Helsinki University Biomedical Dissertations № 25

Genetic Relationships of Saaremaa and Dobrava

Hantaviruses

KIRILL NEMIROV

DEPARTMENT OF VIROLOGY

HAARTMAN INSTITUTE

FACULTY OF MEDICINE

UNIVERSITY OF HELSINKI

ACADEMIC DISSERTATION

To be presented with the permission of the Medical Faculty of the University of Helsinki, for public examination in Small Lecture Hall, Haartman institute, Haartmaninkatu 3, on March 14th, 2003, at 12 o´clock noon.

Helsinki, 2003 2

Supervisors: Docent Alexander Plyusnin Haartman Institute Department of Virology University of Helsinki

Professor Antti Vaheri Haartman Institute Department of Virology University of Helsinki

Reviewers: Professor Ralf Pettersson Ludwig Institute for Cancer Research Stockholm, Sweden

Docent Mika Salminen KTL Helsinki, Finland

Opponent: Professor Richard Elliott University of Glasgow Glasgow, United Kingdom

ISBN 952-10-0985-3 (paperback) ISBN 952-10-0986-1 (e-thesis) ISSN 1457-8433 Yliopistopaino Helsinki, 2003 3

TO MOM AND DAD 4

Contents 5

CONTENTS

I. ORIGINAL PUBLICATIONS 7 II. ABBREVIATIONS 8 III. SUMMARY 9 IV. INTRODUCTION 10 History of hantavirus infections 10 Isolation of hantaviruses 11 Taxonomy 11 Hantavirus structure 15 Genome organization and coding strategy 16 Hantavirus proteins 17 Hantavirus replication 19 Epidemiology of hantaviral diseases 21 HFRS 21 HPS 22 Clinical features of HFRS and HPS 22 HFRS 22 HPS 23 Pathogenesis 24 Treatment 25 Geographic distribution and genetic diversity of hantaviruses 25 Murinae-borne hantaviruses 25 Arvicolinae-borne hantaviruses 29 Sigmodontinae-borne hantaviruses 33 Hantaviruses and their hosts 37 Mechanisms of hantavirus evolution 43 Genetic drift 44 Genetic shift 46 V. AIMS OF THE STUDY 48 VI. MATERIALS AND METHODS 49 Handling of rodent samples and hantavirus-infected cells 49 Immunoblotting 49 MAb reactivity 49 RNA extraction 50 Reverse transcription (RT) and polymerase chain reaction (PCR) 50 Cloning 51 Extraction of mitochondrial (mt) DNA from rodent tissues 51 Sequence and phylogenetic analysis 51 VII. RESULTS AND DISCUSSION 53 Isolation of the virus from lung samples of A. agrarius 53 Antigenic characterization of a new isolate by FRNT and MAbs 53 Characterization of nt and deduced aa sequences of SAAV 54 Genetic diversity and geographic distribution of hantaviruses carried by A. flavicollis and A. agrarius in Europe 56 Evidence that hantaviruses carried by A. agrarius and A. flavicollis represent distinct genotypes 64 6

Host switching 66 VIII. CONCLUSIONS AND FUTURE PROSPECTS 69 IX. ACKNOWLEDGEMENTS 71 X. REFERENCES 73 IX. APPENDIX 1 92

Original Publications 7

ORIGINAL PUBLICATIONS

I. Nemirov K, Vapalahti O, Lundkvist Å, Vasilenko V, Golovljova I, Plyusnina A, Niemimaa J, Laakkonen J, Vaheri A, Plyusnin A. 1999. Isolation and characterization of Dobrava hantavirus carried by the striped field mouse (Apodemus agrarius) in Estonia. Journal of General Virology 80:371-379.

II. Plyusnin A, Nemirov K, Apekina N, Plyusnina A, Lundkvist Å, Vaheri A. 1999. Dobrava hantavirus in Russia. Lancet 353:207.

III. Avsic-Zupanc T, Nemirov K, Petrovec M, Trilar T, Poljak M, Vaheri A, Plyusnin A. 2000. Genetic analysis of wild-type Dobrava hantavirus in Slovenia: co-existence of two distinct genetic lineages within the same natural focus. Journal of General Virology 81:1747-1755.

IV. Papa A, Nemirov K, Henttonen H, Antoniadis A, Vaheri A, Plyusnin A, Vapalahti O. 2001. Isolation of Dobrava virus from Apodemus flavicollis in Greece. Journal of Clinical Microbiology 39:2291-2293.

V. Nemirov K, Henttonen H, Vaheri A, Plyusnin A. 2002. Phylogenetic evidence for host switching in the evolution of hantaviruses carried by Apodemus mice. Virus Research 90:207-215.

VI. Nemirov K, Vapalahti O, Papa A, Plyusnina A, Lundkvist Å, Antoniadis A, Vaheri A and Plyusnin A. 2003. Genetic characterization of new Dobrava hantavirus isolate from Greece. Journal of Medical Virology in press.

VIII. Nemirov K, Andersen H, Lundkvist Å, Henttonen H, Vaheri A, Plyusnin A. Saaremaa hantavirus in Denmark. Submitted.

Abbreviations 8

ABBREVIATIONS aa amino acid(s) ADE antibody-dependent enhancement cRNA complementary (anti-genome sense) RNA CTL cytotoxic T-lymphocyte DM distance matrix DOBV∗ Dobrava hantavirus DDW double-distilled water ER endoplasmic reticulum FRNT focus reduction neutralization test G1, G2 hantaviral glycoproteins G1G2, GPS glycoprotein precursor HFRS hemorrhagic fever with renal syndrome HPS hantavirus pulmonary syndrome HTNV Hantaan hantavirus IFA immunofluorescence assay kb kilobase(s) kDa kilodalton L large RNA segment/protein M medium RNA segment MAb monoclonal antibody ML maximum likelihood MP maximum parsimony N nucleocapsid protein NCR non-coding region NE nephropathia epidemica nt nucleotides ORF open reading frame RT-PCR reverse transcription – polymerase chain reaction S small RNA segment SAAV Saaremaa hantavirus SDS sodium dodecyl sulfate vRNA viral (genome sense) RNA

∗ For a complete list of abbreviations of hantaviruses, see Table 1, page 13 Summary 9

SUMMARY

Hantaviruses represent a group of zoonotic viruses distributed in the world more widely than any other viruses carried by wild animals (97). Since the isolation of the first hantavirus – Hantaan (HTNV) in 1976, the list of newly discovered hantavirus species has been growing and now includes 22 officially recognized hantavirus species and 20 genotypes awaiting recognition (Table 1). Many hantaviruses are pathogenic for humans and cause one of the two clinically distinct syndromes, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The majority of the estimated annual 150 000 cases of HFRS occur in Asia, while HPS occurs only in the Americas, where over 1000 cases of the disease have been reported since its recognition in 1993.

This thesis reports the isolation and genetic characterization of the recently discovered European hantavirus - Saaremaa (SAAV) from striped field mouse (Apodemus agrarius). Geographic distribution and genetic variability of the new virus was estimated through the recovery and sequence analysis of six field strains originated from Estonia, Russia, Slovenia and Denmark. This study has also addressed the question concerning genetic relationships between SAAV and another European hantavirus – Dobrava (DOBV), carried by yellow- necked mouse (Apodemus flavicollis) in the Balkans. Since no complete sequence of DOBV had been previously reported, a new strain of DOBV (DOBV/Ano- Poroia/Af9V/1999, or DOBV-AP for short) was isolated in cell culture and completely sequenced. Comparison of the complete sequences of DOBV and SAAV and the phylogenetic analysis suggested that these viruses may represent two genetically related but phylogenetically distinct virus genotypes. Phylogenetic analysis of the host rodent species based on the sequences of mitochondrial DNA suggested that SAAV and DOBV are ecologically distinct and associated with different rodent species: A. agrarius and A. flavicollis, respectively. The approximate time of evolutionary split between DOBV and SAAV was estimated to be around 2.7-3.4 million years.

Introduction 10

INTRODUCTION

History of hantavirus infections

The earliest description of the disease that resembles HFRS can be found in the Chinese medical records from the 10th century (122). However, the first definite description of HFRS comes from Russian clinical records describing outbreaks of the disease in the Far East of Russia (Amur Basin) in 1913 (24). Later in the 20th century outbreaks were encountered in the same region and also in the European part of Russia where an outbreak of “Tula fever” in 1930- 1934 with 913 cases was reported (53). Since then outbreaks of HFRS were often observed during military activities. Japanese military physicians recorded 12,000 cases of HFRS after the invasion of Manchuria in 1934 (121). The infectious nature of HFRS was proven by independent studies of Soviet and Japanese research groups on human subjects in 1940s. Acute infection was produced in recipients by inoculating them with blood and urine of acutely ill patients. Japanese military researchers also established the possible involvement of rodents in the disease transmission by inoculating prisoners-of- war with tissue suspensions of mites collected from striped field mice (Apodemus agrarius) (7, 107, 113, 234). The mild form of HFRS observed in Europe was first described in Sweden in 1934 and later named nephropathia epidemica (175). During the World War II an epidemic of NE, which coincided with a high population density of lemmings and forest mice, was reported among Finnish and German troops in Finnish Lapland. Interestingly, outbreaks of the disease resembling HFRS have been recorded during other military activities in the past, including the American Civil War and World War I when outbreaks with approximately 14,000 and 12,000 cases, respectively, were described (127). However, western medicine became aware of the diseases only in the 1950s, when the outbreaks of so-called “Korean hemorrhagic fever” characterized by cardiovascular instability, shock, and renal failure were described among 3200 UN troops during the Korean war 1951-1954 (36). By the end of 1950s Soviet researchers noted the similarity between the HFRS-like diseases occurring in the Far East and Europe and suggested the bank vole (Clethrionomys glareolus) as a probable reservoir for the pathogen in nature. Thus, it became evident that the severe disease encountered in Far East and China and the mild disease registered in Scandinavia and Eastern Europe were caused by related pathogens and the term HFRS was suggested as a common name for the disease (53).

Introduction 11

Isolation of hantaviruses

The first successful attempt to identify the causative agent of HFRS was done by Dr. Ho Wang Lee in 1976, when he discovered that sera of HFRS patients reacted in the immunofluorescence assay (IFA) with tissue sections of striped field mice (Apodemus agrarius), but not of other rodents (120). Shortly after this the first hantavirus, Hantaan (HTNV), was isolated from the infected rodents and HFRS patient samples by serial passaging of the virus in colonized Apodemus agrarius (121). The virus was named after the river Hantaan, close to where the first infected rodents were captured and subsequently all hantaviruses isolated later were named after the geographic locations in which they were discovered. Using the same IFA approach Markus Brummer- Korvenkontio and his co-workers discovered hantavirus antigen in lungs of bank voles (Clethrionomys glareolus) captured in the Puumala village, Finland (20). This virus, which was subsequently isolated using cell cultures was named Puumala (PUUV). Two new hantaviruses were isolated in the early 1980s: Seoul virus (SEOV), responsible for urban and laboratory associated outbreaks of HFRS, from rats (Rattus norvegicus and R. rattus) (123); and the first American hantavirus, Prospect Hill (PHV), from meadow voles (Microtus pennsylvanicus) in the United States (125). For a long time only these four hantaviruses were known until in the early 1990s two new viruses were isolated from Asia, Thailand (THAIV) (264) and Thottapalayam viruses (TPMV), and one from Europe, Dobrava virus (DOBV) (11, 12). DOBV, isolated from yellow-necked mouse (Apodemus flavicollis), was later shown to cause severe HFRS in the Balkans (3, 147). The dramatic discovery of Sin Nombre virus (SNV) and the associated disease (HPS) opened a new chapter in hantavirus research (37, 177). Shortly after the discovery and isolation of SNV from the Four Corners region in the United States a whole new group of hantaviruses was characterized throughout the American continent (Table 1). The most prominent of the American hantaviruses is SNV, responsible for the vast majority of HPS cases in North America, and Andes virus (ANDV), which circulates in South America and is capable of human-to-human transmission (139).

Taxonomy

Hantaviruses form a distinct genus Hantavirus within the Bunyaviridae virus family which also include four other genera: Bunyavirus, Nairovirus, Phlebovirus and Tospovirus (40). All viruses are assigned to the Bunyaviridae family based on similarity in the structure of the virion, genome organization and the replication cycle. The main distinguishing feature of Hantavirus from the other Introduction 12

genera is the absence of arthropod vectors in their life cycle. Hantaviruses are carried by distinct rodent species and transmitted to humans via inhalation of contaminated aerosol or direct contact with rodent excreta. Therefore, it has been suggested to classify hantaviruses as roboviruses (derived from rodent- borne viruses), rather than arboviruses (arthropod-borne viruses) (39, 151). Since the first hantavirus, Hantaan virus, was isolated from Apodemus agrarius in 1976 in Korea, at least 22 new hantavirus species have been discovered throughout the world (Table 1; Fig. 1), making hantaviruses a prime example of emerging pathogens.

TULV AMRV

PUUV TOPV KHAV

SNV HTNV

SEOV

NYV MULV SAAV HokkaidoV ISLAV PHV BLLV DOBV BAYV

ELMCV BCCV RIOSV Juquitiba

CADV Choclo LNV RMV Maciel Calabazo Oran Hu 39694 Lechiguanas Bermeo Pergamino

ANDV

Fig. 1. Geographic distribution of different hantavirus species. The geographic locations for the original isolation of viruses are indicated. For the abbreviations of

hantavirus names see Table 1.

