A substitution in nsP1 combined with a double substitution in E2 glycoprotein renders Sindbis-like virus XJ-160 fully neurovirulent for adult mice

Sindbis virus (SV) can be rendered neurovirulent for adult mice by a double substitution within the E2 glycoprotein, including replacing Gln at position 55 of E2 with a His (E2-55: Gln-His) and E2-70: Glu to Lys. However, the mutant Sindbis-like virus XJ-160 with the double substitution (BR-E5570) does not show neurovirulence for adult mice, although the mutant apparently reduced the average survival time of neonatal mice. To produce an XJ-160 virus neurovirulent for adult mice, the BR-E5570 virus containing the double substitution was provided with another substitution in the nsP1 region (nsP1-173: Thr-Ile), which could enhance viral infectivity and neurovirulence for neonatal mice. The mutant containing these three substitutions was accordingly designated as BR-5570-ns173. Like the BR-XJ160 virus derived from the wild-type clone, BR-E5570 and BR-E5570-ns173 were both virulent for newborn mice, between which BR-E5570-ns173 virus showed the greatest neurovirulence. Furthermore, only BR-E5570-ns173 virus was fully neurovirulent for 14-day-old mice, and this fatal adult mouse-virulence was dependent on the E2 double substitutions at positions 55 and 70. Compared with BR-XJ160, both the mutants showed a higher capacity for propagation both in cultured cells and in the mouse brain. In particular, BR-E5570-ns173 virus showed a more persistent existence and higher titer in the brains of 7-day-old mice. These findings indicate that the substitution at nsP1-173 combination with a double substitution in the E2 region renders the XJ-160 virus fully neurovirulent for adult mice, and this neurovirulence may be related to the increased efficiency and persistence of propagation of this virus.