Defective interfering RNAs of Japanese encephalitis virus found in mosquito cells and correlation with persistent infection.
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.
Tsai
Kuen-Nan
KN
Institute of Biotechnology, Department of Life Science, National Dong Hwa University, Taiwan, ROC.
Tsang
Shih-Fang
SF
Huang
Chung-Hao
CH
Chang
Ruey-Yi
RY
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
D008562:Membrane Glycoproteins; C100315:NS1 protein, Flavivirus; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; C080628:glycoprotein E, Japanese encephalitis virus