Hantavirus species are currently defined by the following four criteria adopted by the Bunyaviridae working group in the International Committee on Taxonomy of Viruses (40): (i) “Ecologic criterion”: hantavirus species are found in a unique ecological niche, i.e. in a different primary rodent reservoir species or subspecies; (ii) “Genetic criterion”: different species exhibit at least a 7% difference in amino acid (aa) identity on comparison of the complete sequences of glycoprotein precursor (GPS) and nucleocapsid protein (N); Introduction 13

(iii) “Antigenic criterion”: hantavirus species show at least a four-fold difference in two-way cross-neutralization tests; (iv) “Criterion of reproductive isolation”: different hantavirus species do not naturally form reassortants with other species.

In most cases these criteria could be efficiently employed to make the distinction between hantavirus species (in every case, the decision rests with the Group’s experts). However, a number of newly discovered viruses do not meet some of the criteria, e.g., show less than 7% differences in their aa sequences with sequences of known hantaviruses (166, 200, 201).

Table 1. Hantaviruses. Officially recognized hantavirus species are shown in bold.

Virus Host Distribution Disease Isolation in Complete Reference (origin) cell culture sequence

Murinae-borne hantaviruses

Hantaan (HTNV) Striped field mouse Asia HFRS + + (121) (eastern isolate) (Korea)

Da Bie Shan Niviventer confucianus Asia NR + + (260) (Cnina)

Seoul (SEOV) Rat Worldwide HFRS + + (123) (Rattus rattus, (Korea) R. norvegicus)

Dobrava (DOBV) Yellow-necked mouse Europe HFRS + + (11,12) (A. flavicollis) (Slovenia)

Saaremaa (SAAV) Striped field mouse Europe HFRS + + (199, (I)) (western isolate) (Estonia) (A. agrarius)

Thailand (THAIV) Bandicoot rat Thailand NR + - (264) (Bandicota indica)

Amur (AMRV) Korean field mouse Asia HFRS - - (137, 269) (A. peninsulae) (Russian Far East, China)

Avricolinae-borne hantaviruses

Puumala (PUUV) Bank vole Europe HFRS + + (20) (Clethrionomys glareolus) (Finland)

Hokkaido (HOKV) Red bank vole Asia NR - + (103) (C. rufocanus) (Japan)

Tula (TULV) European common vole Europe NR + + (193) (Microtus arvalis) (Russia)

Introduction 14

Virus Host Distribution Disease Isolation in Complete Reference (origin) cell culture sequence

Prospect Hill (PHV) Meadow vole North America NR + - (124,125) (M. pennsylvanicus) (USA)

Bloodland Lake (BLLV) Prairie vole North America NR - - (236) (M. ochrogaster)

Isla Vista (ISLAV) Californian vole North America NR - - (236) (M. californicus) (USA)

Khabarovsk (KHAV) Reed vole Asia NR + - (91) (M. fortis) (Far East Russia)

Topografov (TOPV) Lemming Siberia NR + - (195) (Lemmus sibiricus) (Russia)

Vladivostok (VLAV) Reed vole Far East Russia NR - - (105) (M. fortis) (Russia)

Sigmodontinae-borne Hantaviruses

Sin Nombre (SNV) Deer mouse North America HPS + + (37, 177) (Peromyscus maniculatus) (USA)

Monongahela (MGLV) Deer mouse North America HPS - - (237) (P. maniculatus nubetirrae) (USA)

New York (NYV) White-footed mouse North America HPS + - (75, 237) (P. leucopus) (USA)

Blue River White-footed mouse North America NR - - (169) (P. leucopus) (USA)

Limestone Canyon (LCV) Brush mouse North America NR - - (216) (P. boylii) (USA)

Bayou (BAYV) Rice rat North America HPS + - (168) (Oryzomys palustris) (USA)

Black Creek Canal (BCCV) Hispid cotton rat North America HPS + - (207, 210) (Sigmodon hispidis) (USA)

Muleshoe (MULV) Hispid cotton rat North America NR - - (210) (S. hispidus) (USA)

Andes (ANDV) Long-tail pigmy rice rat South America HPS + - (138) (Oligoryzomys longicaudatus) (Argentina)

Lechiguanas (LECV) Rice rat South America HPS - - (139) (O. flavescence) (Argentina)

Oran O. longicaudatus South America NR - - (129) (Argentina)

Bermejo O. chacoensis South America NR - - (129) (Argentina)

Hu39694 unknown South America HPS - - (129) (Argentina)

Introduction 15

Virus Host Distribution Disease Isolation in Complete Reference (origin) cell culture sequence

Choclo (CHOV) Pygmy rice rat Central America HPS - - (259) (O. fulvescence) (Panama) Calabazo Zygodontomys brevicauda Central America NR - - (259) (Panama)

Laguna Negra (LANV) Vesper mouse South America HPS + - (95) (Calomys laucha) (Paraguay)

Rio Mamore (RIOMV) Small-eared pygmy rice rat South America NR + - (16, 77) (O. microtis) (Bolivia)

Caño Delgadito (CADV) Cane mouse South America NR + - (52) (Sigmodon alstoni) (Venezuella)

El Moro Canyon (ELMCV) Western harvest mouse North America NR + - (76) (Reithrodontomys megalotis) (USA)

Rio Segundo (RIOSV) Mexican harvest mouse South America NR - - (79) (R. mexicanus) (Costa Rica)

Maciel (MACV) Dark field mouse South America NR - - (129) (Necromys benefactus) (Argentina)

Pergamino (PERV) Grass field mouse South America NR - - (129) (Akodon azarae) (Argentina)

Juquitiba unknown South America HPS - - (257) (Brazil)

Araraquara unknown South America HPS - - (96) (Brazil)

Castelo dos Sonhos unknown South America HPS - - (96) (Brazil)

Insectivorae-borne Hantaviruses

Thottapalayam (TPMV) Shrew Asia NR - - (23) (Suncus murinus) (India)

Hantavirus structure

Under the electron microscope, hantaviruses appear as spherical particles about 100-120 nm in diameter with a distinguished bilayered envelope and granulofilamentous interior (157, 222). Inside the virus particle they contain an RNA genome of negative polarity, surrounded by the molecules of nucleocapsid protein. Dimerized surface glycoproteins form short projections or “spikes”, about 6 nm long, protruding from the viral surface (Fig. 2).

Introduction 16

Genome organization and coding strategy

The hantavirus genome consists of three segments: large (L), medium (M) and small (S) (40, 197). They respectively encode an RNA-dependent RNA- polymerase (L protein), responsible for the transcription and replication of hantaviral RNA genome; the two glycoproteins, G1 and G2, that recognize cellular receptor(s); and nucleocapsid protein (N) that encapsidates viral RNA (Fig. 2). Heterodimer of glycoproteins (G1, G2) Nucleocapsid (N) protein S M

L Genomic RNA

Polymerase (L protein) Lipid envelope

Fig. 2. Schematic structure of the virion

Similarly to nairoviruses and in contrast to the other three genera of the Bunyaviridae family, nonstructural proteins have not been described for hantaviruses (40). Interestingly, all hantaviruses carried by Sigmodontinae and Arvicolinae rodent families have an open reading frame (ORF) for a putative nonstructural protein NSs of 63-90 aa (201). A functional ORF of bunyaviruses of similar size, located in approximately the same region, encodes the NSs protein, which functions as an antagonist of alpha/beta interferon (262). The low frequency of nucleotide substitutions in the NSs ORF of TULV and PUUV and substantial number of conserved aa residues observed in the putative NSs protein of all hantaviruses suggest that the NSs ORF may be functional (201). The 5’ and 3’ termini of all hantavirus RNA segments are highly conserved and complementary to each other, a hallmark of the Bunyaviridae family (38). The terminal sequences of the genome segments are genus-specific and members of every genus have their own specific “signatures” comprising short stretches of identical terminal nucleotides (nt). In all known hantaviruses 14 terminal nt of the S segment, 17 terminal nt of the M segment and 15 terminal nt of the L segment are identical (197). This characteristic was one of the basic features for Introduction 17 the recognition of Hantavirus as a separate genus within the Bunyaviridae family in 1985 (218). Inverted complementary 5’ and 3’ terminal sequences are capable of forming specific structures through base-paring, which are thought to be involved in the regulation of transcription and replication (51, 206). Although direct evidence for these structures in hantaviruses has not been obtained, they were visualized by electron microscopy in other members of Bunyaviridae. Usually they are described as “panhandles”, but the precise structure of these regions is not known. Mutational analysis of terminal genomic regions of influenza A virus, influenza C virus, and thogoto virus (all from Orthomyxoviridae family), which are also highly conserved and complementary to each other, suggested that these regions can form an alternative “corkscrew” structure (51). Theoretically, similar structures could be formed by viruses from four genera of the Bunyaviridae (all, except genus Phlebovirus) and also by tenuiviruses. However, detailed mutational studies of promoter region were reported only for phleboviruses (51, 205), and thus at present there is no experimental evidence for corkscrew promoter structures in the viruses of Bunyaviridae family. The 5’ and 3’ non-coding regions (NCRs) of each of the three genome segments vary in length from approximately 40-50 nt (5’ NCRs of all three genomic segments and 3’ NCR of the L segment) to 300-700 nt (the 3’ NCR of the S segment) (201). Although these regions are not particularly conserved between different hantaviruses (especially 3’ NCRs of S and M segments, which can not be reliably aligned outside of 100 terminal nt), they are relatively conserved within specific hantavirus types, and also contain a number of strictly conserved motifs (197). Apart from the terminal regions mentioned above many hantaviruses contain a nt motif that resembles a hypothetical signal for mRNA termination found in the 3’ NCR of HTNV S segment (3’ CCCCACCCAGUCA 5’) (32). Another motif 3’ GAUGGAGU 5’ with unclear function is present in a single, or multiple copies in all hantaviruses close to the highly conserved 5’ terminus of the S segment (207).

Hantavirus proteins

Hantavirus N protein, consisting of ~430 aa (~50 kDa), is abundantly expressed in infected cells and has been reported to form large granular to filamentous aggregates (209). The main function of the N protein is encapsidation of viral RNA, which is thought to be promoted by the highly conserved carboxyl-terminal 93 aa section, and the sequences at 5’ end of viral genomic (vRNA) and antigenomic or complementary RNA (cRNA) molecules (64). The N protein has been shown to have strong preference for binding Introduction 18 vRNA over nonviral RNA and moderate binding preference over viral mRNA that lacks 3’-terminal sequences. In a recent study on HTNV N protein the minimal region for vRNA-binding was mapped between aa 175 and 217 (265). The current general model proposed for the encapsidation of hantaviral RNA suggests that interaction of N protein with the specific sequence and/or structure located at the 5’ termini of hantavirus vRNA and cRNA initiate polymerization of N protein molecules and subsequent specific and/or non- specific binding along the RNA molecule (98). In support of this model, N protein was shown to oligomerize and form trimers that could be intermediates in nucleocapsid assembly (1, 108). N protein was also shown to interact with cellular structural proteins, such as actin (209). Recent data indicate that N protein may have other functions in the infected cells as it was shown to interact with the apoptosis regulator Daxx (135). The precise role of this interaction has to be clarified. The N protein is highly immunogenic and major B-cell epitopes have been mapped in its N-terminal third (1-120 aa) (145, 254, 271), while most T-cell epitopes are found in the central region of the protein (43, 185, 251, 252).

The envelope glycoproteins G1 and G2 are synthesized in the form of a glycoprotein precursor (GPS), 1132-1148 aa in length (195). GPS is cotranslationally processed immediately after the WAASA motif at the C- terminus of the G1 protein, to yield the two glycoproteins (140). The glycoproteins form a heterodimer in the endoplasmic reticulum (ER), and are targeted to the Golgi compartment (215, 229). The G1 glycoprotein contains a predicted signal peptide of 16-23 aa (5, 220), followed by a 450-aa external domain, a ~75-aa hydrophobic membrane-associated domain, a cytoplasmic tail, and a short ~20-aa domain which may serves as the signal peptide for G2 (186, 215). The C-terminus of G2 protein contains a 30-aa transmembrane domain conserved between the sequences of different hantaviruses (195). Both the G1 and G2 glycoproteins contain glycans, primarily of high-mannose, endoglycosidase H sensitive type (215, 219) The glycans are added co- translationally following translocation of the nascent chain through the ER membrane. The consensus aa sequence for the glycosylation sites is the tripeptide Asn-X-Ser/Thr, where X is any aa except Pro (14). It was also shown that Trp in the middle of tripeptide is associated with very low level of glycosylation (less than 10%) (226). Two putative glycosylation sites located in the ectodomain of G1 are conserved in all hantaviruses and one site is conserved in all of them, except KBRV. Two putative glycosilation sites were identified in the G2 protein (one of them probably cannot be used). Some hantaviruses have additional sites for glycosylation (222). G1 and G2 proteins Introduction 19 are the most variable among hantavirus proteins, probably because they are targeted by neutralizing antibodies; all existing neutralizing monoclonal antibodies (MAbs) are directed against surface glycoproteins (30, 142, 143, 144, 214). Both glycoproteins have a high content of conservative cysteine residues, which suggests similarity in the tertiary structures of G1 and G2 of different hantaviruses. Although hantavirus protein(s) responsible for the attachment to the cell have not been determined, G1 and G2 are most likely to be involved in that process as they are the only proteins exposed on the viral surface. The G1G2 heterodimer seems to have quite complex three-dimensional structure and many of exposed antigenic sites contain conformational non-linear epitopes (70, 92, 261). Interactions between dimerized hantavirus glycoproteins seem to play a particularly important role for the immunogenicity of hantaviruses, as suggested by studies on HTNV MAb escape mutants: mutant that lost an Ab recognition site located on the G1 protein could not be recognized by G2-directed MAb and vice versa (261).

