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.