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Complete gene sequence of a human Puumala hantavirus isolate, Puumala Umeå/hu: sequence comparison and characterisation of encoded gene products.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases. (Clas Ahlm)
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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.

Place, publisher, year, edition, pages
2004. Vol. 105, no 2, 147-155 p.
Keyword [en]
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
Identifiers
URN: urn:nbn:se:umu:diva-15180DOI: 10.1016/j.virusres.2004.05.005PubMedID: 15351488OAI: oai:DiVA.org:umu-15180DiVA: diva2:154852
Available from: 2008-01-08 Created: 2008-01-08 Last updated: 2017-12-14
In thesis
1. Genetic and serologic characterization of a Swedish human hantavirus isolate
Open this publication in new window or tab >>Genetic and serologic characterization of a Swedish human hantavirus isolate
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hantaviruses are found practically all over the world and cause hemorrhagic fevers in man. Each year about 150,000 people are hospitalized in these zoonotic infections which can be of two types: hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS), depending on the infecting virus. Hantavirus infections are emerging infectious diseases. That is, the number of reported cases of hantaviral disease is increasing, new hantaviruses are discovered continually, and already known hantaviruses are expected to spread to new areas. Therefore, knowledge and monitoring of these viruses are imperative from a public health perspective.

In this thesis, the characterization of a local human Puumala (PUUV) virus isolate is described. Genetic and serological relationships to other hantaviruses are investigated and the viral protein interactions, critical for genome packaging and assembly, are studied. We found that the nucleotide and amino acid sequences of the local PUUV strains are significantly different from the PUUV prototype strain Sotkamo, a difference that indicates that there might be a risk of misdiagnosing PUUV infected patients when using reagents derived from the prototype strain. These data contributed to the introduction of locally derived diagnostic tools to the Laboratory of Clinical Virology at the Umeå University hospital, which is the reference centre for hantaviral diseases in Sweden. Furthermore, when studying the underlying mechanisms of genome packaging, we identified several regions and amino acids absolutely required for nucleocapsid protein interactions. Also, a region that appeared to regulate this interaction was discovered. Finally, the serological immune responses in DNA-vaccinated mice and PUUV infected patients were investigated. We found that the cross-reactive antibody response in vaccinated mice and in infected individuals was unique and independent of homologous titres. Furthermore, four immunodominant epitopes with specific cross-reactive characteristics were identified.

Our findings have highlighted the complexity of the serological immune responses to hantavirus infections, and they emphasize the importance of customizing the diagnostic tools and performing clinical analyses on locally derived strains. In conclusion, we believe that these results are valuable in the development of new serological, genetic, and epidemiological tools.

Place, publisher, year, edition, pages
Umeå: Infektionssjukdomar, 2008. 72 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1204
Keyword
hantavirus, puumalavirus, diagnostics, HFRS, nucleocapsid protein, B-cell epitopes, epitope-mapping, protein interactions, glycosylation, antibody response, cross-reactivity
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-1878 (URN)978-91-7264-636-0 (ISBN)
Public defence
2008-10-24, Sal D, 1D, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2008-10-10 Created: 2008-10-10 Last updated: 2009-05-04Bibliographically approved
2. Implications of Local Puumala Hantavirus Genetics and Epidemiology for Diagnostics and Vaccine Development
Open this publication in new window or tab >>Implications of Local Puumala Hantavirus Genetics and Epidemiology for Diagnostics and Vaccine Development
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.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 964
Keyword
Communicable diseases, hantavirus, Puumala virus, nephropathia epidemica, genome sequence, glycosylation, linear DNA vaccine, microarray, identification, genetic variability, Infektionssjukdomar
National Category
Infectious Medicine
Research subject
Medical Virology
Identifiers
urn:nbn:se:umu:diva-532 (URN)91-7305-878-5 (ISBN)
Public defence
2005-06-03, A05, 6A, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2005-05-09 Created: 2005-05-09 Last updated: 2009-11-16Bibliographically approved

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