The L protein of hantaviruses ~2150 aa (approximately 240 kDa) is the only hantavirus protein with known enzymatic functions. This protein is thought to mediate viral transcription and replication and thus to have a number of putative functions such as endonuclease, transcriptase, replicase, and possibly, RNA helicase activities (98). Since the mutation frequency reported for hantaviruses (~1×10-3) is similar to the mutation frequency of other viral RNA polymerases without proof-reading activity, hantaviral L protein most probably lacks this function as well (196). The nature of L protein functions also suggests the presence of structural domains that could bind template RNA and cap structures of mRNA. Although information on the structure and functional relationships of the L proteins of Bunyaviridae viruses is not available, studies of other polymerases have identified several structural motifs essential for the polymerase activity (203, 174), also preserved in the Bunyaviridae family.

Hantavirus replication

Early events of hantavirus cell cycle include attachment of the virus to the cell surface and penetration into the host cell. As recently shown, hantaviruses enter the cell by receptor-mediated endocytosis using the clathrin-dependent pathway (94). After entry, virions are targeted to early endosomes, followed by progression to late endosomes and lysosomes. Viral nucleocapsids are presumably released into the cytoplasm at the late stages of the entry process, at least after the stage of early endosomes, through the fusion of viral and endosomal membranes. Studies on hantavirus entry demonstrated that β3 Introduction 20 integrins found e.g. on endothelial cells, platelets, and macrophages mediate the entry of pathogenic viruses, while the entry of the apathogenic PHV and

TULV is mediated by β1 integrins (57, 58). In addition, a 30-kDa protein of unknown function located on the surface of Vero E6 cells was recently suggested as a candidate receptor for HTNV (110). Primary transcription, which starts after viral nucleocapsids are released into the cytoplasm, produces mRNAs for hantavirus protein synthesis. Primers for the synthesis are obtained from cellular mRNAs in the course of a “cap- snatching” process similar to the one described for influenza virus (117). During this process the viral polymerase cleaves short (7-18 nt) capped fragments from the 5’ termini of cellular mRNAs and uses them to initiate synthesis of viral mRNAs (54). Initiation of both transcription and replication presumably follows the “prime-and-realign” model. According to the model, the G residue located at the 3’ end of every primer aligns with the third nucleotide of RNA template and activates the polymerase; after short initial synthesis the newly produced molecule “slips” three nt backward and realigns, thus creating an exact copy of the 5’ end of viral RNA molecule. The precise moment and factors responsible for the switch from transcription to replication are not known (98). It is thought that N protein, synthesized in sufficient amounts during primary translation encapsidates vRNA molecules thus forcing the polymerase to skip mRNA termination signals and synthesize full- lenght cRNA molecules, similar to what has been shown for vesicular stomatitis virus (184) and influenza virus (85, 134). This is followed by the synthesis of new vRNA molecules and secondary transcription that results in the amplified synthesis of mRNA and subsequent translation of more virus proteins. Translation of S and L mRNAs takes place on free ribosomes, whereas M segment mRNA are translated on membrane-bound ribosomes. The G1 and G2 proteins of HTNV have been shown to dimerize in the ER shortly after synthesis, which is thought to be necessary for the glycoprotein transport from the ER to the Golgi complex (215, 229). Since no matrix protein is encoded by hantaviral genome, direct interaction between the nucleocapsids and the membrane bound glycoproteins is assumed to take place during the assembly (98). For members of Bunyaviridae family, packaging of nucleocapsids into virions occurs by a budding process in the Golgi complex (190). The same might be true for hantaviruses since their glycoproteins are retained in the Golgi where maturation and budding are assumed to take place, at least for Old World hantaviruses (98, 241). However, some Sigmodontinae-carried hantaviruses (e.g. BCCV and SNV) have been shown to bud at the plasma membrane (62, 208, 209).

Introduction 21

Epidemiology of hantaviral diseases

HFRS. Four hantaviruses are responsible for the majority of HFRS cases in Eurasia: HTNV, PUUV, DOBV, and SEOV. Most of the severe hantaviral infections that occur in Far East Asia (including Korea, China and Japan) and Far East Russia are caused by HTNV (127, 133, 245, 269). The yearly number of HFRS cases in Asia has been estimated to be approximately 100 000-150 000, with the majority of cases occurring in China (266). DOBV is responsible for severe HFRS registered in the Balkan area (3, 13, 147, 155, 183). PUUV appears to be the major hantaviral pathogen in Europe, as it is responsible for more than 6000 cases of mild HFRS (also known as nephropathia epidemica (NE)) every year (151). The extent of infections induced by SEOV is not exactly known, but it is thought to cause HFRS of moderate severity in urban areas throughout the World and in rural areas in Asia. Cases of HFRS caused by SEOV have been registered in Korea, China, Russia and Japan (6, 9, 111, 245, 269). Although HTNV is considered the dominant hantaviral pathogen in China, the distribution of HTNV- and SEOV-induced infections varies throughout the country, and SEOV was shown to be a frequent cause of infection in some provinces (245). In Korea SEOV infections are five times less frequent than infections with HTNV and most SEOV-induced cases occur during summer time (111). Most of HFRS cases caused by PUUV occur in Russia (about 3000 cases), followed by Finland (about 1000 cases), Sweden (about 300 cases), Germany, France and Belgium (about 100 cases each). Due to the extensive serological surveys performed in the recent years in many European countries, PUUV is increasingly recognized as an important pathogen throughout Europe. Cases of PUUV-induced HFRS have been reported from Estonia, Latvia, Norway, Bosnia-Herzegovina, Slovenia, Slovakia, Greece and Denmark (151). Moreover, data on the average PUUV seroprevalence collected in Finland and Sweden suggest that the actual rate of PUUV infections is much higher than that determined from hospital admissions and 75-90% of infections appear to be subclinical, mild or atypical (253). Thus, the actual incidence of PUUV-induced infections in Europe is probably in the range of 25 000 – 65 000 cases per year. As described above, seasonal and annual oscillations in rodent dynamics have a very high impact on PUUV epidemiology, and may lead to repeated epidemics in the endemic areas. Three epidemics of PUUV-induced HFRS were registered at the French- Belgian border in 1990, 1993 and 1996 (73). Another example is given by the hundreds of HFRS cases (mostly caused by PUUV) reported from former Yugoslavia during the late 1990s, which are thought to be associated with the Introduction 22 presence of military camps and war-related activities in the area (88). DOBV is responsible for outbreaks of the severe HFRS in the Balkan area including Greece, Albania, Bosnia-Herzegovina, Slovenia and Croatia (3, 13, 147, 155, 183). Although the scale of DOBV-caused infections is thought to be much smaller than that of PUUV, the disease is much more serious and characterized by a fatality rate of 9-12% (13). SAAV is a newly discovered hantavirus, related to DOBV, which has been shown to cause human infections in Central and Eastern Europe. Although the scale and severity of SAAV-related infections is not known, the absence of severe HFRS cases in the areas where only SAAV was found (Estonia, Russia, Slovakia, Germany and Denmark) indicates that these infections are considerably less severe than those caused by DOBV (63, 148, 158, 225, 232). Two other European hantaviruses, TULV and TOPV, have not been associated with any disease. However, FRNT analysis of 315 sera from healthy blood donors living in Moravia identified one serum with a 16-fold higher titer to TULV than to other hantaviruses, suggesting that TULV can infect humans (255). Most recently an anecdotal case of TULV infection after a rodent bite was described in Switzerland (224). TOPV was discovered in the Northern part of Eurasia on Taimyr Peninsula, Russia. This virus was suspected as a causative agent of HFRS epidemic among Finnish and German troops during the war in Finnish Lapland in 1942. However, this link remains elusive since TOPV has not yet been found in Northern Europe (195, 256). Beside the four major hantavirus pathogens endemic in Eurasia, AMRV recently discovered from A. peninsulae was shown to cause HFRS in the Russian Far East and China (137). However, the seroepidemiology of AMRV-associated infections remains to be clarified. HPS can be caused by a number of pathogenic hantaviruses in the American continent, but the majority of cases are due to SNV and ANDV in North and South America, respectively. Approximately 300 cases of HPS caused by SNV have been registered in the United States (mostly from the southwestern states) and 30 cases in Canada. A small number of HPS cases caused by BAYV and BCCV has been reported from the southeastern United States (25, 46, 258). In South and Central America, HPS cases have been reported from Argentina, Brazil, Bolivia, Chile, Uruguay, Panama and Paraguay. The majority of the cases were reported from Argentina, Chile, Brazil and Paraguay with approximately 300, 200, 170, and 70 cases respectively (182).

Clinical features of HFRS and HPS

HFRS. The clinical severity of HFRS in humans varies from asymptomatic infections to severe form of the disease with mortality up to 15% (153, 223, 228). Introduction 23

The degree of severity is clearly associated with the nature of the causative agent. HTNV and DOBV are associated with severe HFRS (fatality rate of 3- 15%) (127), while PUUV causes a mild form with a case fatality rate of 0.1% (154). The most prominent clinical symptoms of HFRS include fever, acute renal failure and in severe cases – hemorrhagic manifestations. The clinical course of HFRS is characterized by five phases, some of which are not clearly recognizable in the mild forms: febrile, hypotensive, oliguric, polyuric, and convalescent phases (100). The incubation period varies from 2 to 3 weeks and the disease usually starts with sudden fever, intense headache, followed by muscular and abdominal pain, chills and nausea (227). Following the febrile period the blood pressure decreases and in severe cases shock may occur. One third of HFRS patient deaths are associated with irreversible shock at this stage. Transient visual disturbances including myopia are present in approximately one third of NE patients. The second phase of the disease develops during the first week and is associated with transient impairment of renal function characterized by decrease in urine output, azotemia, microscopic haematuria, and proteinuria. Severe cases may require transient hemodialysis treatment (100). The hypotensive phase is also associated with thrombocytopenia and hemorrhagic manifestations that appear as petechiae on the skin and mucous membranes in mild cases, or life-threatening internal bleedings in severe forms of the disease. In NE the bleeding tendency is generally mild, but is seen in almost all hospital-treated patients. During the second week the disease progresses into the polyuric phase characterized by the improvement of renal function and elevated urinary output. This phase is followed by clinical recovery although impaired tubular function may result in complications seen long after the infection is cleared. The two-week period of clinical disease is normally followed by a recovery phase that can last for weeks or months until normal renal function is restored. Long-term sequelae of HFRS include predisposition to hypertensive renal disease and occasionally hypophyseal insufficiency. HPS. The main characteristic symptoms of HPS are high fever and respiratory distress and, in contrast to HFRS, the disease can be divided in four phases: febrile, cardiopulmonary, diuretic and convalescent (44). The prodromal symptoms are similar to HFRS and include fever, myalgia, headache, and nausea. After 3-6 days the patients develop progressive cough, tachypnea, tachycardia and hypotension shortly thereafter followed by respiratory distress and pulmonary edema (34). The second phase of HPS that develops within the first 6-8 days from the onset of the disease is associated with the highest mortality due to respiratory failure, incurable shock and Introduction 24 myocardial dysfunction. Once pulmonary edema is present, the disease proceeds fast and 30-40% patients die within 24-48 hours. If the patient survives, resolution of pulmonary edema, fever and shock takes place during the diuretic phase. The convalescent phase can be extended to several weeks or months until full recovery is finally achieved (109, 189). In spite of the differences in the clinical symptoms of HFRS and HPS, these syndromes have several common pathogenic features. In both diseases capillary leakage syndrome and vasodilatation mainly contribute to the pathogenesis and lead to hemorrhagic nephritis in HFRS and pulmonary edema in HPS. Both syndromes have a prominent hypotensive phase and are characterized by hemoconcentration. Thrombocytopenia, proteinuria and leukocytosis are commonly observed in both syndromes (100, 272). Moreover, pulmonary involvement has been described in both HFRS and NE (99, 136, 153, 171), while HPS marked by renal involvement is caused by at least two viruses, BAYV and BCCV (188).

