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Implications of Local Puumala Hantavirus Genetics and Epidemiology for Diagnostics and Vaccine Development
Umeå University, Faculty of Medicine, Clinical Microbiology, Virology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Puumala viruses, a member of the Hantavirus genus in the Bunyaviridae family, are enveloped by a lipid bilayer and possesses a tripartite single stranded RNA genome with negative polarity. The hantaviruses encode four proteins: a nucleocapsid protein (N), two membrane spanning glycoproteins (GN and GC) and a RNA dependent RNA polymerase (RdRp). Hantaviruses cause two forms of diseases, hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia, and hantavirus pulmonary syndrome (HPS) in the Americas. The hantaviruses are mainly rodent borne, and humans are mostly infected by inhalation of aerosolized rodent secrete. Human Puumala virus infection results in nephropathia epidemica (NE), a mild haemorrhagic disease.

It is of importance to have a good understanding of the epidemiology and genetics of these viruses for the development of new diagnostic methods and for future vaccine development.

In this thesis we determined the complete viral genome sequence and characterized the structural proteins based on studies of expression and glycosylation patterns, for a unique human virus isolate; performed a genomic analysis of local Puumala viruses and their individual rodent host, Clethrionomys glareolus, from six different locations was performed. It was seen that the virus genetic variation between different locations could be stable over relatively large distances while there could be large variation over a short distance. For the bank voles no such variation could be seen; developed and evaluated Genetic vaccines, based on PCR-generated linear DNA. We showed that it was important to protect these fragments against nuclease degradation at that attachment of a nuclear localization signal peptide further improved the immune response. We also designed, fabricated and evaluated a 2000 probe cDNA-microarray for identification and differentiation of hantaviruses. The chips was based on 12 different strains of six hantaviruses and could differentiate between both different hantaviruses and strains within one hantavirus serotype.

Place, publisher, year, edition, pages
Umeå: Klinisk mikrobiologi , 2005. , 97 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 964
Keyword [en]
Communicable diseases, hantavirus, Puumala virus, nephropathia epidemica, genome sequence, glycosylation, linear DNA vaccine, microarray, identification, genetic variability
Keyword [sv]
National Category
Infectious Medicine
Research subject
Medical Virology
URN: urn:nbn:se:umu:diva-532ISBN: 91-7305-878-5OAI: diva2:143732
Public defence
2005-06-03, A05, 6A, Umeå Universitet, Umeå, 09:00 (English)
Available from: 2005-05-09 Created: 2005-05-09 Last updated: 2009-11-16Bibliographically approved
List of papers
1. Complete gene sequence of a human Puumala hantavirus isolate, Puumala Umeå/hu: sequence comparison and characterisation of encoded gene products.
Open this publication in new window or tab >>Complete gene sequence of a human Puumala hantavirus isolate, Puumala Umeå/hu: sequence comparison and characterisation of encoded gene products.
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2004 (English)In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 105, no 2, 147-155 p.Article in journal (Refereed) Published
Abstract [en]

Puumala virus is a member of the hantavirus genus in the Bunyaviridae family, and the major causative agent of haemorrhagic fever with renal syndrome in Europe. This study was conducted with a human Puumala virus isolate (PUUV Umeå/hu), and contains the determination of the first complete PUUV sequence from a human source. When the relationship to other Puumala viruses was analysed, a possible RNA segment exchange between two local strains of PUUV was noticed. Furthermore, the coding regions of PUUV Umeå/hu S- and M-segments were cloned, and a large set of gene products were expressed in mammalian cells. In addition, postulated N- and O-linked glycosylation sites in the two envelope proteins (Gn and Gc) were investigated individually by site-directed mutagenesis followed by gel-shift analysis. Our data demonstrate that N-linked glycosylation occurs at three sites in Gn (N142, N357 and N409), and at one site in Gc (N937). Also, one possible O-glycosylation site was identified in Gc (T985). We conclude that the diversity between different Puumala virus isolates is high, and consequently characterization of local PUUV isolates is important for clinical diagnostic work. Finally, the obtained results concerning the encoded gene products are of great importance for the design of new vaccines.

