University of Veterinary Medicine Hannover
Molecular evolution of the bank vole-borne Puumala hantavirus in Central Europe
Thesis
Submitted in partial fulfilment of the requirements for the degree -Doctor of Veterinary Medicine-
Doctor Medicinae veterinariae (Dr. med. vet)
By Hanan Sheikh Ali Khartoum, Sudan Hannover 2015
Academic supervision:
Prof. Dr. Martin H. Groschup, University of Veterinary Medicine Hannover, Friedrich- Loeffler-Institut, Isle of Riems.
1. Referee: Prof. Dr. Martin H. Groschup, University of Veterinary Medicine Hannover, Friedrich-Loeffler-Institut, Isle of Riems
2. Referee: Prof. Dr. Paul Becher, University of Veterinary Medicine Hannover, Institute of Virology.
Day of the oral examination: 11th of March 2015
This work was supported by German Academic Exchange Service (DAAD) desk number 413, Eastern and Southern Africa; code number A/09/ 90015.
To my Family
Contents
List of figures ... I List of tables ... II Summary ... III Zusammenfassung ... V
1. Introduction ... 1
1.1 Discovery and classification of hantaviruses ... 1
1.1.2. Hantavirus structure and genome organization ... 2
1.1.2.1. Virion structure ... 2
1.1.2.2. Genome organization ... 4
1.1.5. Hantavirus evolution... 10
1.2. Human hantavirus infections ... 12
1.2.1 Clinical features of human hantavirus infections: HFRS and NE ... 12
1.2.2. Diagnosis of hantavirus infection in humans ... 13
1.2.3. Prevention and therapy of hantavirus infection ... 14
1.3. Bank vole-associated PUUV ... 20
1.3.1. PUUV and related arvicoline-borne hantaviruses ... 20
1.3.2. Phylogeography of bank vole ... 22
1.4. PUUV and other hantaviruses in Germany ... 24
2. Objectives ... 26
3. Publications ... 27
3.1. Paper I ... 27
3.2. Paper II ... 28
3.3. Paper III ... 54
4. Results and Discussion ... 85
4.1. cyt b analysis for molecular species identification and differentiation of bank vole lineages ... 85
4.2. PUUV in Central Europe ... 89
4.3 Genome organisation of a PUUV strain from Central Europe ... 91
4.4 Spatial and temporal evolution of PUUV ... 97
5. Conclusions and outlook ... 100
References ... 103
List of Publications ... 130
Presentation at Scientific meetings and workshops ... 131
Affidavit ... 133
Acknowledgement ... 134
List of figures
Figure 1: Structure of hantavirus ... 3
Figure 2: Steps of hantavirus replication in the host cell ... 7
Figure 3: Distribution of bank vole lineages in Europe ... 23
Figure 4: Phylogenetic tree of bank vole-based cyt b gene sequences ... 88
Figure 5: Human hantavirus cases in Lower Saxony ... 90
Figure 6: Complete genome organization and structure of the bank vole-derived PUUV strain Astrup. ... 91
Figure 7: Prediction of the secondary structure of N protein from strain Astrup ... 92
Figure 8: Amino acid alignment of motif D/E in RdRp ... 94
Figure 9: Hydropathy plot of the predicted N protein, GPC and RdRp of PUUV strain Astrup ... 95
List of tables
Table 1: Consensus 3´ and 5´ terminal nucleotide sequences of bunyavirus genomes ... 2
Table 2: Hantavirus seroprevalence and clinical cases in Europe ... 17
Table 3: Cyt b-based identification of small mammal species in Germany ... 86
Table 4: Detection and prevalence of PUUV in bank voles from Central European countries ... 90
Table 5: Pairwise nucleotide (above the diagonal) and amino acid (below the diagonal) sequence identity of novel strains and other PUUV strains ... 97
II
Hantaviruses are initially thought to be rodent-borne pathogens, recently novel hantaviruses have been discovered in shrews, moles and bats. The genome of hantaviruses consists of three genome segments, small (S), medium (M) and large (L) segments. Currently, only certain rodent-borne hantaviruses are known to be pathogenic to human. Puumala virus (PUUV) is causing a mild to moderate course of Haemmorhagic fever with renal syndrome in Europe, with an average of 10,000 human cases reported annually. The main host of PUUV is the bank vole (Clethrionomys glareolus), a cricetide rodent distributed in almost all parts of Europe.
The study aimed to develop tools for a parallel analysis of the phylogeography of PUUV and the bank vole in Central Europe, and Germany in particular, on a large and small geographical scale.
Therefore, novel methods for molecular rodent species identification and bank vole lineage discrimination as well as for the molecular differentiation of PUUV strains should be established.
A novel molecular assay using cytochrome b (cyt b) gene-derived degenerated primers was developed for molecular identification of small mammals. The assay was validated for small mammals in Germany comprising representatives of the orders like Rodentia, Soricomorpha, Erinaceomorpha, Lagomorpha, Carnivora and Chiroptera. Thereafter, this method was successfully applied for small mammal specimens from other parts of Europe, Asia and Africa. In addition, the novel assay also was used to differentiate the genetic bank vole lineages in Central Europe. In north-eastern Poland bank voles belong to the Carpathian and Eastern lineages. The investigation of bank voles from Germany revealed the presence of three different lineages, the Western, Eastern and Carpathian lineage with different, but partially overlapping distribution in Germany. Future studies have to prove the distribution of the different bank vole lineages in more details to understand their recolonization routes in Europe and the pathogen association.
The first complete genome sequence of a PUUV strain from Central Europe was determined by a combination of a primer-walking and RNA ligation approach. The length of the S, M and L segments of the bank vole-derived PUUV strain from Astrup, Lower Saxony, were found to be 1,828, 3,680 and 6,550 nucleotides, respectively. The novel strain showed a similar genome organization like other PUUV strains with an identity of 80.1% to 84.7% at the level of the Hanan Sheikh Ali
Molecular evolution of the bank vole- borne Puumala hantavirus in Central Europe
nucleotide sequence, and between 89.5% and 98.1% for the deduced amino acid sequences. A sliding-window analysis of PUUV strain sequences revealed different level of sequence variability along the whole genome. The highest sequence variability was found at the 3´NCR of N protein and GPC, whereas regions between residues 876 and 1258 in RdRp showed high sequence conservation. The identification of regions with different levels of sequence variability will allow a future rational design of RT-PCR assays to study the molecular evolution of PUUV at different scales, i.e. within the host, in the same populations and in different populations.
The investigations of PUUV in bank voles from Germany and north-eastern Poland confirmed the presence of PUUV in almost all parts of Europe, but with very different prevalences and a non-homogenous distribution. The initial study in Poland indicated the presence of PUUV infections in bank voles from the Eastern and Carpathian lineage. In contrast to results from different endemic parts of Germany the prevalence of PUUV infections in the bank voles from north-eastern Poland was rather low. An initial study of the PUUV sequence variation within the same animal was performed for bank voles from north-eastern Poland and the district Osnabrück.
Nucleotide sequence determination of cloned S and M segment derived PUUV sequences suggest the presence of a quasispecies, although the frequency of nucleotide substitutions was found to be low. Future investigations will have to combine the ongoing bank vole field study approach with in vitro and animal model studies on the susceptibility of the different bank vole lineages for PUUV infection. In addition, the long-term study in the district of Osnabrück should be used for analysis of the spatial and temporal microevolution of PUUV and to prove the presence and variation of the PUUV quasispecies within persistently infected voles. These studies will profit from the future application of Next Generation Sequencing approaches.
IV
Hantaviren wurden ursprünglich für ausschließlich Nagetier-assoziierte Pathogene gehalten.
Jedoch wurden in den vergangenen Jahren viele neue Hantaviren in Spitzmäusen, Maulwürfen und Fledermäusen entdeckt. Das Genom der Hantaviren besteht aus drei Genomsegmenten small (S), medium (M) and large (L). Gegenwärtig sind nur Nagetier-übertragene Hantaviren als humanpathogen bekannt. Das Puumala virus (PUUV) verursacht eine milden bis
moderaten Verlauf des Hämorhagischen Fiebers mit renalem Syndrom in Europa, mit durchschnittlich 10.000 humanen Fällen jährlich. Der Reservoirwirt des PUUV ist die Rötelmaus (Clethrionomys glareolus), eine Wühlmaus, die in fast ganz Europa vorkommt.