Pathogenesis

Vascular dysfunction characterized by vasodilatation and increased vascular permeability is thought to be the main factor in the pathogenesis of HFRS and HPS (29, 100). Both human pathogenic (HTNV and PUUV) and apathogenic (PHV) hantaviruses readily infect endothelial cells, macrophages and kidney glomerular cells in vitro without causing any visible cytotoxic effect (187, 267). Although in vitro studies do not necessarily adequately reflect events in vivo, this evidence suggests that functional rather than morphological changes may contribute to the hantavirus pathogenesis. Recently, apoptosis induced by a HTNV infection in cell culture was demonstrated, but it is unclear if apoptosis contributes to the pathogenesis in vivo (101). As endothelial cells do not appear microscopically damaged in HPS, subtle functional changes disturbing the capillary integrity, rather than direct damage of the cells is suspected to increase vascular permeability. This view is supported by the rapid nature of onset and the reversibility of hypotension (100). A widely accepted view is that human immune mechanisms, and especially cytotoxic T-lymphocytes probably play an important role in pathogenesis. Endothelial cells regulate the vascular tone and contraction of smooth muscle cells via paracrine secretion of chemical and hormonal mediators. Activation of monocyte/macrophages and subsequent release of biochemically active substances including TNF-α, interleukins, interferons, proteases, leukotrienes, reactive oxygen and nitrogen oxide may cause local tissue damage and affect the vascular tone and blood clotting mechanisms (100). Remarkably, macrophage-mediated permeability of Introduction 25 endothelial cells was demonstrated in SNV infections in vitro (48). Antibody- dependent enhancement (ADE) of hantavirus infection has been observed in macrophage-like cell lines, but whether ADE has a role in the pathogenesis in vivo is not known (268). Increasing evidence indicates that the patient’s genetic background may be important for the severity and outcome of hantavirus infections. Thus, severe NE associated with shock and requirement for dialysis was associated with an extended HLA haplotype containing HLA B8, DR3 and DQ2 alleles (172). Patients with HLA B8 DR3-haplotype also had higher than normal levels of viral RNA in the blood and urine (198). In contrast, the HLA B27 allele was associated with a mild course of NE among hospitalized patients (173). Animal models for HFRS and HPS have been developed only recently. Cynomolgus macaques infected with wild-type PUUV developed characteristic symptoms and immune responses of HFRS-like disease (114), and Syrian hamsters infected with ANDV developed HPS-like disease that often resulted in death (84). These disease models will be very useful in future studies on hantavirus pathogenicity.

Treatment

No specific treatment for HFRS or HPS is available at present and supportive treatments are administered to the patients. Both diseases require careful fluid management and control of the blood pressure. About 60% of patients with HPS require mechanical ventilation, while dialysis may be necessary in severe cases of HFRS (189). Early administration of ionotropic drugs is encouraged for HPS treatment. Application of ribavirin has been shown to have anti-hantaviral effect in vitro and in vivo and, according to one study, significantly decreased the mortality and severity of HFRS symptoms if used early in the infection (87). Interferon treatment was reported to have a positive antiviral effect in vitro with PUUV and HTNV and also extended the survival rate of HTNV-infected mice (244, 246). However, in human trials treatment of HFRS patients with INF-α had no noticable effect (68).

Geographic distribution and genetic diversity of hantaviruses

Murinae-borne hantaviruses

HTNV. Sequences of HTNV have been recovered from both A. agrarius and HFRS patients from Korea, China and the Far East of Russia (131, 260, 269). The genetic divergence of HTNV in Korea based on the complete M/G1G2 sequences of three strains (the prototype strain originated from A. agrarius Introduction 26 coreae and two strains from Korean HFRS patients) was in the range of 2-5%/2- 3% for nt/aa sequences, respectively (260). Comparison of the Korean sequences with two complete M/G1G2 sequences from China, closely related to each other (2%/1%), reveled 15-16% nt and 5-6% aa divergence between the Korean and Chinese strains. Another comparison based on partial M segment sequences of nine isolates from China (two of mouse and seven of human origin) demonstrated 17-22% nt divergence between the Chinese strains and 14- 23% with the prototype Korean strain (131). Thus, sequences originated from China seem to be much more diverse that those from Korea. This observation was also supported by the phylogenetic analysis of different hantavirus strains (260). A phylogenetic tree based on the partial M segment sequences of 43 HTNV strains originated from Korea (seven strains) and nine provinces of China (36 strains) identified nine genetic lineages. Seven Korean sequences formed a single genetic lineage where the Hojo and Lee strains originated from HFRS patients were placed apart from the other 5 strains including one originating from North Korea. The monophyly of all Korean strains strongly suggested a common origin. Phylogenetic relationships of Chinese strains were less resolved which could be reasonably explained by the fact that they were collected over a wide geographic area. All Chinese strains clustered in eight separate lineages following to their geographic origin. However, the relationships between different lineages could not be determined with certainty, i.e. they showed a star-like pattern of distribution suggesting that different lineages evolved independently from a single common ancestor (151). An additional genetic lineage of HTNV was recently described in Far East Russia (269). Analysis of 24 partial M and 14 partial S segment sequences recovered from Russian HFRS patients showed 9.8-12%/0-3% of nt/aa diversity in the partial M sequences and 11-13%/1-2% diversity in the partial S sequences with the prototype strain of HTNV (76-118). The intra-lineage diversities determined for the M and S sequences were up to 6%/4% and 6%/1%, respectively. All strains from Russian Far East formed a distinct cluster in the S and M segment-based phylogenetic trees, clearly separated from the HTNV strains from Korea (269, Fig. 3). SEOV is associated with two species of rat, Rattus norvegicus and Rattus rattus. Since rats are nowdays distributed worldwide and can be found in any major harbor of the world, SEOV is also thought to have a worldwide distribution. So far, sequences of SEOV have been recovered from Asia (South Korea, China and Japan), Africa (Egypt), America (USA and Brazil) (264), and Europe (191). The phylogenetic tree calculated based on the partial M segment sequences revealed five genetic lineages (260). Four of them are formed by strains originated only from China, while the last lineage included strains from Introduction 27

TOPV 100 SAAV/Kurkino 44 Russia SNV 97 SAAV/Kurkino 53 100 73 SAAV/Slovakia-856Aa Slovakia Apodemus agrarius SAAV/Slovakia-862Aa 62 SAAV/Loll-1403 Denmark (western form) 100 100 SAAV/Saaremaa-90 SAAV/Saaremaa-160v Estonia 89 DOBV/AF442622 Russia Apodemus sylvaticus 100 DOBV/Slovenia Slovenia 100 DOBV-AP Apodemus flavicollis DOBV/Greece 13 Greece SEOV/Gou-3 Eastern China Rattus rattus 55 SEOV/Z37 100 SEOV/zy27 North-Eastern China 100 62 SEOV/Pf26 100 57 SEOV/IR461 UK 82 SEOV/TCH 100 SEOV/Sapporo Rat Japan Rattus norvegicus 56 SEOV/Hb8610 99 100 SEOV/K24-v2 SEOV/K24-e7 Eastern China 55 66 SEOV/R22 SEOV/L99 100 HTNV/NC167 Niviventer 60 HTNV/AH09 Eastern China HTNV/AH211 confucianus HTNV/Maaji-1 100 Korea HTNV/Liu China 100 100 HTNV/AP70 66 HTNV/AP1168 100 100 HTNV/AP1371 Russian Apodemus 100 AMRV/AP6 Far East peninsulae AMRV/AP6 100 HTNV/HTN261 North-Eastern China 100 HTNV/AA1028 60 100 100 HTNV/AA1719 Russian Far East HTNV/AA2499 100 HTNV/Maaji-2 100 HTNV/CFC94-2 100 HTNV/76-118 95 HTNV/CUMC-B11 Korea 98 69 HTNV/S85-46 HTNV/LR-1 100 HTNV/Z10-AF159370 69 HTNV/Z10-AF184987 100 HTNV/A9 100 HTNV/Hu China 100 HTNV/A16 HTNV/Q32 100 84 HTN/Chen4 Apodemus HTNV/84FLi 100 HTNV/E142 agrarius HTNV/SN7 (eastern form) HTNV/RG9

Fig. 3. Phylogenetic tree of Murinae-borne hantaviruses based on the complete coding region of the S segment. The tree was estimated based on the pairwise distance matrix calculated using the Kimura two-parameter evolutionary model. Tree topology was estimated using the Neighbor-Joining method (500 bootstrap replicates). Bootstrap support values of greater than 50% are shown at the appropriate branch points. Gray and white boxes outline strains carried by different rodent hosts. Latin names of each rodent host species are indicated within the boxes. The black arrow shows an apparent virus host switching suggested by the clustering of DOBV, carried by A. flavicollis, with SAAV, carried by A. agrarius. Prototype strains of each hantavirus species are shown in bold. For the abbreviations of hantavirus names see Table 1. Introduction 28

China, Japan and USA. Such clustering is somewhat unusual for hantaviruses, which in most cases demonstrate geographic clustering of genetic variants. SEOV has the rare opportunity to spread inter-continentally with its natural host, e.g. in ships to harbors, and phylogenetic relationships of different SEOV strains most probably indicate the routes of rat migrations (151). THAIV is represented by a single strain Thai749 recovered in 1995 from Bandicota indica and is characterized only by a complete sequence of the M segment and a partial S segment sequence. The M segment sequence demonstrated a similar level of nt divergence with the M segments of SEOV and HTNV, 25% and 28%, respectively, and more prominent divergence with other viruses (264). New hantaviruses carried by Murinae rodents. Most recently, new hantavirus genotypes were recovered from several other species of Murinae rodents, previously unknown to harbor hantaviruses (137, 260, 269). Although these new genotypes have not been completely characterized, prominent genetic divergence from the known hantaviruses and serological data (in one case) suggest that they might represent novel hantavirus species. Hantavirus isolate NC167 recovered from Niviventer confucianus in China is currently classified as a sub-type of HTNV (260), as it is most closely related to this virus. However, genetic comparison of the two viruses demonstrated a difference of 8% and 16% in the complete sequences of N and G1G2 proteins, respectively, suggesting that NC167 represents a unique hantavirus genotype. Comparison of NC167 and HTNV in focus reduction neuralization test (FRNT) showed a 32-fold difference in the reactivity of anti-NC167 serum to NC167 genotype than to HTNV, while the level reactivity of anti-HTNV serum with HTNV was only 2-fold higher than with NC167. Thus, based on currently available genetic and serologic data, as well as on maintenance of NC167 by a unique rodent host, it was suggested that NC167 may represent a new hantavirus type with the proposed name Da Bie Shan virus (J. Arikawa pers. comm.). Another new genotype designated Amur was recovered from Apodemus peninsulae in Far East Russia (269). A recent study showed 7-13% diversity between the deduced N protein sequences of Amur and HTNV and 14-21% diversity between the G1G2 sequences. Moreover, sequences derived from A. peninsulae were closely related to the sequences recovered from two Russian and one Chinese patient with severe HFRS, thus suggesting that Amur virus is pathogenic for humans (137). Phylogenetic analysis demonstrated that all A. peninsulae-derived strains and sequences from patients, all originating from distant geographic areas, have clustered together and separately from HTNV strains, indicating a host-dependent clustering of Amur strains (269, Fig. 3.). Introduction 29

Although the Amur genotype has not been isolated in cell culture and its serologic comparison with HTNV has not been done, the present data indicate that a new pathogenic hantavirus species is harbored by A. peninsulae in the Far East. Most recently hantavirus sequences have been recovered from wood mouse (A. sylvaticus) and also from the blood of an HFRS patient in Krasnodar, Russia (248). Genetic comparison with the sequences of known hantaviruses demonstrates that these new sequences are most closely related to that of DOBV (see Results section).

Arvicolinae-borne hantaviruses

PUUV is carried by the bank vole Clethrionomys glareolus, which is spread across most of Europe excluding the Mediterranean coast and northernmost areas. In Asia, C. glareolus is found in Russia (Southern Siberia from Ural Mountains to Lake Baikal), northern Kazakhstan, and the Altai and Sayan Mountains (170). Genetic analysis of PUUV strains originating from different parts of Europe and Russia showed that PUUV is the most variable of known hantavirus species: the genetic diversity on the nt level is up to 20 and 17% for the coding sequences of M and S segment, respectively, and even higher values (37 and 30%) in the non-coding regions (150). Phylogenetic analysis performed on the complete S segment sequences of 42 PUUV strains from Eurasia demonstrated that the PUUV-based clade consists of seven genetic lineages originating from Finland, Russia, Northern Scandinavia, Southern Scandinavia, Estonia, Denmark and Belgium (233, Fig. 4). Genetic clustering of the geographic variants could be clearly seen within the lineages, but the overall phylogeny appeared to be “star-like”, suggesting an early split and independent evolution of the genetic lineages. The only exception was given by closely related genetic lineages from Finland and Russia. The nt diversity between different lineages of PUUV was in the range of 15-27%, with the smallest values observed between the lineages from Finland and Russia. The intra-lineage diversity ranged from 0.3 to 9% in all the lineages except the lineages from Southern Scandinavia and Russia. Both of those lineages included strains originating from a wide geographic area and thus high intra- lineage diversity, 13% and 16%, was observed for Southern Scandinavian and Russian lineage, respectively. The pattern of geographic distribution of PUUV in Europe is complex and probably reflects the history of post-glacial recolonization of Europe by the mammalian species (200). PUUV-like hantaviruses. Two strains of PUUV-like virus have been recovered from C. rufocanus trapped in Hokkaido, Japan (105). Their sequences Introduction 30

