Adaptation of Tick-Borne Encephalitis Virus to BHK-21 Cells Results in the Formation of Multiple Heparan Sulfate Binding Sites in the Envelope Protein and Attenuation In Vivo

Surface models of wild-type and mutant E proteins colored by electrostatic potential. The viewing angle is the same as in Fig. 1B. Positively charged surfaces are shown in blue, and negatively charged surfaces are red. The yellow ovals indicate areas of increased positive charge relative to wild-type protein E.

Infectivity titers in BHK-21 (open bars) and CE (solid bars) cells. Titers were determined by endpoint dilution experiments (see Material and Methods), starting with 10⁴ PFU of each virus. Values shown are the means of four independent experiments (with error bars representing standard deviations).

Growth curves obtained by infecting BHK-21 cells grown under normal conditions (●) or under conditions that prevent sulfation of proteoglycans (○). Cells were infected with 10 infectious units of wild-type or recombinant mutant virus, and release of virus into the supernatants was monitored by a protein E ELISA (for details, see Materials and Methods). A representative example of several experiments is shown. hpi, hours postinfection; OD, optical density.

Inhibition of virus growth by soluble heparin. Heparin concentrations added to the virus and growth medium are as follows: ●, no heparin; ○, 50 μg/ml; □, 150 μg/ml; ∗, 500 μg/ml. Cells were infected with 10 infectious units of wild-type or recombinant mutant virus, and release of virus into the supernatants was monitored by a protein E ELISA (for details, see Materials and Methods). A representative example of several experiments is shown. hpi, hours postinfection; OD, optical density.

Binding of wild-type and mutant viruses to BHK-21 cells. Equal amounts (500 ng) of purified virus were added to undigested BHK-21 cells (−) or cells that were predigested (+) with heparinase III (top) or chondroitinase ABC (bottom). The amount of cell-bound virus was quantified by fluorescence-activated cell sorter analysis (see Materials and Methods). Median fluorescence intensities are plotted as arbitrary units (a.u.). A representative example of several experiments is shown.