Animals, COS Cells, Cercopithecus aethiops, Cloning; Molecular, DNA; Complementary, Electrophoretic Mobility Shift Assay, Gene Expression, Genes; Viral, Genome; Viral, Glycosylation, Hemorrhagic Fever with Renal Syndrome/virology, Humans, Molecular Sequence Data, Mutagenesis; Site-Directed, Phylogeny, Protein Processing; Post-Translational, Puumala virus/classification/*genetics/isolation & purification, RNA; Viral/genetics/isolation & purification, Recombination; Genetic, Sequence Analysis; DNA, Viral Envelope Proteins/chemistry/*genetics/metabolism
urn:nbn:se:umu:diva-15180 (URN)10.1016/j.virusres.2004.05.005 (DOI)15351488 (PubMedID)
Available from: 2008-01-08 Created: 2008-01-08 Last updated: 2012-03-09
2. Genetic variability among Puumala viruses and bank voles in an endemic area (Northern Sweden)
Open this publication in new window or tab >>Genetic variability among Puumala viruses and bank voles in an endemic area (Northern Sweden)
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Manuscript (Other academic)
urn:nbn:se:umu:diva-4574 (URN)
Available from: 2005-05-09 Created: 2005-05-09 Last updated: 2010-01-13Bibliographically approved
3. PCR-generated linear DNA fragments utilized as a hantavirus DNA vaccine
Open this publication in new window or tab >>PCR-generated linear DNA fragments utilized as a hantavirus DNA vaccine
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2002 (English)In: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 20, no 27-28, 3379-3388 p.Article in journal (Refereed) Published
Abstract [en]

The field of DNA vaccines has grown rapidly, and since most such vaccines involve the inoculation of large circular DNA molecules previously propagated in bacteria, several inconveniences (e.g. the presence of antibiotic resistance genes, impurities from bacterial cultures or inefficient uptake of the large and bulky plasmid DNA molecules to the nucleus) are debated. In this study, we have explored the possibility of using smaller and more flexible PCR-generated linear DNA fragments instead. Phosphorothioate (PTO)-modified primers were used successfully to protect the PCR-generated DNA fragments from exonuclease degradation, and by using a nuclear localization signal-peptide to target the linear DNA to the nucleus the immune response against the encoded antigen was further improved. This approach was tested in cell culture using a sensitive reporter system and in vivo with DNA encoding the amino-terminus of the Puumala hantavirus nucleocapsid protein. Our results indicate that linear DNA fragments have a great potential as a genetic vaccine and phosphorothioate modification in combination with a nuclear localization signal peptide increase the stability and targets the linear DNA molecules to the nucleus resulting in an improved biological response examined both in vitro and in vivo.

urn:nbn:se:umu:diva-4575 (URN)10.1016/S0264-410X(02)00265-7 (DOI)12213408 (PubMedID)
Available from: 2005-05-09 Created: 2005-05-09 Last updated: 2012-03-09Bibliographically approved
4. Microarray technology for identification and distinction of hantaviruses.
Open this publication in new window or tab >>Microarray technology for identification and distinction of hantaviruses.
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2004 (English)In: Journal of Medical Virology, ISSN 0146-6615, Vol. 72, no 4, 646-655 p.Article in journal (Refereed) Published
Abstract [en]

DNA microarrays combine high-precision technology with advanced molecular biology to achieve high-throughput screening of DNA fragments. In this study, we investigated the potential of the cDNA microarray technique to identify and discriminate PCR derived amplicons from genetically highly similar viruses. The wide range of sequence variation among hantaviruses makes them suitable as a model for this purpose. The hantaviruses, carried by rodents, cause several hundred thousand cases of severe human disease every year in many parts of the world. A hantavirus-specific microarray, including DNA fragments from 12 viral isolates of six different hantaviruses, was designed. The S and M genome segments were represented by 500-nucleotide overlapping and 250-nucleotide non-overlapping fragments. A considerable ability to distinguish between different hantaviruses was demonstrated using a novel analysis method. Even different isolates of a single virus, were identified correctly despite 90% sequence similarity. The distinction ability was accompanied by a tolerance for smaller sequence differences, which makes the microarray suitable for testing samples containing unknown viruses. Viral genetic material found in samples from the lungs of bank voles caught in the wild was identified precisely, which demonstrated further the potential for this technology.

Animals, Arvicolinae/*virology, Genes; Viral, Genome; Viral, Hantaan virus/classification/genetics, Hantavirus/*classification/*genetics/isolation & purification, Hantavirus Infections/veterinary/virology, Lung/virology, Molecular Probe Techniques, Nucleic Acid Hybridization/*methods, Oligonucleotide Array Sequence Analysis/*methods, Polymerase Chain Reaction, Puumala virus/classification/genetics, RNA; Viral/chemistry/isolation & purification, Rodent Diseases/virology, Seoul virus/classification/genetics, Sin Nombre virus/classification/genetics, Transcription; Genetic, Variation (Genetics)
urn:nbn:se:umu:diva-20713 (URN)10.1002/jmv.20041 (DOI)14981768 (PubMedID)
Available from: 2009-03-24 Created: 2009-03-24 Last updated: 2009-11-16

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