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

•Construction of recombinant infectious clone.•Production of mutant virus.•Detection of viral growth capacity.•Investigation of Sindbis viral neurovirulence.

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.