Das Ziel der Untersuchungen bestand in der Entwicklung von Methoden für eine parallele Analyse der Phylogeographie des PUUV und der Rötelmaus in Mitteleuropa, und
insbesondere Deutschland, in großem und kleinem geografischen Maßstab. Deshalb wurden neue Methoden sowohl für die molekulare Identifikation von Nagetierarten und die Unterscheidung von Rötelmauslinien als auch die molekulare Differenzierung von PUUV- Stämmen etabliert. Hierfür wurde zunächst ein neuer molekularer Test auf der Basis Cytochrom b (cyt b)-spezifischer degenerierter Primer zur molekularen Identifikation von Kleinsäugern etabliert. Dieser Assay wurde für Kleinsäuger aus Deutschland validiert, die den Ordnungen Rodentia, Soricomorpha, Erinaceomorpha, Lagomorpha, Carnivora und Chiroptera angehören. Anschließend wurde die Methode erfolgreich für Kleinsäugerproben aus anderen Teilen Europas, Asiens und Afrikas angewendet. Daneben wurde der neue Test zur Differenzierung von genetischen Linien der Rötelmaus in Mitteleuropa angewendet.
Rötelmäuse aus Nordost-Polen wurden der Karpatischen und der Östlichen Linie zugeordnet.
Die Untersuchung von Rötelmäusen aus Deutschland zeigte das Vorkommen von drei genetischen Linien, der Westlichen, Östlichen und Karpatischen Line, mit einer unterschiedlichen, aber partiell überlappenden Verbreitung in Deutschland. Für das
Verständnis der Rekolonierungsrouten der Rötelmauslinien und der mit ihnen assoziierten Krankheitserreger sind zukünftige detailliertere Untersuchungen der verschiedenen Rötelmauslinien erforderlich.
Zusammenfassung Hanan Sheikh Ali
Moleculare Evolution der Rötelmaus getragenen Puumala Hantavirus in Mitteleuropa
Die erste komplette Genomsequenz eines PUUV-Stammes aus Mitteleuropa wurde durch eine Kombination von Primer-Walking und RNA-Ligation bestimmt. Die Länge der S-, M- und L- Segmente des PUUV-Stammes au seiner Rötelmaus aus Astrup, Niedersachsen, betragen – 1828, 3680 bzw. 6550 Nukleotide. Der neue Stamm zeigte eine zu allen anderen PUUV-Stämmen ähnliche Genomorganisation mit einer Identität von 80,1% bis 84,7% auf Nukleotidsequenzebene und zwischen 89,5% und 98,1% auf der Aminosäuresequenzebene. Eine sliding-window-Analyse von PUUV-Sequenzen zeigte entlang der drei Genomsegmente Regionen mit unterschiedlicher Variabilität. Die höchste Sequenzvariabilität wurde in der 3´-NCR gefunden, während die RdRp- Region zwischen den Aminosäuren 876 und 1258 hochkonserviert ist. Die Identifikation von Regionen mit unterschiedlichem Niveau der Sequenzvariabilität wird zukünftig ein rationales Design von RT-PCR-Assays für die Untersuchung der molekularen Evolution des PUUV auf unterschiedlichen Stufen, im Reservoirwirt, in der gleichen Population oder zwischen Populationen, erlauben.
PUUV-Untersuchungen in Rötelmäusen aus Deutschland und Nordostpolen bestätigte das breite geografische Vorkommen des Virus in fast ganz Europa, aber mit sehr verschiedenen Prävalenzen und einer inhomogenen Verbreitung. Die Pilotstudie in Polen zeigte das Vorkommen von PUUV-Infektionen in Rötelmäusen der Östlichen und Karpatischen Linie. Im Gegensatz zu Ergebnissen in verschiedenen Endemiegebieten in Deutschland war die Prävalenz der PUUV-Infektionen in Rötelmäusen aus Nordostpolen relativ gering. Eine initiale Studie der PUUV-Sequenzvariation in einzelnen Tieren wurde für ausgewählte Rötelmäuse aus Nordostpolen und aus dem Landkreis Osnabrück durchgeführt. Die Nukleotidsequenzbestimmung von klonierten PUUV-S- und -M-Segment-Sequenzen deutet auf das Vorkommen von Quasispezies hin, obgleich die Häufigkeit von Nukleotidsubstitutionen gering war. Zukünftige Untersuchungen sollten Feldstudien an Rötelmäusen mit in vitro- und Tiermodell-Studien kombiniert werden, um die Suszeptibilität der verschiedenen Rötelmauslinien für eine PUUV-Infektion zu überprüfen. Außerdem sollte eine Longitudinalstudie im Landkreis Osnabrück für die Analyse der räumlichen und zeitlichen Mikroevolution des PUUV und das Vorkommen und die Variation einer PUUV-Quasispecies in
VI
persistent infizierten Rötelmäusen genutzt werden. Diese Studien werden von einer zukünftigen Anwendung der Next Generation Sequencing-Technologie profitieren.
1. Introduction
1.1 Discovery and classification of hantaviruses
Diseases with symptoms related to hantaviruses have been retrospectively identified in earlier times, mainly during military conflicts (Johnson, 2001). Such a disease is the Korean hemorrhagic fever (KHF) described during Korean War in the 1950s among UN soldiers. The causative agent of KHF was discovered in 1978 from the lung tissue of a striped field mouse, Apodemus agrarius, and was designated as Hantaan virus (HTNV), after a small river in south Korea (Lee et al., 1978). Further, in late 1970s in the tissue of a bank vole (Clethrionomys glareolus), from an endemic area of nepropathia epidemica (NE) in Finland, a novel hantavirus was identified. This newly found virus was designated Puumala virus (PUUV), after the small town in south eastern Finland, where the vole was captured (Lahdevirta, 1971; Brummer- Korvenkontio et al., 1980). Later, Seoul virus (SEOV) was discovered in lung tissue specimens from urban rats, Rattus norvegicus and Rattus rattus, captured near to Seoul city (Lee et al., 1982). Another hantavirus was isolated and partially characterized from lung tissue of meadow voles (Microtus pennsylvanicus) on the American continent, and named Prospect Hill virus (PHV) (Lee et al., 1985). Another murinae-borne hantavirus was isolated from a yellow-necked field mouse Apodemus flavicollis in Slovenia, former Yugoslavia (Avsic-Zupanc et al., 1992).
This virus species was later designated by the International Committee of Taxonomy of Viruses Dobrava-Belgrade virus (DOBV). Likewise, a presumptive arbovirus was isolated from a shrew (Suncus murinus) in India (Carey et al., 1971), but only later on found to represent a shrew-borne hantavirus (Yashina et al., 2010).
In northern America, after the death of two young adults from Navajo nation near to New Mexico, the genetic investigation of these patients and rodent tissue (Peromyscus maniculatus) revealed a new virus related to hantaviruses. This hantavirus causing a pulmonary syndrome was finally named Sin Nombre virus (SNV) (Nichol et al., 1993). Later, further hantaviruses were discovered from additional rodent species causing a similar disease in Argentina and Chile (Levis et al., 1997; Toro et al., 1998). The causative agent of this disease in southern America was named Andes virus (ANDV), worth noting, that this virus was reported to be transmitted from
person-to-person (Padula et al., 1998). Recently, additional hantaviruses have been discovered in shrews, moles and bats, but their pathogenicity to human remains unknown (Yashina et al., 2010;
Sumibcay et al., 2012; Weiss et al., 2012; Guo et al., 2013)
Hantaviruses represent the genus Hantavirus within the Bunyaviridae family, which contains four additional genera: Tospovirus, Nairovirus, Phlebovirus, and Orthobunyavirus. Hantaviruses can be distinguished from other family members by the consensus terminal nucleotide sequences of the three segments which are AUCAUCAUCCUG at 3’ and UAGUAGUA at 5’ end (Table 1).
Additionally, the absence of cross-reactivity of hantaviruses with other members of the family made it to be a separate genus (Jonsson and Schmaljohn, 2001).