HTNV SNV TULV-Sw91c Switzerland TULV/g20-s Germany 97 TULV-c109-s TULV/Koziky/5276Ma Croatia 53 TULV/Koziky/5247Ma/94 TULV/Moravia/5302Ma/94 100 100 66 100 TULV/Moravia/5302v C. 53 97 TULVvG 81 TULV/Moravia/5294Ma/94 R. 100 95 74 TULV/Moravia/5286Ma TULV/Moravia/5293Ma 100 TULV/Malacky 370/94 58 Western Slovakia 100 TULV-Serbia TULV/Malacky 32/94 TULV/Kosice 667/Ma/95 Serbia 100 TULV/Kosice144/Ma/95 Eastern Slovakia TULV-Lodz1 Poland 100 100 TULV-Lodz2 TULV-D5-98 100 57 TULV/D63-98 Germany 100 TULV/D17-98 91 TULV/175Ma/87 100 TULV/23Ma/87 Russia TULV/53Ma/87 Microtus arvalis 100 100 TULV/76Ma/87 TULV/249Mr/87 Microtus rossiaemeridionalis ISLAV Microtus californicus BLLV Microtus ochrogaster 74 PHV 100 Microtus pennsylvanicus TOPV Lemmus sibiricus KHAV Microtus fortis 79 95 HOKV/Kamiiso-8Cr-95 100 HOKV/Tobetsu-60Cr-93 Clethrionomys rufocanus PUUV/Vindeln/L20Cg/83 PUUV/Hundberget/Cg36/94 100 100 100 79 PUUV/Tavelsjo/Cg81/94 PUUV/Vranica Sweden 100 PUUV/Mellanse1/Cg47/94 PUUV/Mellanse1/Cg49/94 100 PUUV/Fyn19 Norway 100 100 PUUV/Fyn131 PUUV/Fyn47 Denmark 100 Slo78 Slovenia Slo65 PUUV/Puu/1324Cg/79 PUUV/Sotkamo PUUV/Evo14Cg/93 100 100 100 100 PUUV/Evo15Cg/93 57 PUUV/Evo13Cg/93 63 PUUV/Evo12Cg/93 PUUV/Virrat/25Cg/95 Finland and 51 100 KuhmoX5 KuhmoX11 56 56 Pallas63 Karelia Kolodozero 88 Gomselga Karhumaki 100 PUUV-CG1820 PUUV/Udmurtia/894Cg/91 PUUV/Udmurtia/444Cg/88 100 PUUV/Kaza Russia 96 90 PUUV/Udmurtia/458Cg/88 100 Estonia-205 PUUV/Udmurtia/338Cg/92 Estonia-49 Estonia 100 PUUV/Eidsvoll/Cg1138/87 PUUV/Eidsvoll/1124v 92 PUUV/Solleftea/Cg6/95 Sweden 100 PUUV/Solleftea/Cg3/95 PUUV/Couvin/59Cg/97 100 93 PUUV/Momignies/47Cg/96 100 PUUV/Momignies/55Cg/96 PUUV/Montbliart/23Cg/96 Belgium 100 PUUV/Thuin/33Cg/96 PUUV/Cg13891 Clethrionomys glareolus

Fig. 4. Phylogenetic tree of Arvicollinae-borne hantaviruses based on the complete coding region of the S segment. The tree was estimated based on the pairwise distance matrix calculated using the Kimura two-parameter evolutionary model. Tree topology was estimated using the Fitch-Margoliash method (100 bootstrap replicates). Bootstrap support values of greater than 50% are shown at the appropriate branch points. Gray and white boxes outline strains carried by different rodent hosts. Latin names of each rodent host species are written within the boxes. The black arrows show likely host switching events. Abbreviation C. R. stands for the Czech Republic. For the abbreviations of hantavirus names see Table 1. Introduction 31 are highly divergent from all PUUV strains and form a separate lineage on the phylogenetic trees (Fig. 4), located outside the clade formed by PUUV strains. This virus, provisionally called Hokkaido virus, (HOKV) has not been isolated in cell culture, and thus its serologic and genetic characterization has not been completed. Nevertheless, the absence of PUUV-induced HFRS cases in Japan suggests that this virus may be apathogenic for humans (106). Partial S and M segment sequences of another PUUV-like virus, Muju, were recently recovered in Korea from the vole Eothenomys regulus, which was considered a subspecies C. rufocanus until 1978 (239). However, further investigation is needed to clarify the relationships of this genotype with PUUV and its ability to cause human infections. TULV was discovered in the European common vole (Microtus arvalis) trapped in central Russia (193). Later, this hantavirus was found in M. arvalis in Moravia (Czech Republic) (194), Slovakia (230, 231), Austria (19), Poland (238), and Switzerland (224). As the European common vole is widely distributed across Central and Eastern Europe (165), TULV most likely will be found in other European countries as well. The phylogenetic tree based on complete coding S segment sequences showed all TULV strains to be monophyletic and form two clades. The first clade includes lineages formed by strains from Russia and east Slovakia, and the second includes strains from west Slovakia, Moravia, and Switzerland. Partial sequences available from Austria also belong to the second clade (19). PHV and PHV-like viruses in North America. PHV discovered in the late 1980s from the meadow vole (Microtus pennsylvanicus) was the first hantavirus found in the American continent. Currently, several PHV-like genotypes distinguished from each other by 10-20% of nt divergence, are known to be associated with different species of North American Microtus voles (200). Isla Vista virus (ISLAV) and Bloodland lake virus (BLLV) were discovered from the California vole, Microtus californicus (236), and the prairie vole, Microtus ochrogaster (223), respectively. A few sequences derived from the montane vole, Microtus montanus, could represent yet another PHV-like genotype (166, 200, 213). Although other PHV-like genotypes were described from the California- Nevada region and elsewhere from USA, they were not assessed with separate names. Since PHV-like viruses are thought to be apathogenic to humans they have not been studied in detail and thus the molecular epidemiology and host- associations of these viruses have remained obscure. KHAV discovered in the reed vole (Microtus fortis) from the Far East Russia (35, 91) represents an interesting case in the hantavirus evolution. While all other Microtine-borne hantaviruses - TULV, PHV, BLLV and ISLAV are monophyletic, suggesting that they have evolved from one common ancestor Introduction 32 more than 10,000 years ago (the approximate time of the formation of the Bering strait that separated America from Eurasia). KHAV is paraphyletic with this group and monophyletic with PUUV and Topografov (TOPV), carried by bank voles and lemmings, respectively. Such inconsistence with the general pattern of evolution suggests that KHAV did not co-evolve with Microtines as long as other viruses, but has been acquired from another rodent species (most probably from Lemmus) much later. TOPV was discovered in samples of Siberian lemmings (Lemmus sibiricus), trapped in the basin of the Topografov river in Taimyr Peninsula (195). This area is inhabited by the western type of L. sibiricus, which is found in the Siberian tundra along the arctic coast up to Taimyr. Two other types of L. sibiricus are recognized in Siberia: the central (inhabits a vast area between Lena and Kolyma rivers) and the eastern type (also known as L. trimucronatus, distributed to the east of Kolyma river and in Alaska) (47). Interestingly, the western form of L. sibiricus appears to be more closely related to the Norwegian lemming of Fennoscandia, L. lemmus, than to the central type. Analysis of S segment sequences of two strains originated from the same geographic locality revealed a nt divergence of 8% (256). At the same time, sequence of the third strain recovered approximately 600 km apart from the first two strains was much closer related to one of them (1% nt divergence). This observation indicates that clear geographic clustering seen for many other hantaviruses might not be observed for TOPV. L. lemmus is famous for its mass migrations caused by drastic fluctuations in population density (72), which could be responsible for the redistribution of existing geographic variants. As mentioned above, TOPV is most closely related to KHAV, carried by Microtus fortis. Comparison of the phylogenetic trees of hantaviruses and their rodent hosts revealed two discrepancies: (i) Microtus-derived KHAV clusters with Lemmus- derived TOPV and not with other Microtine-borne viruses; (ii) TOPV/KHAV pair shares a common ancestor with PUUV and this clade has a more recent origin than the clade formed by all other Microtine-borne viruses. Such clustering contradicts to the rodent phylogeny because Clethrionomys and Microtus species are more related to each other than to Lemmus, which occupies the most ancestral position in the phylogenetic tree. Thus the location of an ancestor for TOPV/KHAV pair contradicts to the phylogeny of both rodents and could be explained by two subsequent host-switching events: (i) an original ancestral virus was transmitted from Clethrionomys to Lemmus and eventually became a common ancestor for TOPV and KHAV; (ii) host- switching from Lemmus to Microtus occurred, thus giving rise to KHAV (256). The M segment of TOPV demonstrated 23% nt and 12% aa divergence with KHAV and 24% nt and 18% aa divergence with PUUV. However, the Introduction 33 divergence between S segments of TOPV and KHAV was disproportionately low: 12% at the nt and 4% at the aa level, suggesting that reassortment of S segment could happened in the evolution of these two viruses. VLAV. Recently new hantavirus genotype (proposed name Vladivostok) was identified in Microtus fortis in Far East Russia (105). Although this hantavirus is found in the same rodent species as previously described KHAV, comparison of the complete coding S segment sequences of two genotypes showed 20% and 10% of nt and aa diversity, respectively. This level of diversity is higher than the diversity seen so far between any hantavirus strains that belong to the same species and thus, according to the genetic criteria, the Vladivostok genotype may represent a new hantavirus species. Comparative serology and more detailed epizootiologic studies are needed to define whether this virus is serologically distinct from KHAV and also if M. fortis is its true natural carrier.

Sigmodontinae-borne hantaviruses circulating in North America form three phylogenically distinct groups. Two of them are associated with two distinct genera of Sigmodontinae rodents: Peromyscus and Reithrodontomys, while the third group includes viruses carried be the genera Sigmodon and Oryzomys. Viruses that belong to different groups are distinguished by at least 27% nt differences in the M segment sequences (169).

Peromyscus-associated. Sin Nombre virus (SNV) was discovered as the causative agent of an HPS outbreak in the southwestern United States (Four Corners area) in 1993 (177). The deer mouse (Peromyscus maniculatus) was identified as the primary natural reservoir of this virus. Since then, extensive studies of SNV across North America revealed high genetic diversity of this species and geographic clustering of its genetic variants (71, 75, 166, 213, 240). The complete genomic sequences determined for two SNV strains (one originating from an autopsy of a patient with HPS (NM H10) and another one (NM R11) - from P. maniculatus trapped nearby the patient’s house) were closely related to each other (27, 240). Complete S and M segment sequences were also recovered for two cell culture isolates, Convict Creek 74 and Convict Creek 107, originated from Eastern California (132). Both isolates showed a similar level of nt diversity with the prototype SNV strain, 11-14% and 11-12% in the sequences of S and M segments, respectively. The S segment sequences of the two Californian isolates differed by 13%, while the M segment sequences showed a surprisingly low divergence of 1%, suggesting that reassortment of genomic segments has played a role in the evolution of these strains. As demonstrated later, reassortment between genetic variants of SNV seems to Introduction 34 occur rather frequently. Genetic analysis of hantaviral strains recovered from Peromyscus rodents revealed at least three other genotypes that are often referred to as SNV-like viruses: Monongahela virus (MGLV) from a sub- species of Peromyscus maniculatus, P. maniculatus nubiterrae (cloudland deer mouse), from the Appalachian Mountains (237); New York virus (NYV) (77, 78) and Blue River virus (BRV), both from white-footed mice (Peromyscus leucopus) recovered from the coastal areas of Rhode Island/New York and several locations in the Great Plains, respectively (166, 169). Limestone Canyon virus (LCV), the latest member of this group, was discovered in Arizona from brush mouse, Peromyscus boylii and appears to be distinct from all Peromyscus-borne hantaviruses (216). Analysis of partial (139 nt) sequences of G2 coding region of SNV-like viruses demonstrated up to 10% of nt divergence between all strains of NYV originated from human HPS cases and rodent samples, and at least 12% divergence with SNV sequences (166). Sequences of MGLV showed up to 17% of interstrain divergence in the same region, while at least 11% and 9% nt substitutions distinguished them from SNV and NYV, respectively. BRV is represented by several genetic lineages originated from Oklahoma, Indiana, and Missouri. Genetic diversity between lineages from Oklahoma and Indiana determined for the complete M segment sequence were 4%, and up to 7% between these lineages and viruses SNV and NYV (169). Genetic analysis of the S segment sequence of LCV demonstrated a remarkably high level of sequence divergence with other viruses in the group (14-15% aa differences in the S segment). In fact, genetic comparison and phylogenetic analysis (216, Fig. 5) showed that the S segment of LCV is more closely related to that of Reithrodontomys-associated hantaviruses with 9-11% of aa divergence. Host switching of LCV from Reithrodontomys to Peromyscus was suggested to explain this phenomenon, but without reliable support from the M segment-based phylogenetic analysis, reassortment of the S segment remains an equally likely possibility. Reithrodontomys-associated virus species include: (i) El Moro Canyon virus (ELMCV), carried by the western harvest mouse (Reithrodontomys megalotis), the most common Reithrodontomys species in the western United States and Mexico (76); and (ii) Rio Segundo virus (RIOSV) originated from Costa Rica and carried by the mexican harvest mouse (Reithrodontomys mexicanus). Comparison of the S segment sequences available for ELMCV and RIOSV showed 22% nt divergence (200). Sigmodon/Oryzomys-associated group of hantaviruses include two related species, Black Creek Canal (BCCV) and Muleshoe (MULV), carried by the same rodent species, hispid cotton rat (Sigmodon hispidus). Each virus is Introduction 35

HTNV TOPV BAYV/Louisiana Oryzomys palustris

96 BCCV USA Sigmodon hispidis 60 MULV/SH-Tx-339

Venezuella Sigmodon alstoni CADV/VHV-57 MGHV-1 Peromyscus maniculatus nubetirrae 69 NYV/Convict Creek 107

