Replication of Ljungan virus in cell culture: the genomic 5'-end, infectious cDNA clones and host cell response to viral infections |
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Authors: | Ekström Jens-Ola Tolf Conny Fahlgren Camilla Johansson E Susanne Arbrandt Gustav Niklasson Bo Edman Kjell-A Lindberg A Michael |
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Affiliation: | aDepartment of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden bApodemus, Grevgatan 38, SE-114 53 Stockholm, Sweden |
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Abstract: | 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 18 h 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 48 h p.i., suggesting a, for picornaviruses, not previously described direct cell-to-cell transmission. |
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Keywords: | Infectious cDNA clone 5′-RACE Parechovirus Picornavirus Virus transmission |
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