Table 1: Consensus 3´ and 5´ terminal nucleotide sequences of bunyavirus genomes Orthobunyavirus 3' UCAUCACAUGA ………UCGUGUGAUGA 5'
Hantavirus 3' AUCAUCAUCUG ………AUGAUGAU 5'
Nairovirus 3' AGAGUUUCU ……… AGAAACUCU 5'
Phlebovirus 3' UGUGUUUC ……….. GAAACACA 5'
Tospovirus 3' UCUCGUUA ……….CUAACGAGA 5'
(taken from (Elliott and Blakqori, 2011) with kind permission from the authors)
1.1.2. Hantavirus structure and genome organization 1.1.2.1. Virion structure
Hantaviruses are enveloped viruses with a segmented genome of negative polarity (Fig. 1). The virion is spherical with a diameter range from 120 to 350 nm and a density between 1.15 to 1.18 g/ml (White et al., 1982; Huiskonen et al., 2010). The virion contains three genome segments, which are encapsidated by the nucleocapsid (N) protein and form ribonucleoproteins (RNPs). The N protein assembles into a trimeric form, which is able to discriminate between viral and non- viral RNA molecules. Moreover, it specifically recognizes the terminal panhandle structure and facilitates the function of the viral polymerase to initiate the transcription (Nemirov et al., 2004;
2
Mir and Panganiban, 2006). The trimerization domain is mainly localized in the N-terminal 77 amino acids (aa) and highly conserved among all hantaviruses (Alminaite et al., 2006).
Figure 1: Structure of hantavirus
Schematic structure (A) and (B) electron microscopic image of TUV-infected Vero E6 cells. M-RNP= M-segment- Ribonucleoprotein. L-RNP= L-segment ribonucleoprotein. S-RNP= S-ribonucleoprotein. Gc and Gn= glycoproteins.
The figure was taken from (Schlegel et al., 2014), Copyright (2014), with permission from Elsevier.
Additionally, the amino acid terminal domains stretches from residues 1- 34 and 38- 80 were predicted to form coiled-coil structures which contributed to N-protein trimerization and also has been shown the C- terminal half of the N protein plus the 40 residues of the N-terminal act as interaction domain in the hanatavirus N protein (Alfadhli et al., 2001). The N protein and the RNA-dependent RNA-polymerase (RdRp) are enclosed inside a host cell-derived lipid envelop (Hepojoki et al., 2012). The 5 nm thick envelop contains protruding spikes derived from the glycoproteins (Gn and Gn) and extending 7 nm from the membrane (Goldsmith et al., 1995;
Hussein et al., 2011; Spiropoulou, 2011). These membrane-embedded spikes are ordered in
(A) (B)
lattice form and each spike associated with large cytoplasmic extensions and form also a lattice on the inner surface of the viral membrane (Battisti et al., 2011).
1.1.2.2. Genome organization
The general genome organization looks like that of representatives of other Bunyaviridae genera.
The genome consists of three segments: Small (S) segment, medium (M) segment and Large (L) segment (Elliott, 1990). The terminal sequences of each segment are highly conserved and complementary to each other suggesting that they form a panhandle-like structure (Plyusnin et al., 1996c; Jonsson and Schmaljohn, 2001)
The size of the S segment ranges from 1530 to 2078 nucleotides (nt). It encodes a N protein of 428 to 434 aa. The non-coding region (NCR) of the S segment is at 3´-end longer (200-800nt) and more variable than the 5´ end (40-70nt) (Plyusnin et al., 1996c; Sironen and Plyusnin, 2011).
The N protein is translated from the first mRNA that accumulates in the cells after infection (Kariwa et al., 1995a; Hutchinson et al., 1996). A second overlapping reading frame was observed on the S segment of hantaviruses carried by representatives of the rodent subfamilies Arvicolinae, Sigmodontinae and Neotomininae. This ORF was predicted to encode a putative non-structural protein (NSs) of 88-95 aa (Parrington and Kang, 1990; Plyusnin, 2002). This ORF might represent an interferon antagonist, but might be functional only in the reservoir host (Ulrich et al., 2002). The NSs is reported to be involved in suppression of the host innate immune response, as shown in COS-7 cells expressing TULV and PUUV NSs. The activities of the interferon beta (IFN-beta) promoter, nuclear factor kappa B (NF-kappaB) and interferon regulatory factor-3 (IRF-3) responsive promoters were inhibited in these cells (Jaaskelainen et al., 2007). Interestingly, this ORF is missing on the S segment of hantaviruses carried by representatives of the Murinae subfamily.
The size of the M segment is 3613 to 3801 nt. The M segment encodes for a glycoprotein precursor of 1131 to 1148 aa (Schmaljohn et al., 1987; Giebel et al., 1989; Vapalahti et al., 1992).
The 5´-NCR sequence is similar in length to those from S and L segments, while the 3´-NCR of the M segment is approximately 200-250 nt long (Sironen and Plyusnin, 2011). The glycoprotein
4
precursor (GPC) is cotranslationally cleaved to yield the two mature proteins Gn and Gc; the cleavage site is located directly downstream the conserved aa motif WAASA (Lober et al., 2001).
The hydrophobicity and hydrophilicity polt of the glycoprotein shows the Gn begins at the 649th codon of the open reading frame follows hydrophic leader sequence as well as Gc which begins at the 18 aa beyond the first start codon of open reading frame. (Schmaljohn et al., 1987). Gn and Gc have to be coexpressed to form a heterodimer to be transported out of the endoplasmic reticulum (ER) to the Golgi complex (Ruusala et al., 1992). In the Gn protein there are three asparagine linked glycosylation sites at positions 142, 358 and 410, whereas in Gc is only one at position 939. These glycosylation sites are conserved among hantaviruses (Parrington et al., 1991). The transmembrane regions in Gn and Gc extend from aa residues 452 to 526 and 1108 to 1138, respectively (Spiropoulou et al., 1994). Both glycoproteins have a C-terminal hydrophobic anchor domain, with the N-terminus facing towards the ER and their C-terminal part located in the cytoplasm. It has been suggested that the lengthy Gn cytoplasmic tail substitutes for the function of a matrix protein, lacking in hantaviruses, and is involved in virus budding (Spiropoulou, 2001). The functional analysis of HTNV glycoprotein-specific monoclonal antibodies revealed that there are three neutralizing epitopes, one on Gn and two on Gc (Arikawa et al., 1989).
The size of the L segment is 6474 to 6581 nt. It encodes for the RNA-dependent RNA polymerase (RdRp) of 2149 to 2156 aa (Schmaljohn, 1990; Stohwasser et al., 1991; Chizhikov et al., 1995; Piiparinen et al., 1997). The L segment sequences of hantaviruses are the most conserved within the genome. The maximum sequence diversity is found to be 40% for the most distantly related hantaviruses (Sironen and Plyusnin, 2011). The RdRp has transcriptase, replicase and endonuclease functions. The endonuclease activity is needed for acquisition of 5´- end capped primers from host mRNA to initiate the transcription (Plotch et al., 1981). Recently, it has been shown that the hantavirus RdRp possesses a highly active endonuclease function at the N-terminus (Morin et al., 2010; Reguera et al., 2010; Heinemann et al., 2013). Like other bunyaviruses, hantavirus RdRp possesses six conserved motifs that fold into a palm-like structure, which are premotif A (aa 884-902), motif A (aa 964-980), motif B (aa 1050-1077),
motif C (aa 1091-1101), motif D (aa 1152-1164) and motif E (aa 1171-1181) (Poch et al., 1989;
Muller et al., 1994; Jonsson and Schmaljohn, 2001; Kukkonen et al., 2005).
1.1.3. Replication
Hantaviruses use cell surface receptor β1 and β3 integrins of the endothelial cells for the attachment via the viral glycoprotein (Gavrilovskaya et al., 1998). The β3 was reported to be utilized by pathogenic hantaviruses, whereas the β1 was found to mediate the entry of non- pathogenic hantaviruses (Gavrilovskaya et al., 1999; Larson et al., 2005). Additionally other cellular receptors like gC1qR/p32, a receptor for the globular head domain of the complement C1q, and Decay-accelerating factor DAF/ CD55 have been suggested to serve as alternative receptors for hantaviruses (Choi et al., 2008; Krautkrämer and Zeier, 2008). After the attachment (Fig. 2), the virus enters the cell via clathrin-dependent receptor-mediated endocytosis and uses low pH-dependent intracellular compartment to cause the infection (Jin et al., 2002). The low pH of endosome triggers the fusion of viral and endosomal membranes which is mediated by the viral glycoprotein and results in the release of the viral RNA into the cytoplasm (Ogino et al., 2004; Tischler et al., 2005). After the release of the three RNPs, the transcription starts in the cytoplasm where the P bodies serve as a pool of primers during the initiation of viral mRNA synthesis (Mir et al., 2008). The initiation of viral transcription follows the cap snatching process to give S, M, L mRNA by using the viral polymerase , in which the RdRp acquired the capped 5’
prime from cellular mRNA and protect it from degradation (Mir et al., 2008) and also prime- realign mechanism has been suggested in which the transcription initiates with G-terminated host cell primer alignment at the third nucleotide C residue of the template RNA (Garcin et al., 1995;
Vaheri et al., 2013b). The signal for transcription termination for each segment is unknown. But a CCC-rich motif has been suggested as a signal for terminating S segment transcription (Hutchinson et al., 1996). S and L mRNA are translated by free ribosomes, while the GPC is synthesized by membrane-bound ribosomes (Spiropoulou, 2011). Immediately after the initial transcription the viral polymerase switches to replication of the S, M and L genomic RNAs (Jonsson et al., 2010), and then the newly synthesized viral RNAs are encapsidated by N protein.