100 NYV/U29210 Peromyscus leucopus

100 100 NYV/RI-1

75 SNV/Convict Creek 74 100 SNV/NM H10 USA Peromyscus maniculatus 100 SNV/NM R11 LCV/68273 Peromyscus boylii

98 RIOSV/RMx-Costa-1 Reithrodontomys mexicanus

ELMCV-RM-9 Reithrodontomys megalotis 96 99 ELMCV/NM-164 Neotoma mexicana

host is not known 63 Hu39694 BERV/Oc22531 Oligoryzomys chacoensis 100 100 LECV/22819 Oligoryzomys flavescence

OranV/22996 Argentina Oligoryzomys longicaudatus 78 100 AND Nort Oligoryzomys chacoensis

ANDV/AH-1 96 100 ANDV/9718133 Chile 100 ANDV/Chile-9717869 Oligoryzomys longicaudatus MACV/13796 Argentina Necromys benefactus 95 58 PERGV/14403 Akodon azarae

RIOMV/Om-556 Bolivia Oligoryzomys microtus

99 LANV/510B Paraguay Calomys laucha

Fig. 5. Phylogenetic tree of Sigmodontinae-borne hantaviruses based on the complete coding region of the S segment. The tree was estimated based on the pairwise distance matrix calculated using the Kimura two-parameter evolutionary model. Tree topology was estimated using the Fitch-Margoliash method (100 bootstrap replicates). Bootstrap support values of greater than 50% are shown at the appropriate branch points. Gray and white boxes outline strains carried by different rodent hosts. Latin names of each rodent host species are written within the boxes. Arrows indicate contradictions in the host-dependent clustering of hantaviruses, suggesting possible host switching events. For the abbreviations of hantavirus names see Table 1. Introduction 36 associated with distinct geographic populations of Sigmodon hispidus in the southern United States, which are thought to be morphologically and phylogenetically distinct rodent subspecies (210). The two viruses demonstrated 20% nt diversity in the complete coding sequences of the S segment. Bayou virus (BAYV) carried by the marsh rice rat, Oryzomys palustris, clusters with Sigmodon-associated viruses on the phylogenetic trees and is more closely related to them than to other North American hantaviruses (116, 168, 250). Remarkably, some phylogenetic analyses place BAYV and BCCV together and apart from MULV, suggesting that a host switching event could have been involved in their evolution. (178). This group also includes one South American virus, Caño Delgadito (CADV), which was recovered from cane mouse (Sigmodon alstoni) in Venezuela. Although this virus has been characterized only with partial sequences of S and M segments, phylogenetic analysis indicated that its S segment could have originated from the Reithrodontomys- associated hantavirus lineage (52). Hantaviruses circulating in South America are associated with three major South American tribes of Sigmodontinae: Phyllotini, Acodontini and Oryzomyini. All currently known South American genotypes are monophyletic and share from 75% to 90% S- and M-genome segment nt sequence identity (130, 200). Oryzomyini-associated viruses include the majority of recently discovered South American hantavirus genotypes and represent the most extensively characterized group from that continent. This group includes six genotypes of which only Andes virus (ANDV) has been characterized by a complete genome sequence and recognized as a distinct hantavirus species (18, 159). After the discovery of ANDV in 1996 from Argentina (138), different strains of the virus were recovered from patients with HPS from Argentina and Chile and also from the long-tail pigmy rice rat (Oligoryzomys longicaudatus) (130, 138, 139, 180, 181; 249). Partial M segment sequences of different strains showed up to 11% and 4% of the nt and aa divergence, respectively (138). Sequences of hantaviruses Lechiguanas (LECV), Bermejo (BMJV), Oran (ORNV) and Hu39694 were recovered from different species of Oryzomyini rodents in Argentina (see Table 1), except for Hu39694, which was recovered from the autopsy of an HPS patient (130). These viruses are closely related to ANDV and form a distinct group of ANDV-like genotypes on the phylogenetic trees (18, Fig. 5) Genetic divergence within this group determined for the ORF of S segment and N protein is up to 18% and 4%, respectively. The last virus in this group, Rio Mamore (RIOMV) from O. microtus (16, 204), is phylogenetically distinct from others and instead clusters with Phyllotini-associated LNV, suggesting a possible host switching event. Introduction 37

Akodontini-associated. This group is currently represented by two genotypes: Pergamino (PERV) from Akodon azarae and Maciel (MACV) from Necromys benefactus (129). Phylogenetic analysis showed them to be monophyletic with Oryzomyini-borne viruses (18, Fig. 5) or even indistinguishable from them on some phylogenetic trees. The S segment/N protein sequences of the two viruses showed 19% nt and 4% aa differences, respectively, and up to 21% nt and 6% aa divergence with Oryzomyini-borne hantaviruses. Phyllotini-associated. The vesper mouse (Calomys laucha) is the only Phyllotini species known to harbor hantaviruses. This species carries Laguna Negra virus (LNV) (95), which demonstrates 23-25% nt and 10-12% aa differences with other South American viruses, except for RIOMV (20% nt and 7% aa differences, respectively).

Hantaviruses and their hosts

Phylogenetic analysis of hantavirus species reveals three distinct clades formed by hantaviruses carried by Murinae, Arvicolinae, and Sigmodontinae rodent sub-families (Fig. 6). The only known hantavirus carried by the order Insectivora, Thottapalayam virus (23), is quite distinct from other hantavirus species and is located apart from the rodent-borne hantaviruses on phylogenetic trees. Such a clustering of hantavirus species is observed irrespective of their geographic origin and mirrors the phylogenetic relationships of their hosts, suggesting that hantaviruses have co-evolved with their carriers for about 100 MYA (the approximate diversification time of the orders Rodentia and Insectivora estimated from fossil records) (197). Rodents (order Rodentia) represent the largest order of mammals with 39% of the generic and 43% of the specific global mammalian diversity, including 443 genera and 2021 species, respectively (31). The biggest share of this generic and specific diversity (63% and 66%, respectively) is confined within a single rodent family – Muridae. Members of this family are spread worldwide and occupy essentially all major habitats. The population structure within the family is highly variable: some species experience drastic oscillations in the population size while others maintain relatively stable populations over the years (82). The specific patterns of population behavior can have important consequences for the epidemiology of hantaviruses. For instance, bank voles in Northern Europe experience regular oscillations of the population density that occur every 3-5 years (population cycles). In contrast, in temperate Europe populations of bank voles are relatively stable but occasional “mast years” (abundant crops of beech and oak seeds) can result in the drastic increase of the Introduction 38

SEOV HTNV AMRV Arvicolinae-borne Murinae-borne ISLAV SAAV KHAV BLLV DOBV PHV PUUV TOPV HOKV TPMV

TULV

MULV LECV BCCV

BermejoV BAYV Hu39694 OranV ELMCV PERVA NDV SNV RIOSV MACV LANV NYV RIOMV MGHV Sigmodontinae-borne

Fig. 6. Phylogenetic tree of hantavirus species. The tree was estimated based on the complete coding region of the S segment by calculating a pairwise distance matrix (Kimura two-parameter evolutionary model). Tree topology was estimated using the Fitch-Margoliash method (500 bootstrap replicates). For abbreviations see table 1. population size and lead to HFRS outbreaks (45, 74, 172). It has been noted that rapid fluctuations in population densities of murid rodents are particularly common in semi-arid and arid ecosystems but may occur in any habitat in favorable conditions. Thus, increased precipitation during El Ninõ Southern Oscillation in South and North America (82) has been suggested as the main reason for an increase in rodent population densities, and consequently for increased caseload of HPS (81). Remarkably, different murid species in a given locality may respond differently to the same environmental changes (82). Human activities can play an important role in re-structuring of rodent populations and subsequently influence rodent evolution. Antropogenic changes of natural ecosystems, e.g. deforestation, often favor murid rodents over other animal species and result in decreased variety of species, but higher densities of those that persist. Many hantavirus-carrying species are thought to be particularly favored in the less-species rich and ecologically less complex communities as they inhabit biotopes similar to those created by man and can easily utilize manmade buildings and storage places. Agricultural human activities such as fall harvests have been accompanied by the increase in the Introduction 39 disease rate in Argentina (162) and Korea (119). House-keeping activities such as work in barns and other storage areas during winter time is one of the main risk factors of PUUV infection in Europe (253). As noted before (56), hantaviruses are primarily carried by abundant, widespread rodent species that dominate local rodent communities. This suggests that rodent-to-rodent transmission of hantaviruses is favored in the large continuous rodent communities, while in smaller communities with discrete populations it is restricted and can lead to hantavirus extinction (200). This feature of hantavirus-rodent relationship can explain the fact that only some of the closely related rodent species carry hantaviruses, while others appear to be hantavirus-free. For example, while P. maniculatus and P. leucopus in North America both carry SNV-like viruses, two sister species P. polionotus and P. gossipinus appear to be hantavirus-free (200). Comparison of species diversity of rodents and hantaviruses suggests that adaptive radiation and formation of new species can occur at different rates, depending on the ecological factors. Thus, the genetic variability of North American hantaviruses within single host species appears to be much higher than the intraspecies variability of South American hantaviruses (82). Sigmodontinae rodents invaded South America relatively recently, between 9 and 3 MYA (reviewed in 42), and thus the timescale provided for radiation of species in South America was much smaller than that of North American hantaviruses. It is thought that the main principle of hantavirus evolution, i.e. co- speciation with the radiating genetic lineages of rodents, is most clearly observed when hantaviruses are associated with well-established, genetically stable rodent species. At the same time, fast diversification of “old” rodent species associated with the adaptive radiation and establishment of new species (which most probably has taken place in South America) may be accompanied by fast evolution of hantaviruses (82). The spreading of diverse rodent species to new geographic areas and their competition for new ecological niches is also likely to create a specific ecologic environment associated with high frequency of spill-over infections and resulting in the increased amount of virus host switching. Although apparently every hantavirus has a predominant rodent carrier this association is sometimes compromised by the transmission of hantaviruses to non-specific hosts (spill-over infections). Spill-over infections are thought to occur in nature fairly often. For example, SNV could be frequently detected in a number of different rodent species including P. truei, P. boylii, Mus musculus and some others (178). Hantaviruses carried by harvest mice (Reithrodontomys) have been encountered in deer mice (Peromyscus) and vice versa, and vole-borne hantaviruses have been found in Sigmodontinae rodents (79, 210, 236, 250). The main difference of spill-over infections from Introduction 40 productive infections of natural hosts is the apparent inability of spillover virus to establish a long-term carrier state in the non-specific host. The relatively small number of host switching events observed in hantavirus evolution suggests that there is a principal difference between viral ability to infect a non- specific host and its ability to establish a long-term carrier state in another rodent species. The reasons for the resistance observed in non-specific hosts are not clear. One possible explanation is that multiple specific changes in the viral genome are required for the efficient replication in non-specific host. The low frequency of those changes and/or low rate of transmissions between the species may be the main reason why host switching is uncommon (82). Still, one could consider spill-over infections as an interesting opportunity in hantavirus evolution: trnsmission of the viruses to non-specific hosts in favorable conditions leads to host switching events. From this perspective, host switching, along with the co-speciation, may represent another general mechanism of hantavirus evolution. The current number of proven or suspected cases of host switching in hantavirus evolution is close to 10 (Fig. 3, 4 and 5). The first convincing example was provided by NYV in white-footed mouse (Peromyscus leucopus), that is genetically distinct from BRV, carried by the same host species, and instead demonstrates close genetic ties with SNV and MGHV viruses, carried by Peromyscus maniculatus. As P. maniculatus and P. leucopus have been sharing a part of their geographic range for more than 10,000 years, NYV is thought to have originated by a host switching of ancestral MGHV into Peromyscus leucopus (169). Another prominent case of host switching is given by the close genetic relationships between TOPV and KHAV, carried by Lemmus sibiricus and Microtus fortis. It was suggested that an ancestral PUUV was first transmitted to lemmings (Lemmus sp.) approximately 1.5 million years ago yielding TOPV, which then performed a more recent switching to Microtus producing KHAV (256). Hantavirus infections of the host species are thought to be persistent and generally non-pathogenic, although two studies have provided evidence of lung edema in the wild-caught SNV-infected Peromyscus rodents (152, 176). The data concerning longevity of hantaviral infections in the rodent hosts are controversial: although infections are generally believed to be life-long, studies on SEOV and PUUV have suggested that in some cases infected animals can clear the infection (104, 160). The universal persistent infection caused by hantaviruses in the rodent hosts and in cell culture includes a short acute stage (from 7 to 14 days), marked by high levels of infectious virus, and followed by a prolonged chronic stage when the infection is productive but associated with low levels of infectious virus, fluctuating cyclically. During the chronic phase viral antigens can be detected in different tissues and organs including lungs, Introduction 41 kidneys, liver, spleen, salivary and parotid glands and brown fat. Infectious virus can also be recovered from the urine, feces and saliva. Persistence of hantaviruses within rodent hosts is not well understood but is thought to depend on both virus-mediated modification of host immune response and alteration of viral replication (98). The latter might be achieved through the accumulation defective viral RNA segments, carrying short terminal deletions (160). As it has been shown recently, the length and concentration of the L RNA deletions varied during the persistence of SEOV and correlated with reduced levels of viral replication (161). Maintenance of hantaviruses in rodent populations is poorly understood. It has been shown that hantavirus seroprevalence may substantially change within the population with time and across space. For instance, infection rates in populations of P. maniculatus can vary from 0 to 47% (164). For most rodents the seroprevalence is higher in males than in females and also increases with animal age and body mass (15, 79, 163). Such a picture suggests that increased activity (e.g. territorial behavior) of rodents may lead to increased numbers of rodent-to-rodent contacts and consequently to a high risk of infection. In Rattus norvegicus seroprevalence between males and females is equal (26); higher seroprevalence is mostly associated with increased body mass and also commonly observed in animals with deep wounds (59). The last observation led to the hypothesis that aggressive behavior common for both genders of the brown rat can be a risk factor for hantaviral transmission and that hantaviruses can be transmitted through biting. Direct evidence supporting this hypothesis is missing because it seems reasonable that rodents with aggressive behavior are also more likely to acquire the virus through the inhalation of infected aerosols. One reason for the correllation between increased seroprevalence and rodent age is that no vertical transmisson has been observed for any studied hantaviruses (HTNV, PUUV, and SEOV), and newborn animals are thought to be protected from infection by maternal antibodies (167, 273). Due to the absence of arthropod vectors and moderate efficiency of rodent- to-rodent transmission hantavirus evolution is efficiently restricted to the local populations of the host species. The long-term association and co-evolution of hantaviruses and rodent populations have resulted in remarkable similarity of rodent and hantavirus phylogenetic trees at all levels. The highest level of genetic association is provided by the division of hantaviruses into three main clusters according to the division of the Muridae rodent family into three sub- families: Murinae, Arvicolinae and Sigmodontinae (197). This division is independent of geographic distribution and reflects the macroevolution of big rodent taxa over millions of years. Recent, small-scale, evolutionary events such as geographic and ecologic isolation of rodent populations and formation of Introduction 42 new sub-species are reflected in co-speciation of hantaviral genotypes with genetic or geographic races of rodent species. For instance, phylogenetic analysis of P. maniculatus has revealed four phylogenetic super-groups within the species: central, northeastern, northwestern, and southwestern (200). This division agrees well with the morphological division of P. maniculatus into “grassland” and “forest” forms, as most of the rodents in the central super- group belong to the “grassland” form while the three other super-groups include rodents of the “forest” morphological form from different geographic regions. It was shown that two closely related hantavirus genotypes, SNV and MGLV, both carried by P. maniculatus, are associated with phylogenetically and morphologically distinct forms of that species, the grassland form and the eastern forest form, respectively (200). Another clear pattern of co-evolution is provided by the relationship between the white-footed mouse P. leucopus and the associated hantavirus genotype BRV. Molecular phylogenetic of P. leucopus shows that it is divided into four distinct genetic clades: eastern, central, northwestern and southwestern (169). Comparison of the phylogenies inferred for P. leucopus and BRV demonstrated that two genetically related but distinct lineages of BRV from Indiana and Oklahoma states are associated with two distinct mtDNA haplotypes (and distinct contemporary chromosomal races) of rodents, northeastern and southwestern, respectively (200). The above- mentioned examples are only the best-characterized cases of what seems to be the most general mechanism of host-associated hantavirus evolution. Long-term co-evolution of hantaviruses and rodents also provides insights into past events of rodent evolution, such as mass migrations. The last large- scale migration of rodent species in Europe occurred after the retreat of the Weichselian ice sheet (8.000-13.000 years ago). At that time the territory of Fennoscandia was re-colonized by C. glareolus by two different routes. One route came from the south through the land bridge existing at that time between Denmark and Sweden into the southern part of Scandinavia, while another one came from the northeast through northern Finland and Russia. Populations of bank voles that came by the two routes met in the central Sweden and at present are still divided by a contact zone of 50 km (93, 247). The Northern and Southern populations divided by that zone are distinguished by mtDNA sequences. Comparison of the phylogenetic data of C. glareolus with that of PUUV demonstrated that viral strains circulating on different sides of the population border belong to distinct genetic sub-lineages (North- Scandinavian and South-Scandinavian) (8, 90, 150). Similar “population borders” that distinguished different mitochondrial DNA types have been found in Finland (distinguished the same two mtDNA races as in Sweden) and Russia. The Russian border is located in PUUV endemic region near Ural Introduction 43

