Adaptation of tick-borne encephalitis virus to BHK-21 cells results in theformation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo

Propagation of the flavivirus tick-borne encephalitis virus in BHK-21 cells selected for mutations within the large surface glycoprotein E that increa sed the net positive charge of the protein. In the course of 16 independent experiments, 12 different protein E mutation patterns were identified. The se were located in all three of the structural domains and distributed over almost the entire upper and lateral surface of protein E, The mutations re sulted in the formation of local patches of predominantly positive surface charge. Recombinant viruses carrying some of these mutations in a defined g enetic backbone showed heparan sulfate (HS)dependent phenotypes, resulting in an increased specific infectivity and binding affinity for BHK-21 cells, small plaque formation in porcine kidney cells, and significant attenuatio n of neuroinvasiveness in adult mice. Our results corroborate the notion th at the selection of attenuated HS binding mutants is a common and frequent phenomenon during the propagation of viruses in cell culture and suggest a major role for HS dependence in flavivirus attenuation. Recognition of this principle may be of practical value for designing attenuated flavivirus st rains in the future.