Rhinovirus plaque formation in WI-38 cells with methylcellulose overlay.

A sensitive, reliable plaque assay system is described for five rhinoviruses using freshly prepared methylcellulose overlay and human embryonic diploid cells. Circular plaques with irregular edges, 2 mm in size, were formed by rhinoviruses 1A, 2, 6, and 13 after 6 or 7 days of incubation. A fifth rhinovirus, 17, formed a 1- to 2-mm feather plaque after 14 days of incubation. Plaque counts of rhinoviruses 1A and 13 were not affected by varying the pH of the overlay from 6.9 to 7.5. Plaque sizes and plaque-forming unit values of high passage rhinoviruses 1A and 13 were equivalent when tested at 26, 31, or 36 C. The rhinoviruses tested were sensitive to incubation at 40 C or heating at 50 C. Enhancement of plaques was observed when Mg(++) was incorporated into agar overlays, but enhancement did not occur when Mg(++) was added to methylcellulose overlays.

Use of zonal ultracentrifuge systems for biophysical studies of rhinoviruses.

This paper reports the use of zonal ultracentrifuge techniques to conduct biophysical studies of rhinoviruses grown with WI-38 cells. Good clean-out of infectivity from rhinovirus harvests was obtained with the continuous-flow B-V and B-IX rotors. Use of the B-V rotor resulted in the successful concentration of rhinovirus infectivity and antigenicity. Additional purification was achieved by the combined use of continuous-flow centrifugation and isopycnic banding procedures. Two particle sizes were found to be associated with the virus-infected cell harvests. The infectious 22-nm particle banded in density ranges of 1.38 to 1.40 g/cm(3) in CsCl and 1.26 to 1.27 g/cm(3) in potassium citrate. The 8.0 nm capsomere was composed of 2.0 nm subunits and banded with a density of protein at 1.28 g/cm(3) in CsCl. Equivalent sedimentation coefficients of 155 or 185, depending on particle density in sucrose, were calculated from rate zonal experiments by use of the B-IV zonal rotor.

Effect of complement and viral filtration on the neutralization of respiratory syncytial virus.

The addition of 10 hemolytic units of guinea pig complement has been shown to enhance the neutralizing capacity of respiratory syncytial (RS) immune sera produced in guinea pigs and ferrets. This same immune sera, when tested without complement, had little or no neutralizing capacity. The addition of complement to RS immune horse serum did not significantly increase its neutralizing capacity. Immune horse serum effectively neutralized RS virus without complement. Other studies indicated that a 50% tissue culture infective dose of between 30 and 100 should be used in RS serum neutralization tests and that incubation should be for 90 to 105 min at room temperature. The neutralizing capacity of guinea pig immune serum was not increased by the use of filtered virus. The rate of virus neutralization, however, was increased with the addition of 10 hemolytic units of complement. The neutralizing capacity of RS immune horse serum was much greater for filtered than for unfiltered RS virus. The addition of complement increased the rate of virus neutralization but did not increase the neutralizing capacity of the horse immune serum.

Propagation of Rhinovirus on WI-38 Cell Monolayers in Rolling Bottles.

Techniques are described for the propagation of rhinoviruses on WI-38 monolayers in rolling bottles. High yields of viruses were obtained, as indicated by infectivity titers and electron microscopy. When crude harvests were subjected to low-speed centrifugation and then filtered through a 0.45-mu membrane filter, little or no loss in infectivity titer was observed. However, electron microscopic examination indicated that the concentration of viral physical particles was reduced below detectable levels after filtration. The guinea pig potency test on the lot of unfiltered rhinovirus 14 vaccine prepared in rolling bottles indicated that this vaccine stimulated higher reciprocal serum-neutralizing titers than a rhinovirus 14 vaccine prepared in stationary monolayers.