Detection of replicating negative-sense RNAs in CaCo-2 cells infected with human astrovirus.
Human astrovirus (HAstV) is the second most important cause of viral diarrhea and acute gastroenteritis in infants under five. However, determination of the infectivity of clinical isolates is difficult, and the replication cycle of HAstV is not yet fully understood. In this study, it was attempted to detect negative-sense (-)RNAs generated during the replication of RNA viruses. We used clinical isolates of HAstV to infect CaCo-2 cells. Reverse transcription using only a sense primer followed by PCR using both sense and antisense primers showed that (-)RNAs were first detected in CaCo-2 cells between 9 and 12 h postinfection (p.i.). However, these (-)RNAs were not detected when cells were treated with the protein synthesis inhibitor cycloheximide during HAstV infection. Next, RT with only an antisense primer followed by PCR was performed to detect (+)RNA of HAstVs after production of (-)RNAs during replication. RT-PCR results using the antisense primer revealed that the amount of (+)RNA began to increase starting 9 h p.i., indicating an accumulation of the newly synthesized (+)RNA genome. Cycloheximide was observed to abrogate the increase of newly made (+)RNA during HAstV infection. In conclusion, the use of sense or antisense primers during the RT reaction together with cycloheximide enabled us to quantitatively detect (-)RNAs, and this proved to be an useful tool in understanding the replication cycle of HAstV.
Jang
So Young
SY
Department of Microbiology, College of Natural Sciences, Chungbuk National University, Cheongju, Chungbuk, South Korea.
Jeong
Weon Hwa
WH
Kim
Mi Suk
MS
Lee
Young Min
YM
Lee
Jae In
JI
Lee
Gyu Cheol
GC
Paik
Soon Young
SY
Koh
Gwang Pyo
GP
Kim
Jong Min
JM
Lee
Chan Hee
CH
D019350:Astroviridae Infections / Q000175:diagnosis / Q000821:virology*; D018938:Caco-2 Cells; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000821:virology; D006801:Humans; D001257:Mamastrovirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics*; D014779:Virus Replication
D017931:DNA Primers; D012367:RNA, Viral