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Figure 2: Steps of hantavirus replication in the host cell
(1) Attachment between the virus and the host cell surface through integrin receptors and viral glycoprotein interaction. (2) Entry via receptor-mediated endocytosis and thereafter uncoating and release of viral genomes. (3) Transcription of complementary RNA from the viral RNA genome through the prime-realign mechanism. (4) Translation of the three mRNAs to the corresponding proteins. (5) Replication and amplification of vRNA, assembly with N protein and transportation to the Golgi complex. (6) Assembly of all virus components at the plasma membrane (A), or at the Golgi complex (B). (7) Viral egress via the fusion of the Golgi vesicle harboring the mature virion particles with the plasma membrane. (The figure was modified from Jonsson et al 2010) with permission from the publisher).
.
Gn/Gc
Gn/Gc A
B 5
The signal for encapsidation was found to be within the NCR and the packaging appear to be independent for each genomic RNA (Schmaljohn and Nichol, 2007). Precisely, the trimeric N protein exhibits a genome segment specific recognition of the viral RNA panhandle (Severson et al., 2001; Mir et al., 2006). The maturation of Gn and Gc and formation of a Gn-Gc complex occurs in ER (Antic et al., 1992) and subsequently the two glycoproteins have to be co-expressed and transported to the Golgi complex (Ruusala et al., 1992). The process of hantavirus assembly remains unclear because hantaviruses lack a matrix protein. It has been thought that the aa motif at the Gc-C terminus interacts with N protein and mediates virus packaging and release (Overby et al., 2007a; Overby et al., 2007b; Shi et al., 2007). The assembly of New World hantaviruses has been suggested to occur at the plasma membrane (Goldsmith et al., 1995; Ravkov et al., 1997). In contrast, it has been shown that the assembly of both Old World and other New World hantaviruses occurs in the Golgi complex (Ravkov and Compans, 2001; Spiropoulou et al., 2003;
Ramanathan et al., 2007). Although the New and Old World hantaviruses share a common pathway in their replication cycle, they are obviously differently interacting with host cell machinery (Ramanathan and Jonsson, 2008).
1.1.4. Host association and transmission
Unlike other members of the family Bunyviridae, hantaviruses have been known since long time as rodent-borne pathogens (Henttonen et al., 2008) and the host range include rodents, insectivores and bats (Schlegel et al., 2014). Both human and rodent acquire the infection by virus contaminated aerosol from urine, saliva or feces and less frequently by rodent bite.
Hantaviruses cause undetectable cytopathology in vertebrate cell cultures and cause persistent and non-pathogenic infections in rodent reservoirs (Schmaljohn and Dalrymple, 1983; Plyusnin et al., 2012).
Rodent-borne hantaviruses have been found so far only in representatives of the families Cricetidae and Muridae, both belonging to the suborder Myomorpha. In phylogenetic trees viruses associated to the family Muridae, subfamily Murinae, form a clade that is represented by HTNV, the Rattus-associated SEOV, the Apodemus-associated DOBV, the African Sangassou
8
virus and related viruses. Within the family Cricetidae hantavirues have been identified in representatives of the subfamilies Arvicolinae, Sigmodontinae and Neotominae which form two separate clades of the Arvicolinae- and Sigmodontinae/ Neotominae-associated viruses. PUUV, TULV and related viruses represent the most important Arvicolinae-associated viruses, whereas SNV and ANDV are the most important New World hantaviruses associated with sigmodontine and neotomine rodents (Schlegel et al., 2014). Thottapalayam virus (TPMV) was the first hantavirus identified in a shrew, namely the Asian house shrew Suncus murinus from India (Carey et al., 1971). Recently, additional insectoivore-borne hantaviruses have been detected in shrews of subfamilies Soricinae and Crocidurinae in Africa, Asia and Europe, additional to other hantavirues have been detected in the moles of the family Talpidae (Klempa et al., 2007; Song et al., 2007a; Arai et al., 2008; Kang et al., 2009b; Kang et al., 2009a; Song et al., 2009; Kang et al., 2010; Kang et al., 2011). Further hantaviruses have been identified in the bat species Nycteris hispida, Neoromicia nanus and Neomys fodiens (Sumibcay et al., 2012; Weiss et al., 2012; Gu et al., 2013).
In its natural host, hantaviruses are thought to cause chronic and asymptomatic infections.
Experimentally infected rodents start to shed the virus in the saliva, urine and feces 28- 360 days post infection (Lee et al., 1981; Hardestam et al., 2008). Virus shedding lasts for up to 15 months in naturally infected voles (Yanagihara et al., 1985; Bernshtein et al., 1999). New-borne and young animals with maternal immunity remain resistant to infection for three months (Kallio et al., 2006b). During the chronic infection, the bank voles retain the antibodies, whereas the production, accumulation and horizontal transmission of the virus decreased with the time (Bernshtein et al., 1999). The horizontal transmission of PUUV is compatible with grooming and social behavior of bank voles. The older male showed a higher degree of infection, due to their mobility during reproductive season. All seroconversions occurred during this period (Yanagihara et al., 1985; Verhagen et al., 1986; Bernshtein et al., 1999). In addition the possibility of indirect transmission of PUUV through contaminated environment, burrow and runways, was reported (Sauvage et al., 2003; Kallio et al., 2006a). Furthermore, a fine-scale spatio-temporal survey of kinclustering and dispersal in hantavirus transmission, revealed that PUUV and TULV are preferentially transmitted among relatives of the reservoir vole species. A
MHC II gene polymorphism was found to be involved in susceptibility and resistance to PUUV infection (Deter et al., 2008b; Deter et al., 2008a). Also there is a significant genetic differentiation at MHC class Drb gene between PUUV-seropositive and PUUV–seronegative bank voles in wild populations (Guivier et al., 2010). In contrast, such a kinship does not seem to play a role in transmission of Hokkaido virus (HOKV) in grey red-backed vole (Clethrionomys rufocanus) populations (Iwasa et al., 2004).
Human infections have been found so far only for certain rodent-borne hantaviruses. These hantaviruses are transmitted to human via inhalation of virus-contaminated rodent excreta. In rare cases transmission via bites from infected rodents has been reported (Douron et al., 1984;
Schultze et al., 2002). Human-to-human transmission has been reported for ANDV in Argentina only (Wells et al., 1997; Padula et al., 1998). The main transmission route of hantaviruses between rodent reservoirs is horizontally, either directly by biting or indirectly by inhalation of aerosolized virus-contaminated droppings (Glass et al., 1988; Hinson et al., 2004; Kallio et al., 2006).
1.1.5. Hantavirus evolution
Each hantavirus species is thought to be associated with one or few closely related rodent species.
The persistent infection which is caused by hantaviruses might be the consequence of a long-term co-evolution of the viruses with their hosts (Plyusnin et al., 1996c; Hughes and Friedman, 2000).
In line, phylogenetic analyses of viral and host sequences provide evidence for a virus-host-co- divergence (Jackson and Charleston, 2004). In contrast, a recent reconciliation analysis provided no evidence of a history of co-divergence between hantavirues and their hosts. Furthermore, it was proposed that the similarities between the phylogenies of hantaviruses and their hosts are result of an initial host switching event and a subsequent local host-specific adaptation (Ramsden et al., 2009; Schmidt-Chanasit et al., 2010; Yanagihara et al., 2014). Several hantaviruses seems to be able to infect multiple related rodent species, like TULV, SEOV and DOBV (Plyusnin et al., 1994a; Sibold et al., 2001). In addition, host switch scenarios have been reported for the evolutionary history of some rodent-borne hantaviruses, i.e., DOBV, Topografov virus (TOPV), NewYork virus (NYV) and SNV (Morzunov et al., 1998; Vapalahti et al., 1999; Nemirov et al.,
10
2002; Kang et al., 2009a) and a mole-borne hantavirus Oxbow virus (OXBV) with a close similarity to cricetide-borne hantaviruses (Kang et al., 2009). Moreover, the newfound hantaviruses from soricomorphs and chiropterans are genetically more diverse than those found in rodents, highlighting that shrews and moles may have been the original hosts of hantaviruses (Kang et al., 2011; Yanagihara et al., 2014).
The main mechanism of hantavirus evolution and genetic diversity is genetic drift such as nucleotide substitution, deletion and insertion (Plyusnin et al., 1995b; Lundkvist et al., 1998).
Hantaviruses, like other RNA viruses, are suggested to have a very high error rate during replication due to the absence of a proof reading function of RdRp. The calculation of substitution rates for Araraquara virus, DOBV, PUUV and TULV revealed a high rate of substitutions/site/year in a range from 2.10 x10-2 to 2.66 x 10-4 (Ramsden et al., 2008). In contrast, the substitution rate for hantaviruses has been calculated to be in range from 0.7x10-7 to 2.2x10-6 nt substitutions per site per year (Hughes and Friedman, 2000; Sironen et al., 2001). The mutation frequency results in the appearance of closely related genetic variants of a given virus, forming a quasispecies within the same individual (Plyusnin et al., 1995b; Plyusnin et al., 1996a;
Lundkvist et al., 1997b; Sironen et al., 2008). Another mechanism of genetic diversification like reassortment (genetic shift) and recombination, were reported to occur in nature for SNV (Li et al., 1995) and PUUV (Razzauti et al., 2008; Razzauti et al., 2009). Genetic reassortment processes have been detected between different hantavirus lineages and strains; thus in vitro and in vivo intra-species reassortments have been reported for DOBV (Klempa et al., 2003b;
Kirsanovs et al., 2010). An inter-species reassortment can occur in vivo when an animal get double- infected with two different hantavirus species. This event requires a spillover infection of a non-reservoir animal and its co-infection with the adapted host-specific virus. Inter-species reassortments have been reported after in vitro co-infections with Black Creek Canal virus (BCCV) and SNV (Rodriguez et al., 1998) or PUUV and PHV (Handke et al., 2010), and in nature between HTNV and SEOV (Zou et al., 2008a). Another host switch event has been discussed as an explanation for the contradictory location of TOPV and KHAV, share a common ancestor with PUUV and their host Lemmus and Microtus which are distantly related in the phylogenetic tree (Vapalahti et al., 1999). In addition to reasortment processes, the genetic
alterations of hantaviruses can happen also through intragenomic recombination, as it has been shown for TULV, PUUV, HTNV and ANDV (Sibold et al., 1999; Sironen et al., 2001; Plyusnin et al., 2002; Chare et al., 2003; Medina et al., 2009).
1.2. Human hantavirus infections
1.2.1 Clinical features of human hantavirus infections: HFRS and NE
The infection in human is characterized by an incubation period of about 2 to 4 weeks and abrupt onset of fever and headache and followed by abdominal pain, nausea, vomiting (Settergren et al., 1988a; Vapalahti et al., 2003). During acute phase myopic shift and blurred vision were recorded in 40% of patients due to thickening of the lens (Kontkanen et al., 1994; Hautala et al., 2011).
Central nervous system related symptoms, hypophyseal hemorrhage and inflammation in cerebrospinal fluid are found to be common during or shortly after acute NE (Hautala et al., 2002; Hautala et al., 2010). The typical laboratory findings include leukocytosis, thrombocytopenia, anemia, C-reactive protein and electrolyte abnormalities (Mustonen et al., 1994). The renal involvement manifests clinically by oliguria, anuria, haematuria and a rise in serum creatinine. Acute renal failure is detected in the minority of patients and about 5% of hospitalized cases require dialysis treatment (Settergren et al., 1989; Mustonen et al., 1994;
Rollin et al., 1994; Hukic et al., 2011). Development of anaemia, hypotension, signs of preshock and complicated with spleen and gastropathy hemorrhage has been also reported in NE patients (Nuutinen et al., 1992; Alexeyev et al., 1994). Moreover, severe respiratory involvement and symptoms like in Hantavirus Pulmonary Syndrome (HPS) were also recognized in patients during PUUV infection (Rasmuson et al., 2011; Gizzi et al., 2013).
Hence, the level of host defense including neutralizing antibodies and cellular immune responses may play an important role in the pathogenesis. Immunohistological studies showed increased expression of tumor necrosis factor α (TNF-α), Transforming growth factor (TGF-β) and Platelet- derived growth factor (PDGF) in peritubular area of the kidney (Temonen et al., 1996). Similarly, the acute phase of HFRS is characterized by significantly elevated levels of cytokines in both early and late phase (Sadeghi et al., 2011; Saksida et al., 2011). Moreover, NE patients with borderline or undetectable IgM were found to be highly positive for PUUV-N specific serum IgA
12
(de Carvalho Nicacio et al., 2000). The severity of the disease contributed to a rapid increase and activation of CD8-positive T cells and NK cells during the acute phase of hantavirus infection (Bjorkstrom et al., 2011; Lindgren et al., 2011). Likewise, the more severe clinical outcome was found to be associated with low level of specific IgG and high white blood cell counts, but not with the viral RNA load (Pettersson et al., 2013). Furthermore, the complement system becomes activated via the alternative pathway in the acute stage of NE and the level of activation correlates with disease severity (Paakkala et al., 2000; Sane et al., 2012). It has been suggested that the severe clinical symptoms of NE might be associated with HLA-B8 and the mild symptoms with HLA-B27. In contrast, the severity of clinical symptoms in DOBV-infected patients is associated with HLA-B35 (Mustonen et al., 1996; Mustonen et al., 1998; Korva et al., 2011).
1.2.2. Diagnosis of hantavirus infection in humans
Due to the only short-term detection of virus and viral nucleic acid in infected humans, the diagnostics of hantavirus infections is mainly based on traditional serological assays, such as indirect immunofluorescence assay (Kruger et al., 2001). However, in the recent past, enzyme- linked immunosorbent assays (ELISAs), immunoblotting, and immunochromatographic rapid tests have been developed, allowing the highly sensitive and specific detection of hantavirus infections in humans (Lundkvist et al., 1993; Zoller et al., 1993; Sjolander et al., 1997; Sjolander and Lundkvist, 1999; Hujakka et al., 2003). Furthermore, native viral antigens or recombinant N proteins were employed as diagnostic antigens in tests like monoclonal antibody capture immunoglobulin G (IgG) and IgA and indirect ELISAs. Recombinant antigens were produced in Escherichia coli, yeast or insect cells (Vapalahti et al., 1996a; Meisel et al., 2006; Schmidt et al., 2006). For diagnosis of acute infections, µ-capture IgM ELISAs with N proteins expressed in Drosophila melanogaster Schneider S2 cells (Meisel et al., 2006). The baculovirus-expressed PUUV and DOBV-N based IgG and IgM ELISAs were found to be most suitable for diagnosis, giving the opportunity for earlier diagnosis (Zoller et al., 1993; Kallio-Kokko et al., 1998; Kallio- Kokko et al., 2000). Other serological assays like focus-reduction neutralization test (FRNT) and ELISA based on truncated N protein derivatives were used for serotyping diagnosis (Vapalahti et
al., 1996b; Lundkvist et al., 1997c; Sjolander et al., 1997). Both conventional and real-time RT- PCR methods have been developed, allowing a highly sensitive virus RNA detection in blood, serum, urine, cerebrospinal fluid, or saliva even before detection of virus-specific IgM (Plyusnin et al., 1997; Evander et al., 2007; Mahonen et al., 2007; Pettersson et al., 2008b).
1.2.3. Prevention and therapy of hantavirus infection
Hantavirus infections of are thought to leave a lifelong immunity, preventing a second infection with the same virus. The level of glycoprotein-specific IgG antibodies in sera from NE patients during convalescent phase were considerably increased and lasting for 20 years post infection (Settergren et al., 1991; Lundkvist et al., 1993). In addition, cytotoxic T-cells against PUUV N protein were detected years after PUUV infection (Van Epps et al., 2002).
Both rodent brain- and cell culture-derived inactivated vaccines have been developed, and widely used in Asia (Schmaljohn, 2009). Hantavax is a commercially available vaccine derived from formalin-inactivated HTNV-infected suckling mouse brain. The vaccine induced an immune response in 79% of immunized subjects in 30 days after initial vaccination, however, the booster dose one year later is needed to develop neutralizing antibodies and a protective immunity (Cho and Howard, 1999). In addition recombinant vaccine were developed (Schmaljohn et al., 1990;
Schmaljohn et al., 1992; Xu et al., 1992; Chu et al., 1995). Recombinant proteins have been demonstrated in animal models to induce a protective immunity (Schmaljohn et al., 1990;
Yoshimatsu et al., 1993; Lundkvist et al., 1996; Dargeviciute et al., 2002). To overcome the low immunogenicity of recombinant vaccines autologous or chimeric virus-like particles were generated (Betenbaugh et al., 1995). Moreover, different DNA vaccines were produced to stimulate a strong cellular response (Ulrich et al., 1998; Davis and McCluskie, 1999). Naked DNA vaccines were constructed by subcloning of cDNAs representing M segment or S segment of SEOV into DNA expression vectors (Hooper et al., 1999; Kamrud et al., 1999). A mixed HTNV and PUUV M segment DNA vaccine was developed. Both vaccines induced neutralizing antibodies when given alone but when they were delivered as a mixture, antibodies to only one of the two hantaviruses could be detected (Spik et al., 2008).
14
The treatment of HFRS consists of supportive care with careful management of fluids, control of blood pressure, and dialysis, if required. Early treatment with antiviral ribavirin can reduce mortality and severity of symptoms: A clinical study using intravenous ribavirin for treating HFRS patients in Korea from 1987 to 2005 showed that the earlier intravenous application of ribavirin resulted in a decreased occurrence of oliguria and decreased severity of renal insufficiency (Rusnak et al., 2009). A randomized, double-blind, placebo-controlled trial in China demonstrated that treatment with intravenous ribavirin early in disease is associated with a decrease in morbidity and mortality from HFRS (Huggins et al., 1991). Also the treatment with human and murine interferons inhibited replication and viral protein accumulation in Vero E6 cells and newborn mice (Tamura et al., 1987; Temonen et al., 1995; Frese et al., 1996; Kanerva et al., 1996). Recently, it has been reported the treatment with bradykinin receptor antagonist icatibant inhibited the vascular leakage (Antonen et al., 2013).
1.2.4. Hantavirus epidemiology in Europe
In Europe the average number of annual human cases is about 10,000 (Vaheri et al., 2013a). Most of the cases are caused by PUUV carried by Clethrionomys glareolus and DOBV carried by at least three different species of the genus Apodemus (Klempa et al., 2013b). Although the presence of TULV in the corresponding common vole host in central and eastern Europe and high prevalence of TULV-specific antibodies in human have been reported (Mertens et al., 2011a), only a few human cases have been identified to be caused by TULV. Therefore TULV is thought to be low or almost not pathogenic to human (Schultze et al., 2002; Klempa et al., 2003a;
Zelena et al., 2013).
The HFRS and its mild form NE are common in most countries within Europe (see Table 2). In Fennoscandia the majority of cases are caused by PUUV (Niklasson et al., 1987; Settergren et al., 1988b; Ahlm et al., 1994; Brummer-Korvenkontio et al., 1999). In Estonia, the high incidence of human infections is caused by PUUV and Saaremaa virus (SAAV) (Golovljova et al., 2002). A serosurvey in the Latvian population indicated the presence of PUUV-specific antibodies (Lundkvist et al., 2002). In Germany the majority of human cases are caused by PUUV. Several human infections by DOBV genotype Kurkino in northern, north-eastern and eastern part of
Germany have been reported, while only a single case of TULV infection was identified in eastern Germany (Zoller et al., 1995; Meisel et al., 1998; Sibold et al., 2001; Klempa et al., 2003a; Klempa et al., 2004; Hofmann et al., 2014; Rasche et al., 2014). In Ardennes region at Franco-Belgian border from 1992-1993, human hantavirus infections seem to have been caused by PUUV (Rollin et al., 1994). Moreover, in Belgium during the largest outbreak of hantavirus infection in 1995 and 1996, only PUUV infections could be confirmed by neutralization test (Heyman et al., 1999). In the Netherlands 0.9% of individuals at occupational risk were found to be hantavirus seropositive (Groen et al., 1995). In Austria human PUUV hantavirus infections have been documented (Aberle et al., 1999; Poeppl et al., 2012). In the Balkan region, DOBV causes severe cases of HFRS in Slovenia, Croatia, Bosnia, former Yugoslavia, Albania, Greece, Bulgaria (Chumakov et al., 1988; Gligic et al., 1989; Antoniadis et al., 1996; Lundkvist et al., 1997c; Avsic-Zupanc et al., 1999; Markotic et al., 2002). Interestingly, in Slovenia two genotypes, i.e. Dobrava and Kurkino, are circulating (Avsic-Zupanc et al., 1999; Klempa et al., 2013a). In the European part of Russia, particularly the Republic of Bashkortostan, PUUV has been attributed for the outbreak in 1997 (Nurgaleeva et al., 1999). During winter 1991/1992, DOBV was involved in an outbreak of HFRS in Tula-Ryazan region (Lundkvist et al., 1997a). In Samara region of Russia, a seroprevalence investigation showed blood donor samples equally reactive with HTNV, DOBV and SEOV antigens (Alexeyev et al., 1996) (Table 2). Additionally, some cases of hantavirus infection imported to Germany, France and Italy have been reported (France and Burns, 1988; Bruno et al., 1990; Moulin et al., 1991; Schmidt-Chanasit et al., 2008;
Rovida et al., 2013)
The annual incidence of human cases in some parts of Europe varies in cycles with peaks each second to fourth year (Kallio et al., 2009). The spatial and temporal variation of HFRS occurrence in northern Europe is connected with geographical differences in population dynamics of bank voles in different biomes of Europe (Olsson et al., 2010). In Fennoscandia C. glareolus exhibits population cycles of 3-4 years, and human HFRS epidemics coincide with peak of bank vole population. In temperate Europe the bank vole populations are much more stable, but in occasional “mast years” the rodents become abundant giving rise to HFRS outbreaks (Tersago et al., 2009; Olsson et al., 2010). Based on increasing numbers of bank voles forecasting for an
16
increasing risk of human PUUV infection was made (Olsson et al., 2009). In addition, it has been reported that HFRS outbreaks are preceded by seed mast years of broad-leaf trees like oak and beech which is the main food of bank vole, and significantly related to warmer autumn the year before and hotter summer two years before but also to colder and more moist summers (Clement et al., 2009).
Table 2: Hantavirus seroprevalence and clinical cases in Europe Country Seroprevalence
%
Total Number of clinical cases (year)
incidence in sexes (M:F)*
References
Albania - 1 (1995) - (Antoniadis et al., 1996)
Austria 1.2 37 (1993-1998) - (Aberle et al., 1999; Poeppl et al., 2012)
Belgium 1.35 2,200 (1983-
2007)
- (Clement et al., 2009)
Bosnia and Herzegovina
7.4 (endemic region) 2.4 (non- endemic region )
311 (1999-2000) no difference (Hukic et al., 2003; Hukic et al., 2010)
Bulgaria 90.7 399 (1954-1988) M:93.3% Vasilenko et al., 1990 (Chumakov et al., 1988) Croatia 1.6- 5.4 555 (1987-2002) - (Borcic et al., 1991; Mulic et
al., 2003)
Czech Republic 1- 1.4 18 (2004-2009) - (Pejcoch and Kriz, 2003b;
Pejcoch et al., 2010)
Denmark 1 128 (1987-2000) - (Sironen et al., 2002; Nemirov
et al., 2004)
Estonia 9.1 few cases 1.8: 1.0 (Golovljova et al., 2002)
Finland 5 Annually 957 2: 1 (Brummer-Korvenkontio et al., 1999)
France 1.23 808 (1977-1998) - (Le Guenno, 1998; Sauvage et
al., 2007)
Germany 0.8- 3.12 9300 - (http://www3.rki.de/SurvStat,
October 09, 2014)
Greece 4 200 (1983-
1998)
3: 1 (Antoniadis et al., 1987;
Papadimitriou and Antoniadis, 1994; Papa et al., 1998)
Hungary 1.9- 8.2 136 81950-
1980)
- (Faludi and Ferenczi, 1995;
Ferenczi et al., 2003; Oldal et al., 2014)
Italy 0.2- 7.1 1 - (Nuti et al., 1992; Nuti et al.,
1993; Kallio-Kokko et al., 2006)
Ireland 1.2 0 - (Stanford et al., 1990;
McKenna et al., 1994)
Latvia 4.2 - 1: 2.5 (Lundkvist et al., 2002)
Lithuania 0.7 - 8.2 - 1:1.25 (Sandmann et al., 2005)
Luxembourg - 14 ( 2005) 1: 9 (Schneider and Mossong, 2005)
The Netherlands 0.7 27 (1974-1993) - (Gerding et al., 1995; Groen et al., 1995)
Norway 0.74 Annually 50 2.8- 1 (Sommer et al., 1988;
Lundkvist et al., 1998) Poland 8.6 (high risk
group)
17 (2007) - (Grygorczuk et al., 2008;
Nowakowska et al., 2009)
Romania - 6 since 2008 - (Maftei et al., 2012)
Russia 7.2 annually 5,000- - (Alexeyev et al., 1996; Klempa Table 2 (continued)
18
12,000 et al., 2008; Maftei et al., 2012)
Slovakia 0.54- 1.91 4 (1989) - (Bilcikova et al., 1989;
Alexeyev et al., 1996; Sibold et al., 1999; Klempa et al., 2008)
Slovenia 0- 14.28 511 - (Bilcikova et al., 1989; Avsic-
Zupanc et al., 1999; Sibold et al., 1999; Avsic Zupanc et al., 2014)
Spain 2.2 - 1.5 : 1 (Gegundez et al., 1996; Avsic-
Zupanc et al., 1999; Sanfeliu et al., 2011)
Sweden 5.4 313 in 2007 no difference (Ahlm et al., 1994; Gegundez et al., 1996; Pettersson et al., 2008a; Sanfeliu et al., 2011)
Switzerland 0.0- 1.9 1 - (Ahlm et al., 1994; Schultze et
al., 2007; Fontana-Binard et al., 2008; Pettersson et al., 2008a;
Engler et al., 2013)
Turkey 3.2- 5.2 12 in 2009 6: 1 (Schultze et al., 2007; Fontana- Binard et al., 2008; Ertek and Buzgan, 2009; Gozalan et al., 2013)
United Kingdom 7.6 2 in 1985, 1993 3.5:1 (Walker et al., 1985; Pether et al., 1993; Jameson et al., 2013;
Jameson et al., 2014) As there are no data available for Cyprus and Portugal, these countries are not mentioned in the table. * M: male, F:
female, (-): no data available Table 2 (continued)
1.3. Bank vole-associated PUUV
1.3.1. PUUV and related arvicolinae-borne hantaviruses
Driven by the importance of PUUV as human pathogen in Europe large molecular studies have been performed in different countries. Now more than 600 PUUV sequences are annotated in GenBank indicating a high diversity; in contrast the number of virus isolates and complete genome sequences is rather low (Sironen and Plyusnin, 2011). The analyses of PUUV nucleotide sequences from Finland, Sweden, Norway, Russia, Germany and France illustrates that PUUV is the most variable species of the currently known hantaviruses. The genetic diversity of the coding region of the S segment is about 20%, whereas for the S segment NCR it is about 30% and for the M segment 3´NCR even 37% (Lundkvist et al., 1998). These values are higher than the corresponding ones (13% and 30% respectively) for another hantavirus, like TULV (Plyusnin et al., 1995a). Moreover, the sequences of PUUV strains from different geographical regions demonstrate a high degree of heterogeneity, as shown for example for two PUUV strains circulating in central and north Sweden (Horling et al., 1996). On the other hand the phylogenetic analysis of complete S segment sequences of PUUV strains from several European countries, Russia and Japan revealed seven distinct genetic lineages with typical geographical clustering (Sironen et al., 2001). Furthermore, in individual voles a complex mixture of closely related PUUV variants was found forming a quasispecies (Plyusnin et al., 1995b; Sironen et al., 2008).
Recently, sequences of a PUUV-like virus were discovered in tissue samples of grey red-backed vole (C. rufocanus) trapped in Hokkaido (Japan), China and in Buryatia in Russia (Kariwa et al., 1995b; Zhang et al., 2007; Plyusnina et al., 2008). Unlike to European PUUV, Hokkaido virus (HOKV) appears to be nonpathogenic for human (Kariwa et al., 2000). Likewise, Muju virus (MUJV), another tentative new hantavirus species, was isolated from Korean red-backed vole (C.
regulus) from various regions in Korea. Pairwise analysis of S and M segments of MUJV indicated 77 % sequence identity to corresponding PUUV sequences (Song et al., 2007b).
Another virus called TOPV was detected in two different lemming species, i.e. Lemmus sibiricus and Lemmus lemmus collected near the Topografov River at the Taymyr Peninsula (Plyusnin et
20
al., 1996b). However, TOPV was found to be more closely related to PUUV and Khabarovsk virus (KHAV), another hantavirus, carried by reed vole (Microtus fortis) (Vapalahti et al., 1999).
There are several Microtus-associated hantaviruses, both in the Old and New world. TULV is the most investigated virus of this group. However, it is still unclear if this hantavirus is a low or even non-pathogenic hantavirus (Vapalahti et al., 1996b; Schultze et al., 2002; Clement et al., 2003; Klempa et al., 2003a). It was originally discovered in two sibling species of European common vole, Microtus arvalis and Microtus rossiaemeridionalis, namely in tissue samples of European common vole M. arvalis trapped in Tula town, central Russia , and Malacky, Slovakia (Plyusnin et al., 1994a; Sibold et al., 1995). Later, TULV strains were detected in Moravia (Czech Republic) (Plyusnin et al., 1995b), Austria (Bowen et al., 1997), Poland (Song et al., 2004), Belgium (Heyman et al., 2002) and most recently in different parts of Germany (Klempa et al., 2003a; Schmidt-Chanasit et al., 2010). Initial phylogenetic analyses of TULV sequences indicate that it is most closely related to PHV, PUUV, and Muerto Canyon virus (Plyusnin et al., 1994a). TULV has been also demonstrated in other species such as M. agrestis (Scharninghausen et al., 2002; Schmidt-Chanasit et al., 2010), M. subterraneus (Song et al., 2002) and recently in water vole Arvicola amphibius (Schlegel et al., 2012a), but the main reservoir is thought to be M.
arvalis. KHAV was isolated from Microtus fortis in the Khabarovsk region of Far East Russia and after, KHAV strain named Yakeshi-strains was detected in Microtus maximowiczii in China which is could be the ture host for KHAV (Sironen and Plyusnin, 2011). The both strains have aa sequence identity of more than 98.4% for the S segment and 95.6% for the M segment (Zou et al., 2008b). Additionally, another virus Vladivostok virus (VLAV) was also found to be closely related to KHAV (79% nucleotide and 90% amino acid identities). It was isolated from M. fortis in Vladivostok, Far East Russia (Kariwa et al., 1999).
PHV is another Microtus-associated hantavirus initially discovered in meadow voles (M.
pennsylvanicus) in the New world. (Lee et al., 1985). It is believed to represent a non-pathogenic model virus and has consequently frequently used in in vitro studies (Larson et al., 2005; Handke et al., 2010), but has also found to cause disease in cynomolgus monkeys (Macaca facicularis) after experimental inoculation (Yanagihara et al., 1988). Similarly, serological evidence of PHV infection in American mammologists has been reported previously (Yanagihara et al., 1984). A
similar New world hantavirus is the Isla vista virus (ISLAV) identified initially in the California vole (M. californicus) trapped in North America (Song et al., 1995). ISLAV is genetically clearly distinct from PHV; there are 11% and 19.6% differences at aa level in N and portion of Gc glycoproteins between the two viruses, respectively. A spillover infection with ISLAV was also detected in Peromyscus californicus in Santa Barbara county, California (Song et al., 1995).
1.3.2. Phylogeography of bank vole
The bank vole (C. glareolus) is widely distributed in Europe and Asia from British Isles to Lake Baikal. In Europe it extends from the Arctic Circle to northern Spain, mountains of Italy, and the Balkans region. It is common on islands of the Atlantic and Baltic coasts, however it is absent from the Mediterranean Islands (Mitchell-Jones et al., 1999). In Asia C. glareolus is found in Russia (southern Siberia from the Ural Mountains to Lake Baikal), northern Kazakhstan, and the Altai and Sayan Mountains (Musser and Carleton, 2005). A comparative phylogeographic study of small mammals and plant postglacial colonization showed that there is a clear influence of the Quaternary cold periods on distribution and genetic variation of small mammals (Taberlet et al., 1998). The temperate forest mammal species were found to have a higher genetic variation in the Mediterranean peninsulas compared to northern Europe indicating the role of potential Mediterranean refuges (Michaux et al., 2003). A cytochrome b (cyt b)-based phylogeography study of bank voles collected from 62 localities revealed the presence of six bank vole phylogroups in Europe, three Mediterranean (Spanish, Italian and Balkan) and three continental (western, eastern and 'Ural') (Deffontaine et al., 2005). In addition, there is a mounting of evidence that bank voles apparently survived the Weichselian glaciation 25,000-10,000 years ago in a high-latitude glacial refugium in the Carpathian Mountains and that may give rise to a further genetic lineage (Kotlik et al., 2006; Wojcik et al., 2010); see Fig. 3).
The bank vole population in Fennoscandia originated from recolonization of the territory after the retreat of the Weichselian ice sheet 8,000- 13,000 years ago. This recolonization followed two directions, either south via a land bridge connecting the present Denmark with southern Sweden and called Southern population, or from the northeast via northern Finland and Russia and called
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northern population. (Jaarola et al., 1999). The Northern and Southern bank vole populations could be distinguished by mtDNA sequences, with the north lineage being very similar to that of C. rutilus (Tegelstrom, 1987). A contact zone between the northern and southern populations in Sweden is estimated to be 150-200 km wide (Jaarola and Tegelstrom, 1995; Jaarola et al., 1999).
Noteworthy, PUUV strains circulating in central Sweden are distinct from those in the northern region, and the Norwegian PUUV strain is well clustering with southern Sweden PUUV strains.
This finding could be explained by the postglacial recolonization of Sweden by bank voles (Horling et al., 1996; Lundkvist et al., 1998).
Figure 3: Distribution of bank vole lineages in Europe
Distribution of bank vole lineages in Europe based on previously published data from Deffontaine et al., 2005 and Wojcik et al., 2010. The basic map was provided by Dr. C. Staubach (Friedrich- Loeffler-Institut, Greifswald- Insel Riems, Germany).
Ural Eastern Carpathia n western Balkan Italian Basque Spanish
1.4. PUUV and other hantaviruses in Germany
In Germany human hantavirus infections have been reported since the 1980ies (Antoniadis et al., 1985; Zeier et al., 1986). The geographical distribution and frequency of hantavirus infections in humans is well documented by large seroprevalence studies and recording of clinically apparent infections. Endemic regions are well known in Baden-Wuerttemberg, Bavaria, Lower Saxony and North Rhine Westphalia (Pilaski et al., 1991; Ulrich et al., 2004; Essbauer et al., 2006;
Essbauer et al., 2007; Hofmann et al., 2008). The overall seroprevalence in the German human population was estimated to range between 0.8 in eastern Germany and 3.12% in Swabian Jura (Zoller et al., 1995).
In Germany at least five different hantaviruses are present. Bank vole-borne PUUV causes the majority of human infections in Germany which are characterized by mild to moderate courses (Ulrich et al., 2004). Furthermore, DOBV, genotype Kurkino, was found to cause human infections in the northern and eastern part of Germany (Meisel et al., 1998; Sibold et al., 2001;
Klempa et al., 2004; Ulrich et al., 2004). The reservoir host of this hantavirus is the striped field mouse Apodemus agrarius (Schlegel et al., 2009). Additionally, TULV was molecularly detected in common vole, field vole and water vole from different regions in Germany (Schmidt-Chanasit et al., 2010; Schlegel et al., 2012a). So far only one clinical HFRS case was identified by neutralization assay to be caused by a TULV infection (Klempa et al., 2003a). Recently, two shrew-borne hantaviruses were detected in Germany, Seewis virus in the common shrew Sorex araneus and Asikkala virus in the pygmy shrew S. minutus (Schlegel et al., 2012b; Radosa et al., 2013).
PUUV causes not only the majority of human cases, but is also heavily influenced by bank vole population oscillations. Whereas in 2001, 2004, 2006, 2008, 2009, 2011 and 2013 about 57-233 human cases were annually recorded in Germany, in 2005, 2007, 2010 and 2012 a large increase in the number of PUUV cases was observed, reaching 387, 1,625, 1,873 and 2,370 cases, respectively (Robert Koch-Institut: SurvStat, http://www3.rki.de/SurvStat, data as of 06.11. 2013;
and (Faber et al., 2010). The majority of recorded cases were observed in Baden-Wuerttemberg, Bavaria, North Rhine Westphalia and Lower Saxony. Interestingly, the number of cases is not
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homogenously distributed in each of these federal states. In Lower Saxony the district Osnabrück is a high-endemicity region. During the outbreaks in 2010 and 2012 increased numbers of human cases were for the first time recorded in Hesse, Thuringia and Rhineland-Palatinate (Robert Koch-Institut: SurvStat, http:// www3.rki.de/survstat). Interestingly, the increased number of cases in 2010 could be explained by synchronized outbreaks in different regions in north-western and southern Germany (Ettinger et al., 2012). This outbreak was driven by at least 6 distinct molecular clades of PUUV detected in humans and rodents (Ettinger et al., 2012). A 5-fold increase in notified PUUV cases compared to the previous annual maximum was observed in a part of western Thuringia in 2010 (Faber et al., 2013). Moreover, the molecular investigation of PUUV in bank voles from different regions in Germany revealed a high genetic diversity of PUUV sequences and presence of several distinct subtypes (Heiske et al., 1999; Essbauer et al., 2006; Schilling et al., 2007; Hofmann et al., 2008; Mertens et al., 2011b). Currently, there is no complete nt sequence of a PUUV strain from Germany or Central Europe known. The phylogenetic analysis of PUUV strains, and other hantaviruses as well, is usually done by short S and M segment-derived sequences, based on protocols empirically developed in the past (Escutenaire et al., 2001; Essbauer et al., 2006; Mertens et al., 2011b; Razzauti et al., 2013).
2. Objectives
The major objective of the study was to develop tools for a parallel analysis of the phylogeography of PUUV and the bank vole in Central Europe, and in Germany in particular, on a large and small geographical scale.
For this purpose a novel cyt b PCR assay should be established initially for molecular identification of small mammal species, but then also tested for identification of bank vole lineages in Germany and in north-eastern Poland. For the molecular characterization of PUUV in Central Europe strains in an endemic region in Germany, i.e., the district of Osnabrück, and in the north-eastern part of Poland, where so far no human cases were reported, should be analysed. For the initial molecular investigation partial S-, M- and L-segment sequences should be determined.
To identify regions of different levels of sequence variability a primer-walking-mediated complete genome sequence determination should be performed for a bank vole-derived PUUV strain from the district Osnabrück.
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3. Publications
3.1. Paper I
Schlegel, M., Ali, HS, Stieger, N., Groschup, M.H, Wolf, R and Ulrich, R.G. (2012). Molecular identification of small mammal species using novel cytochrome b gene-derived degenerated primers. Biochem Genet 50 (5-6):440-7.
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Fig. B
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3.2. Paper II
Printed from Viruses. Ali, H.S., Drewes, S., Sadowska, E.T., Mikowska, M., Groschup, M.H., Heckel, G., Koteja, P. and Ulrich, R.G. (2014). First molecular evidence for Puumala hantavirus in Poland. Viruses 6 (1): 340- 53.
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3.3. Paper III
Ali, H.S., Drewes, S., Weber de Melo, V., Schlegel, M., Freise, J., Groschup, H.M., Heckel, G. and Ulrich, R.G. (2014). Complete genome of Puumala virus strain from Central Europe.
Virus Genes
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