Dengue Virus Plaque Formation on Microplate Cultures and Its Application to Virus Neutralization

Abstract

Discussion and Summary

Dengue virus plaque formation on BHK-21 cell micro-plate cultures was described. The clear plaques were visible usually 5 days after incubation in a CO₂ incubator at 37°. The cells cultured in a 3-oz bottle were sufficient to prepare two microculture plates which were usually ready for use after 1-2 days of cultivation in the CO₂ incubator at 37°. The overall procedures were easy and of economic advantage.

It is to be stated in this connection that the affinity of dengue viruses to tissue culture cells is not necessarily high, so that the cell culture systems suitable for dengue virus plaquing have so far been limited; and even in such suitable systems the formation of clear plaques takes a much longer time of incubation than for other kinds of arbo-viruses in general. Some of the difficulties regarding this matter have been overcome by the techniques reported here.

By use of the microplate cultures, in combination with the transfer-plates reported by previous investigators (8), dengue neutralization tests were performed. Technical specifics, such as dilution of serum, transfer of virus-serum mixtures, etc., could be defined with good reproducibility. A sigmoid curve relationship was revealed between the decrease in virus titer and reciprocals of antiserum concentration. Within certain grades of the serum dilution, the same relationship was linear. This is in general accordance with data obtained by other investigators (10) dealing with dengue virus plaques formed in bottle cultures. In our experiments in which the NT titers of particular serum samples measured by this method were compared with the HI titers of the same samples determined by the standard method (9), both values paralleled well with each other, indicating the compatibility of the former with the latter.