title: Defective interfering RNAs of Japanese encephalitis virus found in mosquito cells and correlation with persistent infection.
abstract: Defective interfering (DI) RNAs are deletion mutants of viral genomes that are known in many cases to contribute to persistent infection and modification of viral pathogenesis. Cell type also plays a critical role in the establishment of viral persistence. In this study we have identified for the first time the generation of DI RNAs of Japanese encephalitis virus in C6/36 mosquito cells. A persistent infection was established by replacing growth medium on surviving cells and continued cell passaging. Persistent infection was demonstrated by a continual release of infectious virus, fluorescent antibody staining, and Northern analysis. A population of DI RNAs of approximately 8.2-9.7 kb, not detectable in acutely infected cells, became apparent in the persistently infected cells by 25 days postinfection. Sequence analyses revealed a population of DI RNAs that contained in-frame deletions of 1.3-2.8 kb covering the region of the E gene and some flanking C or prM and NS1 gene sequences. Transcripts from one cDNA clone of a DI RNA replicated in uninfected mosquito cells as demonstrated by RT-PCR. DI RNA-containing virions in supernatant fluids from persistently infected mosquito cells could be used to establish persistent infection in BHK-21 cells. The correlation of DI RNA presence with cell survival suggests that DI RNAs are contributing mechanistically to the establishment of persistent infection in both the mosquito and mammalian cells.
authors.affiliation: Institute of Biotechnology, Department of Life Science, National Dong Hwa University, Taiwan, ROC.
authors.forename: Kuen-Nan
authors.initials: KN
authors.lastname: Tsai
authors.forename: Shih-Fang
authors.initials: SF
authors.lastname: Tsang
authors.forename: Chung-Hao
authors.initials: CH
authors.lastname: Huang
authors.forename: Ruey-Yi
authors.initials: RY
authors.lastname: Chang
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D002470:Cell Survival; D002477:Cells; D006224:Cricetinae; D009033:Culicidae / Q000235:genetics / Q000821:virology*; D003673:Defective Viruses / Q000235:genetics*; D004175:Diptera; D004664:Encephalitis Virus, Japanese / Q000235:genetics / Q000502:physiology*; D007150:Immunohistochemistry; D007239:Infections; D008562:Membrane Glycoproteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D014759:Viral Envelope Proteins / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics
chemical_list: D008562:Membrane Glycoproteins; C100315:NS1 protein, Flavivirus; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; C080628:glycoprotein E, Japanese encephalitis virus