Mountains in western Bashkortostan, Udmurtia and Perm regions. It separates the western (mtDNA race similar to the “southern” bank voles from Fennoscandia) and the eastern (mtDNA race similar to the “northern” bank voles from Fennoscandia) populations of C. glareolus (200). The absence of the arthropod and other vectors in hantavirus evolution means that their evolution is restricted to the host range, i.e. primarily influenced by the genetic and ecological factors that form rodent populations. This has several important consequences: a) diversification of hantavirus species occurs mainly along the lines of adaptations to genetically diverse specific rodent species; b) geographic distribution of existing genetic variants reflects evolutionary events in the rodent history, i.e. speciation, diversification, extinction, migration, that result in circulation of genetically distinct hantaviruses on different continents, geographic clustering of hantavirus variants, and co- circulation of different hantavirus species within the same geographic area; c) epidemiology of hantavirus infections is strictly dependent on the population dynamics of the rodent hosts; d) lower (smaller rate) of genetic variability via genetic shift in comparison with arthropod-borne viruses due to the absence of arthropod vectors which serve as “mixing vessels” for genetic exchange; e) comparatively low rate of viral transmission due to more local distribution of rodent population in comparison to that of flying vectors, e.g. mosquitoes and birds, and also due to the less efficient way of virus delivery: air-borne transmission from the environment (hantaviruses) vs. direct injection of virus into the bloodstream (arboviruses).

Mechanisms of hantavirus evolution

Since hantavirus evolution is closely associated with the evolution of their rodent hosts and human infection represent a mere dead-end event in hantavirus evolution, the persistently infected rodent species should be regarded as the main evolutionary scene for these viruses (197). Genetic analysis of different hantavirus species suggests that genetic drift, i.e. gradual accumulation of genomic changes such as nucleotide substitutions, small deletions and insertions, is the main evolutionary mechanism leading to hantavirus diversification. Persistent long-term infection that hantaviruses establish in the rodent species also creates an opportunity for co-infections of a single rodent by different hantavirus strains, thus allowing genetic shift through reassortment of genome segments or homologous recombination. Introduction 44

Genetic drift. Analyses of genomic pools that comprise PUUV and TULV within their natural hosts indicate that hantaviruses, similarly to many other RNA viruses, exist as mixtures of closely related genetic variants, i.e. quasispecies. In general, quasispecies populations of RNA viruses allow for rapid evolution via selection of pre-existing variants, resulting in the establishment of altered mutant spectra with a higher fitness to a new environment (196). Nevertheless, in the constant environment quasispecies populations may exhibit long periods of stasis if the master genotype has high fitness to it (33, 83). The latter is likely to be the case for hantaviruses. Analysis of the S segments of TULV quasispecies suggested that fixation of neutral mutations that arise due to the error-prone activity of hantaviral polymerase may be the main diversifying factor in hantavirus evolution (196). The relative genetic stability of hantaviruses within the specific rodent populations, e.g. co- circulation of distinct genetic lineages within the same geographic area (213), suggests that in spite of their apparent genetic plasticity hantaviral genomes may remain stable over long periods of time, and be represented by a relatively conserved master sequence. Hantavirus evolution appears to be quite slow, presumably because most hantaviruses adapted to their natural hosts a long time ago and little current positive selection pressure for change is applied to hantaviruses from the rodent hosts (142). The error rate of PUUV and TULV polymerases, estimated as 1 × 10-3 – 3 × 10-3 mis-incorporations per site (196) is close to that of other viral RNA-dependent RNA polymerases lacking proofreading activity and, therefore, should provide sufficient basis for the nt variation. Nevertheless, the actual substitution rate of PUUV approximated from the rodent fossile data is much lower and ranges from 0.7 × 10-7 to 2.2 × 10-6 nt per site per year and from 3.7 × 10-7 to 8.7 × 10-7 nt per site per year for the S and M segment sequences, respectively (233). Such a slow substitution rate in hantavirus evolution is comparable with the rates suggested for other stable RNA viruses, e.g. hepatitis G virus (9 × 10-6) (243), which infect humans persistently. In contrast, the substitution rates estimated for more rapidly evolving RNA viruses such as human immunodeficiency virus and hepatitis C virus are much higher, in the order of 10-2 to 10-5 nt per site per year (2, 128). The extremely high ratio of synonymous vs. non-synonymous substitutions as well as the high proportion of nt substitution in the 3rd position of codon observed between hantaviral strains compared across large geographic area suggests a neutral mode of hantavirus evolution and shows that purifying selection predominantly occurs at the aa level (61, 82, 233). The neutralist view of RNA virus evolution suggests that advantageous mutations are rare and that most of the non-neutral mutations are deleterious (112). This view also agrees with the data concerning the structure of quasispecies population. Studies of Introduction 45

TULV quasispecies existing in the natural host demonstrated that mutations in coding and non-coding regions are generated with equal frequencies and are evenly distributed between the three positions of the codon (196). Thus, although non-synonymous substitutions are frequently generated, they do not become fixed in the hantaviral populations, and strain variation of 18-20% at the nt level often translates to changes of only 2-3% at the aa level (79). Positive selection of beneficial variants may also play a role in hantavirus evolution, especially in the situations when fast adaptation to an altered environment is needed (for example when the virus is adapting to a new host after host switching). In that case new quasispecies with better fitness to the changed environment may quickly emerge and become dominant in the population. This can be illustrated by the adaptation of PUUV to cell culture, when after 11 passages in Vero E6 cells PUUV variants carrying two specific nt substitutions in the NCRs of the S segment emerged and became dominant in the quasispecies population (149). Values of diversity between the nt and aa sequences of distantly related hantaviruses, i.e. hantaviruses carried by different rodent sub-families, appear to be similar. Generally, different hantaviral sero/genotypes demonstrate 30- 40% nt diversity in all genomic segments, while the corresponding values for deduced sequences of N, G1G2 and L proteins are 15-40%, 20-50% and 10-30%, respectively (197). Levels of diversity reported for different strains of the same species vary between different viruses, ranging from 13-14% for the S segment sequences of SNV to 20%-23% of nt divergence in the M segment sequences of PUUV and HTNV, respectively. Sequence variation seems to be unevenly distributed along the genome. The coding sequences of the three hantavirus segments are generally more conserved than their NCRs, probably due to the functional constraints imposed by the protein-coding function. NCRs of different hantaviruses vary substantially both in length and sequence, but also contain very conserved terminal regions that are thought to be involved in the regulation of transcription and replication (reviewed in 98, 197, 201, 222). The most heterogeneous regions are located in the 3’ NCRs (cRNA-sense) of the S and the M segments, between the stop codon and approximately 100 terminal nucleotides. Comparison of different hantaviruses within those regions shows prominent sequence variation with multiple substitutions, deletions and insertions. NCRs are routinely excluded from the phylogenetic analysis of distantly related hantaviruses due to the high variability, but may be useful for clarification of phylogenetic relationships between different genetic lineages of the same species (201). The coding and non-coding regions of hantavirus genes are functioning under different pressures that seem to be unequal for the different parts of the coding region as well. Analysis of PUUV variability Introduction 46 identified two regions with increased number of nt substitutions, which can be approximately mapped to the nt 650-900 and 1200-1600 (192). The N protein also contains a hypervariable region spanning through aa 233-275 that corresponds to the first variable region of the S segment and carries epitopes recognized by both monoclonal antibodies and human patient sera (145, 254). Although the rate of non-synonymous substitutions within this region is unusually high, they seem to be distributed at random (86). Besides, the ratio of conserved/non-conserved substitutions does not exceed 1, suggesting that low functional or structural constraints rather than positive selection are responsible for the variability in this region (233). Genetic analysis of TULV quasispecies existing in the persistently infected natural hosts also demonstrated high nt diversity within the corresponding region of the S segment (nt 400-900) (196). Genetic variability of the deduced G2 protein sequences of two different PUUV strains was found to be two-times higher than that of the N protein and it was relatively evenly distributed along the sequence (192). Genetic shift. The other principal evolutionary mechanism that is responsible for drastic changes in viral genetic background, genetic shift, has been recorded in hantavirus evolution in the form of reassortment and homologous recombination. Reassortment (exchange of genomic segments) is a common evolutionary mechanism operating in segmented RNA viruses, e.g. influenza A virus is well-known for its reassortment that has led to human pandemics. Reassortment provides a potential for rapid transitions across the virus fitness landscapes, which are difficult (or even impossible) to achieve through ordinary genetic drift. This mechanism could allow for rapid expansion of the virus to new ecological niches and fast adaptation to a new environment. Evidence for reassortment can be provided by a discrepancy in the phylogenies inferred for different genomic segments, which suggest different evolutionary histories for different virus genes. In hantaviruses, reassortment was first observed between the genetic lineages of SNV (132). The data suggested that reassortment of the M segment (and to a lesser extent of S and L segments) may occur between different strains of a single hantavirus genotype. Nevertheless, phylogenetic analyses do not exclude the possibility that S and L segments represent one group of genetic complementation and thus undergo reassortment together. The possibility of inter-genotype reassortment was also suggested by phylogenetic analysis of CADV. While this virus is carried by Sigmodon alstoni and clusters with other Sigmodon-borne hantaviruses on the M segment-based phylogenetic tree, its S segment has clearly originated from the Reithrodontomys-borne viruses, currently represented by ELMCV and RIOSV (71, 132). Model experiments aimed at Introduction 47 producing reassortants in vitro demonstrated that intra-genotype reassortants (between SNV strains) of M and S segments appear fairly often and more frequently than that of the L segment (211). In contrast, inter-genotype reassortment (between SNV and BCCV) was very rare and only one such variant was obtained among 294 progenitor plaques. Remarkably, the same study showed that diploid progeny plaques (i.e. containing S or M segment of two parent viruses) were a frequent outcome of double infections (30% of progenitor plaques). While those plaques were not stable and diploid variants were not detected after a few passages, such a prolonged association of two different genotypes (which could be packaged in the same virus particle) is likely to facilitate another type of genetic exchange - recombination. This mechanism was only recently described for hantaviruses and became the first example of recombination registered in negative-strand RNA viruses. Similarly to reassortment, recombination can be detected by phylogenetic analysis. In this case, contradictory phylogenies based on the different regions of a particular genome segment provide the evidence for homologous recombination. Analysis of TULV genetic lineages from Slovakia indicated that the central part of the S segment of East Slovakian lineage (mapped to the region between nt 400-415 and around nt 1200) was more closely related to TULV lineage from Russia, while two other regions (nt 1-400 and 1200-1833) appeared to be more closely related to the lineages from Western Slovakia and Czech Republic (231). Such a mosaic structure was consistent with a hypothesis of homologous recombination between the “Russian” and “Czech” genotypes with two recombination points located between nt 400-415 and around nt 1200, respectively. Recently, experimental evidence for the possibility of homologous recombination in hantaviruses was provided by the in vitro experiments with S segments of two TULV strains (202). Using a transfection-mediated system the authors were able to rescue recombinant S segments from TULV-infected cells. The virus containing the recombinant S segment was passaged several times and grew to similar titers as the wt virus, thus proving its viability and replication competence. In the majority of analyzed recombinant molecules the recombination breakpoint was located quite close to one of the breakpoints mapped in the natural recombinants, i.e. at nt 332-368, demonstrating a possible common mechanism for the recombination.

