vitro in the continuous presence of ganglion neurons. Sciatic nerve Schwann cells were cultured and purified according to the methods of Brockes et al. (1979). A combination of crude glial growth factor and forskolin was found to act synergistically in providing maximal stimulation of Schwann cell proliferation. Sciatic nerve Schwann cells that were continuously expanded for at least 2 months were compared to Schwann cells derived from fetal dorsal root ganglia. The results indicate that the complement of secreted proteins from both cell populations, either in isolation or recombined with neurons, was essentially identical; both cell populations expressed the cell-surface antigens laminin and Ran 1 (217C antibody); after seeding onto DRG neurons, both cell populations associated with neuronal processes with the same time course; and under identical nutrient conditions, both cell populations were observed to exhibit a comparable capacity for myelination of DRG axons in vitro. Thus, methods used to establish primary cultures of rat sciatic nerve Schwann cells and to expand secondary cultures in vitro in the absence of neurons preserve basic Schwann cell functions.

The infectivity of most rotaviruses is enhanced by treatment with trypsin. We studied the mechanism of enhancement by examining the effect of trypsin on rotavirus infectivity, aggregation, early interactions with host cells, and structure. The results indicated that trypsin does not increase levels of infectious virus by dispersion of aggregates or affect the efficiency or rate of attachment of virus to cells. A fraction of virus that was not infectious without trypsin treatment was found to attach to cells, but did not initiate antigen synthesis. When cells were infected with labeled, purified virus, increased levels of uncoated particles were found in cells infected with trypsin-treated virus. Infection of cells with trypsintreated virus also led to greater levels of RNA synthesis early in the infection. The results suggest that trypsin converts a noninfectious fraction of virus into infectious virus by allowing this fraction to uncoat in the infected cell. Trypsin was found to cleave an 88,000-dalton structural polypeptide of bovine rotavirus generating 67,000-and 20,000-dalton cleavage products.

The infectivity of a bovine rotavirus was enhanced 140-, 8-, and 3-fold, respectively, by trypsin, protease, and lactase. Ficin, carboxypeptidases A and B, lysozyme, and ,B-galactosidase had little effect on the infectivity. Chymotrypsin caused a threefold decrease in the infectivity. Trypsin acts directly on the rotavirus and not on the host cell. Previous enzyme studies (9, 13-15) with reoviruses have shown the following: (i) the infectivity of reoviruses can be enhanced by at least eight different proteolytic enzymes; (ii) there are strains of types 1 and 3 reoviruses (called CTstrains) that are inactivated by chymotrypsin; (iii) the infectivity ofthe CTstrains is enhanced by chymotrypsin if ethylenediaminetetraacetic acid (EDTA) is added to the reaction mixture or if the virus is treated in the absence of calcium; and (iv) the enzyme enhances the viral infectivity by removing the outer viral coat. This study was conducted to determine the effect of enzymes on the reo-like rotaviruses. Unlike the reoviruses, the bovine rotavirus was not enhanced by chymotrypsin or ficin. The slight (threeto sixfold) inactivation of rotavirus by chymotrypsin was not prevented by EDTA. On the contrary, EDTA in the absence of enzyme inactivates the virus. Work in progress