A High Titered Hemagglutinin in Tissue Culture Prepared from Japanese B Encephalitis Virus. ∗

Summary

Hemagglutinins (HA) of JBE virus prepared from infected HKC cultures were masked by serum, an essential ingredient of good nutrient medium. By removing the serum containing medium at the first sign of CPE and substituting a serum-free medium 199 at pH 8.0, until harvesting 16 hours later, a high titered HA was obtained. Reducing the amount of culture medium resulted in a further proportional increase in hemagglutinins and an HA titer of at least 1:1024 was easily obtained. Factors such as adaptation of the virus strain to tissue culture, multiplicity of infection as respects the inoculum, and delayed harvest did not seem to play any important role in HA titer. However, the presence of a stabilizer (H.A1.) in the serum-free medium may have a minor effect on HA titer at pH 7.0. A direct relationship was usually found between the infectious and HA titers in a freshly harvested serum-free medium. Stability of hemagglutinin was demonstrated for at least 8 days at 30° and 37°C in medium 199 containing 2% H.A1. at pH 7.8 though the infectivity titer decreased rapidly. At 5°C the HA titer was preserved without detectable decrease for at least 18 months and such stability was not dependent on the pH of the harvest or the presence of any added stabilizer in the medium. Such HA was non-infectious. The simple technique employed to achieve high-titered hemagglutinin from tissue culture and its stability together with loss of infectivity at refrigerator temperature offer a valuable substitute for the suckling mouse brain antigen for this, and at least some other group B arboviruses and probably others.