Replication of Ljungan virus in cell culture: the genomic 5'-end, infectious cDNA clones and host cell response to viral infections.
Ljungan virus (LV) is a picornavirus recently isolated from bank voles (Clethrionomys glareolus). The previously uncharacterised 5'-end sequence of the LV genome was determined. Infectious cDNA clones were constructed of the wild type LV prototype strain 87-012 and of the cytolytically replicating cell culture adapted variant 87-012G. Virus generated from cDNA clones showed identical growth characteristics as uncloned virus stocks. Cell culture adapted LV, 87-012G, showed a clear cytopathic effect (CPE) at 3-4 days post-infection (p.i.). Virus titers, determined by plaque titration, increased however only within the first 18h p.i. Replication of LV (+) strand RNA was determined by real-time PCR and corresponded in time with increasing titers. In contrast, the amounts of the replication intermediate, the (-) strand, continued to increase until the cells showed CPE. This indicates separate controlling mechanisms for replication of LV (+) and (-) genome strands. Replication was also monitored by immunofluorescence (IF) staining. IF staining of both prototype 87-012 and the CPE causing 87-012G showed groups of 5-25 infected cells at 48h p.i., suggesting a, for picornaviruses, not previously described direct cell-to-cell transmission.
Ekström
Jens-Ola
JO
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden.
Tolf
Conny
C
Fahlgren
Camilla
C
Johansson
E Susanne
ES
Arbrandt
Gustav
G
Niklasson
Bo
B
Edman
Kjell-A
KA
Lindberg
A Michael
AM
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003588:Cytopathogenic Effect, Viral; D018076:DNA, Complementary / Q000235:genetics; D030061:Parechovirus / Q000254:growth & development*; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017421:Sequence Analysis; D010948:Viral Plaque Assay; D014776:Virus Cultivation / Q000379:methods*; D014779:Virus Replication
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D012367:RNA, Viral