Two mutations in the envelope glycoprotein E2 of Semliki Forest virus affecting the maturation and entry patterns of the virus alter pathogenicity for mice.

The prototype strain of Semliki Forest virus (SFV) of known sequence and virus produced by the cDNA clone derived from it were lethal following intranasal (i.n.) infection of 40-day-old and intraperitoneal (i.p.) infection of pregnant BALB/c mice; this lethality was related to neuronal necrosis in the central nervous system (CNS). We conclude that the virulence of the prototype strain, and virus from the cDNA clone derived from it, is similar to that of L10 (the original SFV isolate). The effects of two mutations in the p62 envelope protein region of the clone were determined. Substitution of Glu for Lys at position 162 (mut64) extended the mean time of death following i.n. inoculation of 40-day-old mice. Pregnant mice infected with this virus survived but lethal infection of some fetuses did occur. Substitution of Leu for Arg at position 66 (mL), the cleavage site of the E2 and E3 proteins, results in the production of particles containing uncleaved p62. These particles were less virulent than the prototype strain when inoculated i.n. and induced immunity to virulent SFV challenge. The virus also induced the formation of multifocal glial nodules in the CNS of surviving mice. The differences in pathogenicity between the two mutants and the virulent parental virus are probably related to differences in the efficiency of virus multiplication in infected mice. The mut64 mutation attenuated the virus and allowed survival of pregnant mice infected i.p. so that the effects of fetal infection could be detected. The mL mutation allowed survival of i.n.-infected mice so that the later effects of virus multiplication in the CNS could be assessed. In the former case, this is probably a result of reduced virus release, whereas in the latter case it is due to inefficient entry of host cells. The results are consistent with our previous suggestion that lethality for virulent SFV infection results from a lethal threshold of damage to neurons in the CNS and that attenuating mutations may reduce neuronal damage below this threshold level.