ABSTRACT The NTU-LY-1 cell line is a Lymantria xylina cell line; however, during cell passage, obvious cytopathic effects (CPEs) were observed under light microscopy. Round inclusion body (IB)-like particles and a few irregular larger IB-like particles were found in the cytoplasm of NTU-LY-1 cells. To further investigate the CPEs in NTU-LY-1 cells, reverse transcription–PCR screening of cytoplasmic polyhedrosis virus, infectious flacherie virus and Perina nuda virus (PnV) was performed. The results indicated that NTU-LY-1 cells were persistently infected with PnV from days 1 to 15 of culture. NTU-LY-1 cells were further subjected to Western blotting and indirect immunofluorescence assays to confirm PnV infection. Based on transmission electronic microscopy observations, the CPEs in NTU-LY-1 cells were different from those in the PnV-homologous NTU-Pn-HF cell line (a cell line of P. nuda). Membrane-bound IBs were found in newly seeded NTU-LY-1 cells. Lysed NTU-LY-1 cells formed many microsomes after 15 days of culture, and three different types of IBs could be distinguished among the microsomes, including membrane-bound, fibril-bound and sheath-bound IBs. After 15 days of culture, the cytoplasm of un-lysed NTU-LY-1 cells contained zipper-like arrays of non-membrane-bound virogenic stroma and parallel rows of viral particles that were restricted by two membranes. These viral particles were purified and showed a PnV morphology. This is the first study to show persistent PnV infection in a heterologous cell line. The findings suggest a new strategy for microbial control; moreover, the findings could support the development of a model for membrane-associated replication of positive-sense single-stranded RNA viruses.