Persistence of viral RNA in the central nervous system of mice inoculated with MHV-4.
In order to study the role that viral persistence may play in chronic central nervous system (CNS) disease induced by murine coronaviruses, we have used the reverse transcriptase-polymerase chain reaction (RT-PCR) to study viral RNA in the brains of mice after intracerebral inoculation of JHM virus (JHMV or MHV-4). Quantitative RT-PCR showed that JHMV RNA decreased from approximately 2 ng/ug total brain RNA at day 6 post-inoculation (PI) to 0.1 pg/ug total brain RNA at 360 days PI. Double-stranded viral RNA could be detected up to day 20 PI. By the selective use of upstream or downstream primers during the RT step, it was possible to measure negative sense and positive sense JHMV RNA respectively, and we found that there was a marked rise in the ratio of positive to negative sense JHMV RNA after day 13 PI. Analysis of amplified products by dideoxy DNA sequencing showed that the characteristic mutation of our input virus (at position 3340 of gene 3) is maintained to at least day 42 PI. Taken together, these results favor a model of JHMV persistence in vivo in which viral RNA is present as double stranded forms initially and predominantly as single stranded, positive sense forms at late timepoints. Further analysis of this model in quantitative terms may contribute to our understanding of the biological significance of coronavirus persistence in the CNS.
Fleming
J O
JO
University of Wisconsin, Madison 53792.
Houtman
J J
JJ
Alaca
H
H
Hinze
H C
HC
McKenzie
D
D
Aiken
J
J
Bleasdale
T
T
Baker
S
S
D000818:Animals; D001921:Brain / Q000382:microbiology*; D007267:Injections; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D006517:Murine hepatitis virus / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase; D017735:Virus Latency
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase