Dobrava and Tula hantaviruses from Central Europe: molecular evolution and pathogenic relevance

Dissertation

zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie

eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Diplom-Biologe Boris Klempa
geboren am 5. Dezember 1976 in Malacky, Slowakei

Präsident der Humboldt-Universität zu Berlin

Prof. Dr. Jürgen Mlynek

Dekan: Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Thomas Buckhout, PhD

Gutachter/innen:
1. Prof. Dr. Detlev H. Krüger
2. Prof. Dr. Gholamreza Darai
3. Dr. habil. Milan Labuda, DSc.

Tag der mündlichen Prüfung: 09. Dezember 2004

SUMMARY

Hantaviruses are rodent-borne bunyaviruses that cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. The viruses form a genus Hantavirus within the Bunyaviridae family. They are negative-strand RNA viruses with a genome consisting of three different segments, S, M, and L. Hantaviruses belong to the group of “emerging viruses” exhibiting an increasing significance as human pathogens. To cause human disease, the viruses have to be transmitted from their respective hosts to men.

There is an urgent need to acquire substantial knowledge about the epidemiology, molecular evolution and clinical relevance of hantaviruses circulating in Central Europe. This thesis presents novel data about two European hantaviruses, Dobrava virus (DOBV) and Tula virus (TULV).

DOBV is an important etiologic agent of HFRS in Europe. DOBV strains were found to be hosted by at least two different rodent species, yellow-necked mouse (Apodemus flavicollis) and striped field mouse (A. agrarius). According to their natural hosts they form the distinct genetic lineages DOBV-Af and DOBV-Aa, respectively. We have determined and analysed the complete S and M, and partial L segment nucleotide sequences of sympatrically occurring DOBV-Af and DOBV-Aa strains from Central Europe. Molecular phylogenetic analyses gave evidence for genetic reassortment in the evolution of the virus species. It will be important to see whether such reassortment processes (similar to the situation in influenza viruses which carry segmented genomes, too) can change the virulence of hantaviruses towards humans.

Whereas for virus strains of the DOBV-Af lineage their pathogenic potential towards humans was known from studies on the Balkans, such evidence was still missing for the newly discovered DOBV-Aa lineage in Central Europe. We were able to amplify a DOBV-Aa nucleotide sequence from a DOBV-seropositive HFRS patient in Central Europe. This is the first molecular identification of human infection by DOBV in Central Europe and the first direct proof that a virus strain related to the DOBV-Aa lineage, carried by A. agrarius rodents, is able to cause HFRS.

For future studies on the virus-host interactions of DOBV-Aa, it was important to isolate a viable virus strain. This urgency was underlined by the fact that the Central European DOBV-Aa strains were shown to be only distantly related to the existing DOBV cell culture isolates from Estonia, Slovenia and Greece. Therefore, under biosafety level 3 conditions, we have established a DOBV isolate named Slovakia (SK/Aa) from an A . agrarius animal captured in Slovakia. SK/Aa, as the only isolate clearly belonging to the DOBV-Aa lineage, can be taken as the representative of this virus lineage. The new virus isolate, in comparison to a DOBV-Af strain, was used for serotyping neutralising antibodies of HFRS patients in Central Europe by the use of a focus reduction neutralisation assay. Most patients' sera exhibited a higher end-point titer towards SK/Aa suggesting that DOBV-Aa strains are responsible for most of the DOBV HFRS cases in this region.

TULV is carried by European common voles (Microtus sp.). Its pathogenic potential for humans was rather unknown. We have described the first case of HFRS which can be associated with TULV infection. Moreover, TULV strains detected in M. arvalis near the home village of the patient in North-East Germany clustered with strains from Poland and represent a new, well-supported genetic lineage within the TULV species. In addition to DOBV and longer known Puumala virus, TULV is most likely an additional causative agent of HFRS in Central Europe.

ZUSAMMENFASSUNG

Hantaviren sind Erreger, die von Nagetieren auf den Menschen übertragen werden. In Europa und Asien vorkommende Hantaviren lösen Hämorrhagische Fieber mit Renalem Syndrom (HFRS) aus, in den Amerikas zirkulierende Viren das Hantavirus Cardiopulmonale Syndrom. Die Viren bilden ein eigenes Genus Hantavirus innerhalb der Familie Bunyaviridae. Sie sind Negativstrang-RNA Viren, deren Genom aus drei Segmenten (S, M, L) besteht. Hantaviren gehören zur Gruppe der „emerging viruses“, die durch eine zunehmende Bedeutung als allgemeingefährliche Humanpathogene gekennzeichnet sind. Die einzelnen Virusspecies sind mit unterschiedlichen Nager-Wirtsspecies assoziiert und werden von diesen auf den Menschen übertragen, der einen Fehlwirt darstellt.

Es besteht die dringende Notwendigkeit, neue, grundsätzliche Erkenntnisse zur Epidemiologie, molekularen Evolution und klinischen Relevanz der in Mitteleuropa zirkulierenden Hantaviren zu gewinnen. Die vorgelegte Arbeit beinhaltet derartige Ergebnisse zu zwei europäischen Hantaviren, dem Dobravavirus (DOBV) und dem Tulavirus (TULV).

DOBV ist ein wichtiger HFRS-Erreger in Europa. DOBV Stämme kommen in mindestens zwei verschiedenen Nagerspecies, der Gelbhalsmaus (Apodemus flavicollis) und der Brandmaus (Apodemus agrarius) vor. In Übereinstimmung mit diesen unterschiedlichen natürlichen Wirten konnten wir zeigen, dass die Virusstämme zwei genetische Linien innerhalb der DOBV Species bilden: DOBV-Af und DOBV-Aa. Es wurden die vollständigen Nukleotidsequenzen der S- und M-Segmente sowie partielle Sequenzen der L-Segmente von sympatrisch vorkommenden DOBV-Af und DOBV-Aa Stämmen aus Mitteleuropa bestimmt und für molekularphylogenetische Analysen genutzt. Die Analysen zeigten das Vorkommen von Reassortmentprozessen der Genomsegmente während der Evolution der Virusspecies. Für Influenzaviren (die ebenfalls segmentierte Genome besitzen) ist bekannt, dass Reassortment die wichtigste genetische Grundlage für Veränderungen der Pathogenität der Viren ist. Unsere Ergebnisse schaffen nun die Voraussetzung, diese Prozesse ebenfalls für die Hantaviren zu untersuchen.

Während für Infektionen mit Virusstämmen der DOBV-Af Linie in Südosteuropa das Auftreten von mittelschwerem und schwerem HFRS beschrieben wurde, gab es bisher keinen Beweis für die Humanpathogenität der neu entdeckten DOBV-Aa Stämme. Es konnte nun die virale Nukleotidsequenz aus einem DOBV-seropositiven HFRS-Patienten in Mitteleuropa amplifiziert und analysiert werden. Damit wurde erstmalig der molekulare Beweis erbracht, dass DOBV in Mitteleuropa HFRS auslöst und dass die DOBV-Aa Linie aus Apodemus agrarius humanpathogen ist.

Für weitere Forschungen zu den Virus-Wirt-Interaktionen von DOBV-Aa ist das Vorhandensein eines vermehrungsfähigen DOBV-Aa Virusisolates die entscheidende Voraussetzung. Diese Notwendigkeit wird durch die Tatsache unterstrichen, dass die mitteleuropäischen DOBV-Aa Stämme mit den existierenden DOBV Isolaten aus Slowenien, Griechenland und Estland nur relativ entfernt verwandt sind. Unter Laborbedingungen der Sicherheitsstufe 3 wurde aus einer in der Slowakei gefangenen Apodemus agrarius Maus ein neues Virusisolat gewonnen, welches „Slovakia (SK/Aa)“ genannt wurde. SK/Aa ist das bisher einzige Virusisolat, das die DOBV-Aa Linie repräsentiert. Es wurde gemeinsam mit einem Isolat der DOBV-Af Linie zur vergleichenden Typisierung der Antikörper von mitteleuropäischen HFRS-Patienten mittels Fokusreduktionsneutralisationstest eingesetzt. Die Seren der meisten Patienten zeigten die höchsten neutralisierenden Antikörpertiter gegenüber SK/Aa, was die Schlussfolgerung zulässt, dass DOBV-Aa Stämme für die meisten DOBV-Infektionen in Mitteleuropa verantwortlich sind.

TULV wird durch die Feldmaus (Microtus arvalis) beherbergt. Die Übertragbarkeit auf den Menschen bzw. die Fähigkeit zur Auslösung von HFRS waren bisher wenig bzw. nicht bekannt. Wir haben den ersten Fall von HFRS gefunden, der mit einer TULV Infektion assoziiert ist. Aus demselben geographischen Gebiet in Nordostdeutschland, in dem dieser Patient lebt, konnten aus Feldmäusen TULV Nukleotidsequenzen amplifiziert werden. In phylogenetischen Analysen clustern sie mit Stämmen aus Polen und bilden mit diesen gemeinsam eine eigene, neue genetische Linie innerhalb der TULV Species. Ausser dem hier untersuchten DOBV und dem länger bekannten Puumalavirus ist TULV offenbar das dritte Hantavirus, das in Mitteleuropa HFRS hervorruft.

Contents

• 1. INTRODUCTION
  • 1.1 Short historical overview
  • 1.2 Hantaviruses within the Bunyaviridae family
  • 1.3 Genome structure and replication
  • 1.4 Pathogenesis
    • 1.4.1 Infection in natural host vs. humans
    • 1.4.2 Human diseases
    • 1.4.3 Hemorrhagic fever with renal syndrome
    • 1.4.4 Hantavirus cardiopulmonary syndrome
  • 1.5 Virus ecology
  • 1.6 Evolution of hantaviruses
    • 1.6.1 Methods employed in phylogenetic analysis of viral sequences
    • 1.6.2 Phylogenetic analysis of hantaviruses
  • 1.7 Dobrava hantavirus
  • 1.8 Tula hantavirus
  • 1.9 Aims of the study
• 2. MATERIALS AND METHODS
  • 2.1 Trapping of rodents
  • 2.2 Screening of rodents specimens
    • 2.2.1 Enzyme-linked immunosorbent assay (ELISA)
    • 2.2.2 Immunoblotting
  • 2.3 RNA extraction
  • 2.4 DNA extraction
  • 2.5 PCR
    • 2.5.1 Hantavirus initial screening RT-PCR
    • 2.5.2 RT-PCR for the sequencing of DOBV complete S and M segments and partial L segment sequence
    • 2.5.3 PCR of rodent genetic markers
  • 2.6 Cloning and sequencing
  • 2.7 Sequence comparison, phylogeny and recombination analysis
  • 2.8 Virus isolation
  • 2.9 Immunofluorescence assay
    • 2.9.1 Preparation of slides for indirect immunofluorescence assay (IFA)
    • 2.9.2 Staining of slides
  • 2.10 Virus titration
  • 2.11  Chemiluminiscent focus reduction neutralisation test (c-FRNT)
• 3. RESULTS
  • 3.1 Screening of rodents from Slovakia
  • 3.2 Genetic analysis of complete S and M segment sequences from distinct Dobrava hantavirus subtypes of Apodemus agrarius and A. flavicollis
    • 3.2.1 Comparison of S segment sequences
    • 3.2.2 Comparison of M segment sequences
    • 3.2.3 Phylogenetic trees and proof of reassortment
    • 3.2.4 Recombination analysis
    • 3.2.5 Identification of putative host-specific differences in the virus-coded N and GPC
  • 3.3 Genetic diversity of DOBV on single geographical locus
    • 3.3.1 DOBV in Rozhanovce locality, Eastern Slovakia
    • 3.3.2 Rodent genetics
  • 3.4 Isolation of DOBV from A. agrarius captured in East Slovakia
    • 3.4.1 Virus isolation and titration
    • 3.4.2 Sequence analysis of SK/Aa genomic segments
    • 3.4.3 Phylogenetic analysis
    • 3.4.4  In vitro evolution of virus during passaging
    • 3.4.5 Antigenic characterisation of the DOBV-SK/Aa isolate
  • 3.5 First genetically confirmed DOBV infection in Central Europe
    • 3.5.1 Clinical description
    • 3.5.2 DOBV infection confirmed by sequence analysis
  • 3.6 First report of DOBV-Af associated HFRS cases in Slovakia
  • 3.7 Occurrence of renal and pulmonary syndrome in a region of North-East Germany where Tula hantavirus circulates
    • 3.7.1 HFRS clinical case associated with TULV infection
      • 3.7.1.1 Clinical description
      • 3.7.1.2 Serological data
    • 3.7.2 Detection of TULV in Microtus arvalis from North-East Germany
• 4. DISCUSSION
  • 4.1 Phylogenetic classification of Central European DOBV-Aa and DOBV-Af strains
  • 4.2 Genetic reassortment of genome segments in Saa/160V
  • 4.3 Genetic recombination between DOBV-Af and DOBV-Aa lineages
  • 4.4 Role of genetic differences for host adaptation
  • 4.5 Evolution of DOBV and the problem of hantavirus species definition
  • 4.6 Presence of distinct DOBV-Aa strains on single geographical locus
  • 4.7 A new DOBV strain isolated from A. agrarius
  • 4.8 HFRS in West Slovakia associated with DOBV-Af infection
  • 4.9 First direct evidence that DOBV causes HFRS in Central Europe
  • 4.10 Questioning the hypothesis about different pathogenicity of DOBV-Af and DOBV-Aa towards humans
  • 4.11  Does pathogenic TULV circulate in North-East Germany?
• REFERENCES
• Abbreviations
• Acknowledgement
• List of Publications
• Communications to Scientific Meetings
• Selbständigkeitserklärung

List of tables

• Table 1: Family Bunyaviridae. Taxonomical classification according to 7th report of The International Committee on Taxonomy of Viruses (ICTV) (Elliott et al., 1999) and selected significant pathogens
• Table 2: Species in the genus Hantavirus, according to their rodent host.
• Table 3: The current state of the detection of DOBV. The summary of epidemiological and epizootiological data on DOBV from three regions of Europe. The data which were extended by this work are in bold.
• Table 4: List of PCR primers used for amplification of hantaviral RNA and rodent DNA
• Table 5: List of hantavirus strains and their abbreviated names used in sequence analysis
• Table 6: Prevalence of antibodies to HTNV, PUUV and/or DOBV among wild rodents in Slovakia, 1995-2001.
• Table 7: S segment nucleotide and amino acid percent identity of DOBV strains, HTNV, and SEOV
• Table 8: M segment nucleotide and amino acid percent identity of DOBV strains, HTNV, and SEOV
• Table 9: S segment nucleotide percent identity of Rozhanovce-originating and other DOBV strains
• Table 10: M segment nucleotide percent identity of Rozhanovce-associated and other DOBV strains
• Table 11: Apodemusmice used for amplification and sequencing of mitochondrial markers 12S rRNA gene and D-loop
• Table 12: 12s rRNA gene nucleotide sequence percent identity of Apodemus mice trapped on selected localities of Slovakia
• Table 13: Multiple alignment of a partial 12S rRNA gene sequence (372 nt) from Apodemus sp. mice captured in Slovakia; only polymorphic sites are shown.
• Table 14: Multiple alignment of mitochondrial DNA (1167 nt) including D-loop of A. agrariusmice captured in Slovakia; only polymorphic sites are shown.
• Table 15: D-loop nucleotide sequence percent identity of A. agrariusmice trapped on selected localities of Slovakia
• Table 16: Complete S and M segment nucleotide and amino acid sequence identities of SK/Aa with other DOBV, HTNV and SEOV isolates.
• Table 17: Reactivity of DOBV isolates Slovakia and Slovenia in IFA with the panel of anti-N protein monoclonal antibodies.
• Table 18: Comparison of DOBV isolates SK/Aa and Slo/Af with respect to their neutralisation by convalescent sera from Central European HFRS patients.
• Table 19: Selected laboratory and clinical findings of the H169 patient with acute DOBV infection
• Table 20: Partial S segment nucleotide (599 nt, nt position 357-955) and amino acid (71 aa, aa position 115-185) percent identity of patient-derived sequence H169, other DOBV strains, HTNV, and SEOV
• Table 21: Clinical and laboratory data of two DOBV-Af patients according to criteria for estimating severity of the various phases of HFRS
• Table 22: Further laboratory and clinical data of the Trencin patients of acute DOBV infection
• Table 23: Detection of hantavirus-specific antibodies in serum samples of HFRS patients from West Slovakia
• Table 24: Detection of hantavirus-specific antibodies in serum samples of the patient
• Table 25: Percent sequence identities among North-East German and other European TULV strains based on entire S segment and N protein sequences
• Table 26: Amino acid exchanges in N protein of TULV strains

List of figures

• Figure 1: Scheme of a hantavirus particle (own drawing).
• Figure 2: Hantavirus genome structure (adapted from Plyusnin et al., 1996).
• Figure 3: Phylogenetic relationship between the main hantavirus representatives corresponding to the three subfamilies of Murinae rodents.
• Figure 4: Apodemus flavicollis (yellow necked mouse) -above- and A. agrarius (striped field mouse) -below-, the natural hosts of DOBV (own pictures).
• Figure 5: Microtus arvalis (common vole), the natural host of TULV (© Rollin Verlinde – www.natuurbeleving.be)
• Figure 6: Trapping of small rodents using Swedish bridge metal traps.
• Figure 7: Map of Central Europe.
• Figure 8: Phylogenetic ML trees from the complete S segment nucleotide sequences of DOBV and further hantaviruses, computed with TREE-PUZZLE.
• Figure 9: Phylogenetic ML tree of the available complete M segment nucleotide sequences of different members of the DOBV lineage and further hantaviruses computed with TREE-PUZZLE.
• Figure 10: Recombination analysis for the M segment ORFs.
• Figure 11: Putative host specific amino acid exchanges in the N protein (Figure 11A) and the glycoproteins (Figure 11B) of DOBV strains from A. flavicollis vs. A. agrarius hosts.
• Figure 12: Phylogenetic ML tree based on the partial S segment nt sequences (377-935 nt) of Rozhanovce-originating and other DOBV strains, computed with TREE-PUZZLE.
• Figure 13: Map of Slovakia with trapping localities for virus and rodent genetics studies.
• Figure 14: Maximum likelihood phylogenetic trees of DOBV strains based on (a) complete S and (b) complete M segment ORF nucleotide sequences (corresponding to S segment nt sequence position 36 - 1,325 and M segment nt position 41 to 3,445 of SK/Aa, respectively).
• Figure 15: Maximum likelihood phylogenetic trees of DOBV virus isolates based on (a) partial (541 nt, position 109 - 649) L segment sequences, (b) complete S ORF, and (c) complete M ORF nucleotide sequences (corresponding to S segment nt position 36 - 1,325 and M segment nt position 41 to 3,445, respectively).
• Figure 16: Results of diagnostic DOBV S segment specific PCR with samples from patient H169 visualised by electrophoresis in 1.0% agarose gel after 20 min of staining in ethidium bromide (1 μg/ml) .
• Figure 17: Phylogenetic ML tree based on the partial S segment nucleotide sequences (378-934 nt) of H196 and other DOBV strains, computed with TREE-PUZZLE.
• Figure 18: Phylogenetic ML tree based on the partial S segment nucleotide sequences (378-762 nt) of H196 and other DOBV strains, computed with TREE-PUZZLE.
• Figure 19: ML phylogenetic tree of TULV strains based on complete S segment nucleotide sequences
• Figure 20: Schematic illustration of the proposed scenario of genetic reassortment during DOBV evolution. S and L segments of Saaremaa virus are phylogenetically related to their counterparts in DOBV-Af, whereas the M segment (determining the antigenicity of the virus envelope) resembles that of DOBV-Aa.