Suckling mice were used to detect infectious dengue-2 viruses by intracerebral injection of the full-length RNA transcript.
OBJECTIVE
In this study we established a mouse brain injection system to detect infectious dengue-2 virus produced from the full-length RNA transcripts.


METHODS
In vitro transcription was used to synthesize full-length dengue-2 virus RNA from the plasmid pRS424FLDEN2NGC, which was intracerebrally injected into the 6-day-old suckling mice (ICR strains). Engineered dengue-2 viruses were detected in the brain sections using immunohistochemistry staining. RT-PCR followed by restriction endonuclease BstEII digestion was used to confirm the mosquito C6/36 cells cocultured with the mouse brain extract.


RESULTS
The mice inoculated with the full-length dengue-2 viral RNA transcript showed paralysis symptoms and died between day 10 and 13 postinjection. The dengue-2 virus-specific antigens (E, Core and NS1) were detected in all the brain and part of the liver sections of the paralyzed mice by immunohistochemistry staining, indicating the existence of dengue-2 virus in these tissues of the suckling mice. The viruses detected in the brains of suckling mice were indeed infectious, which was further confirmed by coculturing mosquito C6/36 cells with the brain extract of the injected mice.


CONCLUSIONS
We developed an in vivo approach to detect and produce engineered dengue viruses with infectivity from the full-length plasmid cDNA. This suckling mice system will also aid in screening the infectious viruses that are created by site-directed mutagenesis and is useful for the studies of dengue virus gene function and pathogenesis in the host.
Lee
Ying-Ray
YR
Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Huang
Kao-Jean
KJ
Lei
Huan-Yao
HY
Chen
Shun-Hua
SH
Lin
Yee-Shin
YS
Yeh
Trai-Ming
TM
Liu
Hsiao-Sheng
HS
D000818:Animals; D000833:Animals, Suckling; D000956:Antigens, Viral / Q000302:isolation & purification; D001921:Brain / Q000821:virology*; D002478:Cells, Cultured; D009033:Culicidae; D003715:Dengue / Q000473:pathology / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D007150:Immunohistochemistry; D008099:Liver / Q000821:virology; D051379:Mice; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014776:Virus Cultivation / Q000379:methods*
D000956:Antigens, Viral; D012367:RNA, Viral