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.
Chen
L K
LK
Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China. lkchen@ndmc1.ndmctsgh.edu.tw
Lin
Y L
YL
Liao
C L
CL
Lin
C G
CG
Huang
Y L
YL
Yeh
C T
CT
Lai
S C
SC
Jan
J T
JT
Chin
C
C
D000330:Aedes / Q000166:cytology; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003465:Culex / Q000821:virology; D004664:Encephalitis Virus, Japanese / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D004672:Encephalitis, Japanese / Q000821:virology; D005260:Female; D005720:Gamma Rays; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008813:Mice, Inbred ICR; D009154:Mutation; D009928:Organ Specificity; D010537:Peritoneum; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics; D010948:Viral Plaque Assay; D014774:Virulence / Q000235:genetics; D014779:Virus Replication
D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; C080628:glycoprotein E, Japanese encephalitis virus; C074932:prM protein, Flavivirus