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Cell culture propagation and biochemical analysis of the Ljungan virus prototype strain
University of Kalmar, School of Pure and Applied Natural Sciences.
University of Kalmar, School of Pure and Applied Natural Sciences.
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2004 (English)In: Biochemical and Biophysical Research Communication, Vol. 317, no 4, p. 1023-1029Article in journal (Refereed) Published
Abstract [en]

Ljungan virus (LV) is proposed as a potentially important rodent harbored viral human pathogen. Little is known about the biophysical nature of the virus and despite being molecularly characterized, progress in epidemiological and basic biological studies of LV has been hampered by the lack of a robust and reliable cell culture propagation system. Here we report the first description of an efficient lytic multi-cycle cell culture propagation of the LV prototype strain (87-012). Biophysical analysis of gradient purified LV virions generated by this system identified mature infectious virions to possess a sedimentation coefficient of 160S and in agreement with previous molecular prediction, polyprotein analysis suggests that the native virion is composed of only three major structural proteins. The nucleotide composition of the complete genome of the LV cell culture adapted virus was determined and compared to that of the parental prototype LV. Numerous mutations were observed scattered throughout the viral genome and particularly in VP1. The development of this cell culture system for LV should open new avenues in the study of LV biology, structure, pathogenesis, and prevalence of natural infection in the wider community.

Place, publisher, year, edition, pages
2004. Vol. 317, no 4, p. 1023-1029
National Category
Microbiology in the medical area
Research subject
Biomedical Sciences, Virology
Identifiers
URN: urn:nbn:se:hik:diva-592DOI: 10.1016/j.bbrc.2004.03.145OAI: oai:DiVA.org:hik-592DiVA, id: diva2:1836
Available from: 2008-12-11 Created: 2008-11-10 Last updated: 2018-01-13Bibliographically 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
Keyword
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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Publisher's full texthttp://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WBK-4C4WX7J-4-8&_cdi=6713&_user=745831&_orig=search&_coverDate=05%2F14%2F2004&_sk=996829995&view=c&wchp=dGLbVtz-zSkzV&_valck=1&md5=741e3f87767113b478e2c1f58cca9ed8&ie=/sdarticle.pdf

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Johansson, SusanneEdman, KjellLindberg, Michael

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