Conditions for haemolysis by flaviviruses and characterization of the haemolysin

The 17D vaccine strain of yellow fever virus (YF 17D) was used to establish the optimal conditions for lysis of chick erythrocytes. Tissue culture-grown, polyethylene glycol-concentrated virus showed peak activity at pH 5.4 in citrate buffer when incubated at 37 degrees C. A further two- to fourfold increase in titre was obtained by pretreatment of the chick erythrocytes with 250 micrograms/ml trypsin. These conditions were also shown to be optimal for Japanese encephalitis (JE), West Nile (WN) and dengue-2 (den2) viruses. The ratio of haemagglutination (HA) titre to haemolysis (HL) titre approximated to unity, suggesting that the two functions are associated with the same molecule although as separable entities since selective inactivation of the HL activity of the virus was accomplished using 60 micrograms/ml trypsin. HL could be demonstrated at neutral pH if the chick erythrocytes were first subjected to treatment with acidic pH buffer. The effect on the virus envelope is thus not the sole contribution of a low pH environment to optimal HL. Hyperimmune rabbit antiserum prepared against purified YF 17D virions inhibited HA and HL if added before agglutination had occurred by the virus but when added after agglutination had taken place it showed specific anti-HL activity. Monoclonal antibodies that inhibited HA (HAI) by YF 17D did not inhibit HL (HLI) activity when applied after agglutination had taken place. Moreover, monoclonal antibodies specific for the 54K glycoprotein of YF virus but without HAI activity also had no effect on HL when added either before or after agglutination. As yet, we have been unable to identify a monoclonal antibody displaying specific anti-HL activity but all those directed against the 54K envelope glycoprotein possessing HAI activity showed HA to be a prerequisite for HL.