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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.

Persistent infection with Japanese encephalitis virus (JEV) was established in murine neuroblastoma N18 cells, and the persistency has been maintained in cell culture for over 6 months. From the persistently infected cells, a clone named C2-2 was selected and expanded to form a stable cell line. The vast majority of C2-2 cells showed viral protein staining by immunofluorescence and continuously produced low levels of virus (10(3) to 10(4) PFU/ml) without marked cytopathic effects or cyclic variations. In addition to the wild-type viral proteins, truncated forms of the viral nonstructural protein 1 (NS1) as well as its derivative NS1' were produced in C2-2 cells. Both truncated NS1 and NS1' contain deletions at their N-termini; however, the analyses by RT-PCR and direct sequencing of the viral RNA failed to detect any truncations or mutations within the NS1 region, suggesting that NS1 truncation was a result of a unique posttranslational proteolytic cleavage of NS1 in the persistently infected cells. Similar but not identical truncation of NS1 was also observed in two other persistently infected cell lines established in Vero and DBT (murine astrocytoma) cells. However, viruses released from C2-2 cells did not produce truncated NS1 upon infection of N18 cells, suggesting that NS1 truncations were the result of virus-cell interaction in persistently infected cells. These data indicate a strong association between abnormal NS1 expression and JEV persistency. A probable involvement of dysfunctional NS1 in the establishment and/or maintenance of JEV persistency in tissue culture is discussed.

In retroviruses, the pol gene is expressed in the form of a gag-pol fusion protein by the mechanism of ribosomal frameshifting. In studies of the possible mechanism of hepadnaviral pol protein synthesis, recent results have ruled out core-pol fusion protein synthesis by ribosomal frameshifting. In this study, an in vitro transcription and translation coupling system was used to demonstrate that the HBV core and pol proteins could be synthesized independently using the pregenome RNA template. The result has led us to design experiments to distinguish between the involvement of a termination-reinitiation, internal initiation, or leaky scanning mechanism in the pol protein synthesis. In vitro experiments were then carried out to measure the amount of pol proteins being synthesized from (i) the preC mRNA, which contained an extra AUG and seven more nucleotides at the 5'-end in comparison with the pregenome RNA; (ii) the pregenome RNA in the presence of various amounts of antisense RNA annealing to the 5'-end of the pregenome RNA; and (iii) the pregenome RNA with an additional hairpin structure located upstream of the C gene. Results indicated that the synthesis of both core and pol proteins was concomitantly reduced in these three conditions, which suggested that leaky scanning is the most probable mechanism for pol protein synthesis in vitro. To further verify the mechanism in vivo, experiments were performed to assay the activity of DNA polymerase in virions, which were obtained from hepatoma cells transfected by plasmids containing either a wild-type sequence (5'-GGCATGG-3') or an optimal initiation context (5'-ACCATGG-3') of the C gene. Transfection results showed that the plasmid-containing mutations of the C gene significantly decreased the DNA polymerase activity in virions. This observation supports our hypothesis that the leaky scanning model is involved in the synthesis of pol protein.