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| 2. |
- Ekström, Jens-Ola, et al.
(författare)
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Replication of Ljungan virus in cell culture : the genomic 5'-end, infectious cDNA clones and host cell response to viral infections
- 2007
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Ingår i: Virus Research. - : Elsevier BV. - 0168-1702 .- 1872-7492. ; 130:1-2, s. 129-139
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Tidskriftsartikel (refereegranskat)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 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.
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| 3. |
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| 4. |
- Tolf, Conny, et al.
(författare)
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Characterization of polyclonal antibodies against the capsid proteins of Ljungan virus
- 2008
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 150:1-2, s. 34-40
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Tidskriftsartikel (refereegranskat)abstract
- Ljungan virus (LV) is a suspected human pathogen isolated from voles in Sweden and North America. To enable virus detection and studies of localization and activity of virion proteins, polyclonal antibodies were produced against bacterially expressed capsid proteins of the LV strain, 87-012G. Specific detection of proteins corresponding to viral antigens in lysates of LV infected cells was demonstrated by immunoblotting using each one of the generated polyclonal antibodies. In addition, native viral antigens present in cell culture infected with LV strains 87-012G or 145SLG were detected in ELISA and by immunofluorescence using the antibodies against the VP0 and VP1 proteins. The anti-VP3 antibody did not react with native proteins of the LV virion, suggesting that the VP3 is less potent in evoking humoral response and may have a less exposed orientation in the virus capsid. No activity of the antibodies was observed against the closely related human parechovirus type 1. The polyclonal antibody against the VP1 protein was further used for detection of LV infected myocytes in a mouse model of LV-induced myocarditis. Thus, polyclonal antibodies against recombinant viral capsid proteins enabled detection of natural LV virions by several different immunological methods.
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| 5. |
- Tolf, Conny, et al.
(författare)
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Identification of Ljungan virus VP0 and VP1 amino acid residues associated with cytolytic replication in cultured cells
- 2009
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Ingår i: Archives of Virology. - : Springer Science and Business Media LLC. - 0304-8608 .- 1432-8798. ; 154:8, s. 1271-1284
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Tidskriftsartikel (refereegranskat)abstract
- Ljungan virus is a picornavirus isolated from Swedish and North American rodents. Replication of Ljungan virus in cultured cells normally induces a weak and delayed cytopathic effect compared to that of many other picornaviruses. However, efficiently replicating Ljungan virus variants may evolve during serial passages in cell culture. In this study, we evaluate the significance of three substitutions in capsid protein VP0 and VP1 of a cell-culture-adapted variant of the Swedish Ljungan virus 145SL strain. In contrast to the parental strain, this 145SLG variant grows to high titers in green monkey kidney cells and induces a distinct cytopathic effect. Reverse genetic analyses demonstrated that each one of the individual capsid substitutions contributes to lytic replication in cell culture, but also that expression of all three substitutions results in a 100- to 500-fold increase in viral titers compared to viruses encoding single capsid substitutions. In addition, as indicated by detection of activated caspase-3 and DNA fragmentation, there seems to be an association between increased replication efficiency of lytic Ljungan virus variants and induction of an apoptotic response in infected green monkey kidney cells.
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| 6. |
- Tolf, Conny, et al.
(författare)
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Molecular characterization of a novel Ljungan virus (Parechovirus; Picornaviridae) reveals a fourth genotype and indications of ancestral recombination
- 2009
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Ingår i: Journal of General Virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 90, s. 843-853
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Tidskriftsartikel (refereegranskat)abstract
- Ljungan virus (LV) was discovered 20 years ago in Swedish bank voles (Myodes glareolus, previously referred to as Clethrionomys glareolus) during the search for an infectious agent causing lethal myocarditis in young athletes. To date, the genomes of four LV isolates, including the prototype 87-012 strain, have been characterized. Three of these LV strains were isolated from bank voles trapped in Sweden. Sequence analysis of an American virus (M1146), isolated from a montane vole (Microtus montanus) in western USA, indicates that this strain represents a genotype that is different from the Swedish strains. Here, we present genomic analyses of a fifth LV strain (64-7855) isolated from a southern red-backed vole (Myodes gapperi) trapped during arbovirus studies in New York state in the north-eastern USA in the 1960s. Sequence analysis of the 64-7855 genome showed an LV-like genome organization and sequence similarity to other LV strains. Genetic and phylogenetic analyses of the evolutionary relationship between the 64-7855 strain and other viruses within the family Picornaviridae, including previously published LV strains, demonstrated that the 64-7855 strain constitutes a new genotype within the LV species. Analyses also showed that different regions of the 64-7855 genome have different phylogenetic relationships with other LV strains, indicating that previous recombination events have been involved in the evolution of this virus.
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