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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
University of Kalmar, School of Pure and Applied Natural Sciences.
University of Kalmar, School of Pure and Applied Natural Sciences.
University of Kalmar, School of Pure and Applied Natural Sciences.
University of Kalmar, School of Pure and Applied Natural Sciences.
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2007 (English)In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 130, no December 2007, p. 129-139Article in journal (Refereed) Published
Abstract [en]

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 cultureadapted 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 thereplication 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.

Place, publisher, year, edition, pages
2007. Vol. 130, no December 2007, p. 129-139
National Category
Microbiology
Research subject
Natural Science, Biomedical Sciences
Identifiers
URN: urn:nbn:se:lnu:diva-2358DOI: 10.1016/j.virusres.2007.06.004ISI: 000251480800015PubMedID: 17645978OAI: oai:DiVA.org:lnu-2358DiVA, id: diva2:309444
Note

Ingår i avhandling under titeln: "Replication of Ljungan virus in cell culture: properties of the virus-host cell interactions".

Available from: 2010-04-07 Created: 2010-04-07 Last updated: 2017-11-27Bibliographically approved
In thesis
1. Ljungan Virus Replication in Cell Culture
Open this publication in new window or tab >>Ljungan Virus Replication in Cell Culture
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Ljungan virus (LV) is a recently identified picornavirus of the genus Parechovirus. LV has been isolated from voles trapped in Sweden and also in the United States. LV infected small rodents may suffer from diabetes type 1 and type 2 like symptoms, myocarditis and encephalitis. LV has been proposed as a human pathogen, with indications of causing diabetes type 1, myocarditis and intrauterine fetal deaths.

In this thesis, cell culture adapted LV strains were utilised for development and adaptation of several basic methodological protocols to study the LV biology, e.g. real time PCR, highly specific antibodies and a reverse genetics system. These methods allowed detailed studies of this virus and how it interacts with the host cell. The genomic 5'-end was identified and modelling showed unique secondary structure folding of this region. The LV encodes an aphthovirus-like 2A protein with a DvExNPGP motif. This motif was found to mediate primary cleavage of the LV polyprotein in vitro and is proposed to constitute the carboxy terminus of the structural protein VP1 in LV. Rabbit polyclonal antibodies generated against recombinant structural proteins were used to verify that the LV virion is composed of the structural proteins VP0, VP1 and VP3. Cell culture studies showed that LV replicates to low titer with an absent or delayed cell lysis. LV is proposed to be able to spread by a, for picornaviruses, not previously demonstrated direct cell-to-cell transmission. All results taken together suggest a maintenance strategy of LV including low amounts of the LV genome and persistently infected hosts. Stability studies showed that the LV virion not only maintain activity in acidic and alkaline environments but also exhibit resistance to the commonly used disinfectant Virkon®.The results presented in this thesis show that LV has several unique properties, not previously observed for a picornavirus.

Place, publisher, year, edition, pages
Högskolan i Kalmar, 2007
Series
Dissertation series: University of Kalmar, Faculty of Natural Science, ISSN 1650-2779 ; 37
Keywords
Ljungan virus, picornavirus, parechovirus, 5' - RACE, 5' -end, infectious cDNA clone, real time PCR, virus purification, virion stability, disinfection, aphthovirus-like 2A
National Category
Microbiology in the medical area
Research subject
Ecology, Microbiology
Identifiers
urn:nbn:se:hik:diva-10 (URN)978-91-89584-70-9 (ISBN)
Public defence
2007-04-20, 09:30 (English)
Opponent
Supervisors
Available from: 2007-08-27 Created: 2007-08-27 Last updated: 2018-01-13Bibliographically approved

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Ekström, Jens-OlaTolf, ConnyFahlgren, CamillaJohansson, SusanneEdman, KjellLindberg, A. Michael

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