Abstract
We previously showed that the growth ability of the Japanese encephalitis virus (JEV) genotype V (GV) strain Muar is clearly lower than that of the genotype I (GI) JEV strain Mie/41/2002 in murine neuroblastoma cells. Here, we sought to identify the region in GV JEV that is involved in its low growth potential in cultured cells. An intertypic virus containing the NS1-3 region of Muar in the Mie/41/2002 backbone (NS1-3Muar) exhibited a markedly diminished growth ability in murine neuroblastoma cells. Moreover, the growth rate of a Muar NS2A-bearing intertypic virus (NS2AMuar) was also similar to that of Muar in these cells, indicating that NS2A of Muar is one of the regions responsible for the Muar-specific growth ability in murine neuroblastoma cells. Sequencing analysis of murine neuroblastoma Neuro-2a cell-adapted NS1-3Muar virus clones revealed that His-to-Tyr mutation at position 166 of NS2A (NS2A166) could rescue the low replication ability of NS1-3Muar in Neuro-2a cells. Notably, a virus harboring a Tyr-to-His substitution at NS2A166 (NS2AY166H) showed a decreased growth ability relative to that of the parental virus Mie/41/2002, whereas an NS2AMuar-based mutant virus, NS2AMuar-H166Y, showed a higher growth ability than NS2AMuar in Neuro-2a cells. Thus, these results indicate that the NS2A166 amino acid in JEV is critical for the growth and tissue tropism of JEV in vitro.
Highlights
Japanese encephalitis virus (JEV) is a mosquito-borne arbovirus that causes the serious neurological disorder Japanese encephalitis (JE)
Cells were plated into 6-well culture plates and infected with the JEV strains at an multiplicity of infection (MOI) of 0.1 plaque-forming units (PFU)/cell
It was shown that NS2A of Muar was one of the regions responsible for the Muar-specific lower growth ability in murine neuroblastoma cells
Summary
Japanese encephalitis virus (JEV) is a mosquito-borne arbovirus that causes the serious neurological disorder Japanese encephalitis (JE). JE vaccines currently available are derived from GIII strains These JE vaccines were previously reported to potentially exhibit less ability to induce neutralizing antibodies against GV JEV than those against other JEV genotypes [15,16]. IgGs raised against GV JEV XZ0934 were reported to show a poor neutralizing ability against GIII JEV [17] These findings raise the possibility that GV JEV is distinct from other JEV genotypes in terms of antigenicity, and the current GIII-derived JE vaccines might provide inadequate levels of protection against GV JEV. Our findings showed that the 5’-NCR-C-prM-E region of Muar, which is responsible for the high neuroinvasiveness of the strain, does not influence the growth characteristics of Muar in murine neuroblastoma cells [18]. Mie/41/2002 using a reverse-genetics system that we previously established, and analyzed the mechanism for the growth properties of these JEV strains in vitro
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