Semliki Forest virus (SFV) provides a well-characterized model system to study the pathogenesis of virus encephalitis. Several studies have used virus derived from the molecular clone SFV4. SFV4 virus does not have the same phenotype as the closely related L10 or the prototype virus from which its molecular clone was derived. In mice, L10 generates a high-titer plasma viremia, is efficiently neuroinvasive, and produces a fatal panencephalitis, whereas low-dose SFV4 produces a low-titer viremia, rarely enters the brain, and generally is avirulent. To determine the genetic differences responsible, the consensus sequence of L10 was determined and compared to that of SFV4. Of the 12 nucleotide differences, six were nonsynonymous; these were engineered into a new molecular clone, termed SFV6. The derived virus, SFV6, generated a high-titer viremia and was efficiently neuroinvasive and virulent. The phenotypic difference mapped to a single amino acid residue at position 162 in the E2 envelope glycoprotein (lysine in SFV4, glutamic acid in SFV6). Analysis of the L10 virus showed it contained different plaque phenotypes which differed in virulence. A lysine at E2 247 conferred a small-plaque avirulent phenotype and glutamic acid a large-plaque virulent phenotype. Viruses with a positively charged lysine at E2 162 or 247 were more reliant on glycosaminoglycans (GAGs) to enter cells and were selected for by passage in BHK-21 cells. Interestingly, viruses with the greatest reliance on binding to GAGs replicated to higher titers in the brain and more efficiently crossed an in vitro blood-brain barrier (BBB).

IMPORTANCE Virus encephalitis is a major disease, and alphaviruses, as highlighted by the recent epidemic of chikungunya virus (CHIKV), are medically important pathogens. In addition, alphaviruses provide well-studied experimental systems with extensive literature, many tools, and easy genetic modification. In this study, we elucidate the genetic basis for the difference in phenotype between SFV4 and the virus stocks from which it was derived and correct this by engineering a new molecular clone. We then use this clone in one comprehensive study to demonstrate that positively charged amino acid residues on the surface of the E2 glycoprotein, mediated by binding to GAGs, determine selective advantage and plaque size in BHK-21 cells, level of viremia in mice, ability to cross an artificial BBB, efficiency of replication in the brain, and virulence. Together with studies on Sindbis virus (SINV), this study provides an important advance in understanding alphavirus, and probably other virus, encephalitis.