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Efficient replication of recombinant Enterovirus B types, carrying different P1 genes in the coxsackievirus B5 replicative backbone
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. (Picorna)ORCID iD: 0000-0003-4533-9094
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
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2015 (English)In: Virus genes, ISSN 0920-8569, E-ISSN 1572-994X, Vol. 50, no 3, 351-357 p.Article in journal (Refereed) Published
Abstract [en]

Recombination is an important feature in theevolution of the Enterovirus genus. Phylogenetic studies ofenteroviruses have revealed that the capsid genomic region(P1) is type specific, while the parts of the genome codingfor the non-structural proteins (P2–P3) are species specific.Hence, the genome may be regarded as consisting of twomodules that evolve independently. In this study, it wasinvestigated whether the non-structural coding part of thegenome in one type could support replication of a virus witha P1 region from another type of the same species. A cas-sette vector (pCas) containing a full-length cDNA copy ofcoxsackievirus B5 (CVB5) was used as a replicative back-bone. The P1 region of pCas was replaced with the corre-sponding part from coxsackievirus B3Nancy(CVB3N),coxsackievirus B6Schmitt(CVB6S), and echovirus 7Wal-lace(E7W), all members of theEnterovirus Bspecies. Thereplication efficiency after transfection with clone-derivedin vitro transcribed RNA was studied and compared withthat of pCas. All the recombinant viruses replicated with similar efficiencies and showed threshold cycle (Ct) values,tissue culture infectivity dose 50 %, and plaque-forming unittiters comparable to viruses generated from the pCas con-struct. In addition to this, a clone without the P1 region wasalso constructed, and Western Blot and immunofluorescencestaining analysis showed that the viral genome could betranslated and replicated despite the lack of the structuralprotein-coding region. To conclude, the replicative back-bone of the CVB5 cassette vector supports replication ofintraspecies constructs with P1 regions derived from othermembers of theEnterovirus Bspecies. In addition to this,the replicative backbone can be both translated and repli-cated without the presence of a P1 region.

Place, publisher, year, edition, pages
2015. Vol. 50, no 3, 351-357 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biomedical Sciences, Virology
Identifiers
URN: urn:nbn:se:lnu:diva-41538DOI: 10.1007/s11262-015-1177-xISI: 000355233000001PubMedID: 25663145OAI: oai:DiVA.org:lnu-41538DiVA: diva2:800020
Available from: 2015-04-01 Created: 2015-04-01 Last updated: 2016-10-05Bibliographically approved
In thesis
1. Reverse genetic studies of Enterovirus replication
Open this publication in new window or tab >>Reverse genetic studies of Enterovirus replication
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enteroviruses belong to the Picornaviridae family and are small icosahedral viruses with RNA genomes of positive polarity, containing a single open reading frame. They mostly cause mild or asymptomatic infections, but also a wide array of diseases including: poliomyelitis, encephalitis, gastroenteritis, aseptic meningitis, myocarditis, hand-foot-and-mouth disease, hepatitis and respiratory diseases, ranging from severe infections to the common cold. The projects described in this thesis have been carried out through reverse genetic studies of Enterovirus B and Rhinovirus C.

                  In Papers I and II, a cassette vector was used to study recombination and translation of the RNA genome. It was found that the non-structural coding region could replicate when combined with the structural protein-coding region of other viruses of the same species. Furthermore, the genome could be translated and replicated without the presence of the structural protein-coding region. Moreover, it was found that when two additional nucleotides were introduced, shifting the reading frame, the virus could revert to the original reading frame, restoring efficient replication. In Paper III, a vector containing the genome of echovirus 5 was altered to produce an authentic 5’end of the in vitro transcribed RNA, which increased efficiency of replication initiation 20 times. This result is important, as it may lead to more efficient oncolytic virotherapy. An authentic 5’end was further used in Paper IV, where replication of Rhinovirus C in cell lines was attempted. Although passaging of the virus was unsuccessful, the genome was replicated and cytopathic effect induced after transfection. The restriction of efficient replication was therefore hypothesized to lie in the attachment and entry stages of the replication cycle. In Paper V, a cytolytic virus was found to have almost 10 times larger impact on gene expression of the host cell than a non-cytolytic variant. Furthermore, the lytic virus was found to build up inside the host cell, while the non-cytolytic virus was efficiently released.

                  As a whole, this thesis has contributed to a deeper understanding of replication of enteroviruses, which may prove important in development of novel vaccines, antiviral agents and oncolytic virotherapies.

Place, publisher, year, edition, pages
Växjö: Linnaeus University Press, 2015
Series
Linnaeus University Dissertations, 216/2015
Keyword
Enterovirus, picornavirus, coxsackievirus, echovirus, rhinovirus, reverse genetics, replication, translation, transfection, cassette vector.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biomedical Sciences, Virology
Identifiers
urn:nbn:se:lnu:diva-41636 (URN)978-91-87925-55-9 (ISBN)
Public defence
2015-04-24, N2007K, Smålandsgatan 26, Kalmar, 09:00 (English)
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Supervisors
Available from: 2015-04-10 Created: 2015-04-01 Last updated: 2015-04-10Bibliographically approved

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