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Genetic and serologic characterization of a Swedish human hantavirus isolate
Umeå University, Faculty of Medicine, Clinical Microbiology, Infectious Diseases.
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 [en]
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: urn:nbn:se:umu:diva-1878ISBN: 978-91-7264-636-0 (print)OAI: oai:DiVA.org:umu-1878DiVA: diva2:142285
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
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.

Keyword
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: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: 2017-12-14
2. Regions of importance for interaction of puumala virus nucleocapsid subunits
Open this publication in new window or tab >>Regions of importance for interaction of puumala virus nucleocapsid subunits
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2006 (English)In: Virus genes, ISSN 0920-8569, E-ISSN 1572-994X, Vol. 33, no 2, 169-174 p.Article in journal (Refereed) Published
Abstract [en]

Puumala virus (PUUV) is a hantavirus that causes a mild form of hemorrhagic fever with renal syndrome in northern and central Europe, and in large parts of Russia. The nucleocapsid (N) protein encoded by hantaviruses plays an important role in the life-cycle of these viruses, and one important function for the N-protein is to oligomerize, surround and protect the viral RNAs. We have identified amino- and carboxy-terminal regions involved in PUUV N-N interactions, which comprise amino acids 100-120 and 330-405. Our findings strengthen the hypothesis that the amino-terminus of the N-protein of hantaviruses holds a more regulatory function regarding N-N interactions, while conserved residues in the carboxy-terminal region, F335 together with F336 and W392, in concert with Y388 and/or F400 seems to play a more critical role in the PUUV N-N formation. This study provides evidence that the amino-terminal regions involved in the N-N interaction of Puumala virus are similar to those reported for Seoul virus (SEOV) and to some extent Hantaan virus (HTNV), even though the identity between PUUV N and SEOV/HTNV N is markedly lower than between PUUV N and Tula virus (TULV) N or Sin Nombre virus (SNV) N.

Keyword
Hantavirus, Nucleocapsid protein, Yeast two-hybrid, Protein–protein interactions, Puumala virus
National Category
Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-22246 (URN)10.1007/s11262-005-0045-5 (DOI)16972031 (PubMedID)
Available from: 2009-05-04 Created: 2009-05-04 Last updated: 2017-12-13Bibliographically approved
3. Cross-reactive immune responses in mice after genetic vaccination with cDNA encoding hantavirus nucleocapsid proteins.
Open this publication in new window or tab >>Cross-reactive immune responses in mice after genetic vaccination with cDNA encoding hantavirus nucleocapsid proteins.
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2007 (English)In: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 25, no 9, 1690-1699 p.Article in journal (Refereed) Published
Abstract [en]

Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in about 150,000 individuals in Eurasia, and several hundred cases of hantavirus pulmonary syndrome (HPS) on the American continent annually. There is consequently a need for rapid diagnostics and effective prevention of hantaviral infections. In this study we have performed DNA-vaccination of mice with full-length genes encoding the immunogenic nucleocapsid protein (NP) of Puumala (PUUV), Seoul (SEOV) and Sin Nombre virus (SNV). The antibody reactivity towards the NPs, and deleted or truncated variants thereof, were studied to localise and investigate the major polyclonal B-cell epitopes. Our findings clearly show that the antibody reactivity in each immunised mouse is unique, not only in a quantitative respect (titers) but also in cross-reactivity and most likely also in the epitope specificity. Our experimental data in combination with B-cell prediction software indicate that strong homologous virus species specific and cross-reactive epitopes are located around amino acid residue 40 in the nucleocapsid proteins.

National Category
Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-22245 (URN)10.1016/j.vaccine.2006.09.082 (DOI)17110000 (PubMedID)
Available from: 2009-05-04 Created: 2009-05-04 Last updated: 2017-12-13
4. Cross-reactive and serospecific epitopes of nucleocapsid proteins of three hantaviruses: prospects for new diagnostic tools.
Open this publication in new window or tab >>Cross-reactive and serospecific epitopes of nucleocapsid proteins of three hantaviruses: prospects for new diagnostic tools.
2008 (English)In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 137, no 1, 97-105 p.Article in journal (Refereed) Published
Abstract [en]

The diagnosis of infectious diseases is sometimes difficult because of extensive immunological cross-reactivity between related viral antigens. On the path of constructing sero-specific antigens, we have identified residues involved in sero-specific and cross-reactive recognition of the nucleocapsid proteins (NPs) of Puumala virus (PUUV), Seoul virus (SEOV), and Sin Nombre virus (SNV) using serum samples from 17 Nephropathia epidemica patients. The mapping was performed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis on a panel of N protein derivatives and alanine-substitution mutants in the three different hantavirus backgrounds. Four regions with different serological profiles were identified encompassing the amino acids (aa) 14-17, 22-24, 26, and 35-38. One of the regions showed strong cross-reactivity and was important for the recognition of SEOV and SNV antigens, but not the PUUV antigen (aa 35-38). Two regions displayed perceivable SEOV characteristics (aa 14-17 and aa 22-24 and 26) and the combined result of the alanine replacements resulted in a synergetic effect against the PUUV antigen (aa 14-17, 22-24, 26).

Keyword
Hantavirus; Puumala virus; Nucleocapsid protein; Human immune response; Cross-reactivity; Epitope mapping
Identifiers
urn:nbn:se:umu:diva-22247 (URN)10.1016/j.virusres.2008.06.003 (DOI)18620010 (PubMedID)
Available from: 2009-05-04 Created: 2009-05-04 Last updated: 2017-12-13

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