Generation and characterization of organ-tropism mutants of Japanese encephalitis virus in vivo and in vitro

Using gamma-ray irradiation, a pair of virulent (RP-9) and attenuated (RP-2ms) variants of Japanese encephalitis virus (JEV) were generated from a Taiwanese isolate, NT109. The two variants differed in plaque morphology, virus adsorption, and growth properties in BHK-21 cells: (i) RP-2ms produced smaller plaques than RP-9; (ii) RP-2ms adsorbed less efficiently to host cells but yielded a higher virus titer (burst size); and (iii) RP-2ms virions were mostly accumulated intracellularly, whereas RP-9 was released extracellularly. In addition, in an in vitro binding assay, the envelope (E) protein of RP-9, but not that of RP-2ms, bound specifically to a cellular protein of 57-kDa derived from BHK-21 cells. When injected into mice intracerebrally, RP-2ms was much less virulent than RP-9, with 50% lethal doses of >10(7) and 0.4 plaque forming units, respectively. Moreover, when inoculated intraperitoneally, their organ tropism differed in that the main target organ for RP-2ms was liver, whereas that for RP-9 was brain. These results suggest that RP-2ms was less neurovirulent and less neuroinvasive from peripheral routes. Molecular analysis of the virus structural proteins detected only two differences between RP-9 and RP-2ms: one in E protein, Glu-138 in RP-9 and Lys-138 in RP-2ms, and the other in prM, Tyr-43 in RP-9 and His-43 in RP-2ms. Since the N-terminal 92 amino acids of prM are cleaved and not present in mature JEV virions, the single-amino-acid change of the E protein at position 138 may account for the difference between the mutants in the in vitro binding assay, Such mutation in E protein, or perhaps in conjunction with the prM mutation, may be responsible, in part, for the phenotypic differences observed in vitro and in vivo between the two mutants. (C) 1996 Academic Press, Inc.