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Wallis, Craig (Baylor University College of Medicine, Houston, Tex.), Fred Morales, Joycelyn Powell, and Joseph L. Melnick. Plaque enhancement of enteroviruses by magnesium chloride, cysteine, and pancreatin. J. Bacteriol. 91:1932-1935. 1966.-Plaque formation of 21 echoviruses (types 1-6, 9, 13, 15-19, 23-26, 29-32) and 8 coxsackieviruses (B1-6, A7, and A9) was enhanced by increased concentrations of MgCl(2), l-cysteine, and pancreatin in agar overlay medium. In most cases, cationic and anionic polymers (diethylaminoethyl dextran, dextran sulfate, or protamine sulfate) were ineffective. All strains of poliovirus and group B coxsackieviruses were enhanced under agar by MgCl(2). Five of the eight coxsackieviruses tested were also enhanced by cysteine or pancreatin. Certain enteroviruses, which have been difficult to assay by plaque method, can now be quantified effectively by incorporation of additives such as MgCl(2), cysteine, or pancreatin into the overlay medium.

The use of protease inhibitors causes the accumulation of very large polypeptides (polyprotein) in tissue culture cells infected with either poliovirus or echovirus 12. The effectiveness of the inhibitor varies, depending on the cell line chosen. In infected monkey kidney cells, polyprotein is not cleaved when a chymotrypsin inhibitor is added, but in infected HeLa cells a trypsin inhibitor is most effective. Therefore, at least a part of the proteolytic activity is supplied by the host cell. Extracted viral polyprotein can be cleaved in vitro by trypsin or chymotrypsin. As estimated by migration in sodium dodecyl sulfate gels and antigenicity, chymotrypsin cleavage of the poliovirus polyprotein yields fragments which are similar to the in vivo product. The polyprotein is not in soluble form but is attached to a fast-sedimenting, membrane-bound structure. Proteolytic activities in cell extracts were assayed using polyprotein as substrate, and infected and uninfected extracts produced qualitatively dissimilar cleavages.