Aims of the study 48

AIMS OF THE STUDY

By the beginning of the 1990s several reports had suggested that hantavirus(es) other than PUUV can cause human HFRS cases in Europe including the European part of Russia (10, 60, 66, 179). Serological screening of patient sera performed by immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) revealed higher antibody titers to the antigens of Murinae-borne HTNV and SEOV than to Arvicolinae-borne PUUV. In 1992 a new hantavirus, Dobrava (DOBV), was isolated in Slovenia from yellow- necked mouse (Apodemus flavicollis) (11). Soon this virus was linked to outbreaks of severe HFRS in Slovenia, Albania, Greece and Bosnia- Herzegovina (12, 147) and thus was recognized as a pathogen in the Balkan area. Nevertheless, serological studies had shown that antibodies to Murinae- borne virus(es) were detected in humans and rodents in the wide area stretching far outside the Balkans including the Netherlands, Belgium, Germany, former Czechoslovakia and the European part of Russia (55, 65, 67). To answer the question if DOBV, HTNV or other related hantaviruses might cause human infections outside of the Balkan area, A. agrarius and A. flavicollis (natural reservoirs of HTNV and DOBV, respectively) as well as other rodent species from different European countries were screened for the presence of hantavirus antigens. In 1996 hantavirus antigens were detected in lung samples of three A. agrarius trapped on the Estonian island Saaremaa (199). Analysis of partial S segment sequences recovered from two rodents suggested that they are most closely related to the sequence of DOBV. The virus from lung tissue of one A. agrarius was isolated in the cell culture and initially designated DOBV/Saaremaa/160v (SAAV).

The specific aims of the present project were:

(i) to characterize the new virus isolate antigenically and genetically and compare it with other hantaviruses carried by Apodemus mice, DOBV and HTNV;

(ii) to study the genetic diversity and geographic distribution of viruses carried by A. agrarius and A. flavicollis in Europe;

(iii) to analyze phylogenetic relationships of Murinae-borne hantaviruses in Europe. Materials and Methods 49

MATERIALS AND METHODS

Handling of rodent samples and hantavirus-infected cells

Hantaviruses are regarded as Biosafety Level 3 agents when used in cell culture. Therefore, all the work with infected materials that might generate aerosols was performed in a Class II laminar flow biosafety hood. When working with the materials apparently infected with DOBV (rodent or patient tissue sections) protective gear including respirators with appropriate filters was used. All the equipment that came in contact with the infectious material was treated with 10% sodium dodecyl sulfate and disposed as infectious waste.

Immunoblotting

Small pieces of lung tissue specimens (3-4 mm3) were homogenized by sonication in 400 µl of Laemmli sample buffer for 15 minutes, heat-denaturated for 5 min at 95˚C and 10 µl of the resulting mixture were loaded on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. Hantaviral antigens were separated by electrophoresis and immunoblotted with polyclonal rabbit anti- HTNV serum (149) at a dilution 1:200 for 2 h at the room temperature. Swine anti-rabbit horseradish peroxidase conjugate (DAKO) was used for detection of specific antibody binding at a dilution of 1:600 for 1 h at 37˚C. Blocking of the membranes was performed by incubation with phosphate-buffered saline containing 5% non-fat dry milk for 1 h at 37˚C or overnight at 4˚C. Antibodies were diluted in the same buffer containing 1% non-fat dry milk. The washing buffer used was 50 mM Tris-HCl, pH 7.4/ 5 mM EDTA/ 150 mM NaCl/ 0.05% Tween 20. Staining of the membranes was performed using o- phenylenediamine dihydrochloride as substrate.

MAb reactivity

A MAb panel was used in a standard IFA on hantavirus-infected, acetone- fixed Vero E6 cells prepared as described in (141), and consisted of MAbs against HTNV glycoproteins (4), HTNV N protein (271), PUUV N protein (141) and recombinant TULV N protein (146). MAbs were incubated for 30 min at 37 °C. Fluorescein isothiocyanate conjugated rabbit anti-mouse antibodies (DAKO) were used for the detection of specific antibody binding. Antibodies against HTNV N and G1 or G2 (ascitic fluid) were used at dilutions of 1:100 and 1: 200, respectively. For antibodies against PUUV and TULV (hybridoma supernatants), the dilutions were 1:20 and 1:50, respectively, except for the Materials and Methods 50

MAb 4E5, which was diluted 1:5 to demonstrate differences between DOBV and SAAV. For affinity-purified MAbs (1C12, 4C3), a concentration of at least 5 µg/ml was used. In addition, four MAbs (3B6, 8B6, 23G10-2 and 4E5), suspected to have different reactivity for DOBV and SAAV on the basis of preliminary screening, were titrated with dilutions ranging from 1:2 to 1:64.

RNA extraction

Frozen sections of lung, spleen, kidney or heart tissues cut out from euthanized rodents were ground manually in sterile mortars under liquid nitrogen and total RNA was purified by phenol-chloroform extraction followed by ethanol precipitation as described in (28) or with TriPure reagent (Molecular Research Center) as recommended by the manufacturer. To avoid possibily of RNA contamination, a separate set of mortars and pestles was used for each tissue sample. For viruses propagated in cell culture, the cell pellet or virion- containing supernatant was mixed with the required amount of the extracting agent and processed in the same way as tissue sections. Ethanol-precipitated RNA was pelleted, lyophilized and dissolved in 20-50 µl of double-distilled water (DDW).

Reverse transcription (RT) and polymerase chain reaction (PCR)

A sample of total RNA (4.5 µl) was denatured with 0.6 µl of 2.5 mM methylmercuric hydroxide at room temperature for 5 min. Then 1.0 µl of 90 mM β-mercaptoethanol was added prior to the RT reaction. The reaction mixture contained 4 µl of 5× first strand buffer (250 mM Tris-HCl, pH 8.3/ 375 mM KCl/ 15 mM MgCl2/ 50mM DTT), 0.8 µl of 2.5 mM dNTPs (Promega), 1.5 µl of RNAsin (40 units/µl, Promega), 1.5 µl of Moloney murine leukaemia virus reverse transcriptase (200 units/µl, USB Corporation), and 1 µl of each specific primer (100 pmol/µl). For all the RTs aiming at the transcription of complete S segment sequence, the universal primer mixture was used, containing 1 µl of random hexamers (20 pmol/µl) and 3 µl of primers TAGTAGTAGACT (complement to the vRNA of all genome segments) and TAGTAGTATACT (complement to the cRNA of all genome segments) used at equal concentrations (100 pmol/µl). The RT mix was adjusted with DDW to a final volume of 15 µl, combined with denatured RNA and incubated at 37°C for 1h. Five µl of RT mixture was used for PCR. For that, 5 µl of RT mixture was combined with 10 µl of 10× buffer (100 mM Tris-HCl, pH 8.3/ 500mM KCl/ 15 mM MgCl2/ 0.01% (w/v) gelatin), 10 µl of 25 mM MgCl2, 1 µl of each dNTP (2.5 mM), 0.5 µl of each primer (100 pmol/µl), 0.6 µl of Taq DNA polymerase (2.0 units/µl, AmpliTaq, Perkin-Elmer Cetus). The total volume was adjusted with Materials and Methods 51

DDW to 100 µl. The conditions of amplification varied depending on the annealing temperature and the primer sequence. Most often a touchdown PCR was used with the initial denaturation at 95°C for 2 min followed by the annealing for 1 min at X+10°C (where X is the lowest annealing temperature calculated according to the formula: (the total number of G and C bases in the primer sequence) × 3 + (the total number of A and T bases in the primer sequence) × 2) and the extension for 1-2 min at 72°C. The annealing temperature was gradually lowed from X+10°C to X during the first 5 PCR cycles with a 2°C decrease each cycle. After that, 29 more cycles were performed (95°C for 1 min, X°C for 1 min, 72°C for 1 min) with final extension at 72°C for 15 min. PCR products were separated by gel electrophoresis, visualized with ethidium bromide and purified from the gel using Gel Purification Kit (Qiagen).

Cloning

The PCR products were TA-ligated into the pGEM-T vector (Promega) overnight and 5 µl of the ligation mixture was used to transform JM 109 E. coli cells (Promega), according to the manufacturer´s recommendations (blue-white screening on the bacterial plates containing x-gal and IPTG). The plasmid DNA was extracted from white colonies and analyzed in the gel by restriction analysis. Plasmids that contained the insert of expected size were mixed with the vector-based commercial primers M13F and M13R (Promega) and analyzed by automatic sequencing with ABI PRISM M13F and M13R Dye Primer sequencing kit (Perkin-Elmer). Recovered sequences were screened against sequence databases and if they were identified as unique hantavirus sequences further sequencing was done with the specific primers chosen based on the new sequence with ABI PRISM Dye Terminator kit (Perkin-Elmer).

Extraction of mitochondrial (mt) DNA from rodent tissues

Total DNA from rodent tissue samples (lungs, kidneys or fragments of tail) was prepared using routine proteinase K treatment (17) followed by phenol- chloroform extraction and precipitation with ethanol. Alternatively, the TriPure reagent (Molecular Research Center, Inc.) was used to simultaneously extract total RNA and DNA from a tissue samples according to the manufacturer's recommendations.

Materials and Methods 52

Sequencing and phylogenetic analysis

Sequence data obtained from an automatic sequencer was analyzed using the SeqApp (version 1.9.1 for Macintosh) or BioEdit programs (version 5.0.9 for Windows). Sequencing mistakes were checked either by sequencing of another clone (if cDNA fragments were cloned prior to sequencing) or by repeated sequencing of the same PCR product (if PCR fragments were sequenced directly, without cloning). For the regions that could not be resolved by repeated sequencing, the complementary DNA strand was analyzed. Completed and corrected sequences were aligned with sequences of other hantaviruses retrieved from GenBank using PileUp (Wisconsin Package Version 10.2, Genetics Computer Group (GCG), Madison, WI). Nucleotide and aa distances between the newly obtained sequences and those of know hantaviruses were calculated using Distances program, without corrections (Wisconsin Package Version 10.2, Genetics Computer Group (GCG), Madison, WI). The PHYLIP program package (49) was used to perform phylogenetic analysis using Distance Matrix (DM) and Maximum Parsimony (MP) phylogenetic approaches. As a first step of both methods, SEQBOOT program was used to make 500 bootstrap replicates on sequence data. Distance matrices used for the DM analysis were calculated with DNADIST program using Kimura's two-parameter model of substitutions (for nt sequences) or PROTDIST program (for aa sequences) with Kimura's corrections. After that, Fitch-Margoliash (FITCH) or Neighbor-Joining (NEIGHBOR) fitting algorithms were applied to estimate tree topologies. Alternatively, the DNAPARS program was used after the bootstrapping step to create a set of phylogenetic trees with maximum parsimony. As the last step of both methods, consensus trees were calculated and bootstrap values were estimated based on the output files of FITCH or DNAPARS using the CONSENSE program. Reconstruction of phylogenetic trees according to the Maximum Likelihood model was done with the TREE-PUZZLE program (242). Ten thousand puzzling steps were applied using the Hasegawa-Kishino-Yano (HKY) model of substitution (69). The transition/transversion ratio and nucleotide frequencies were estimated from the data sets. Rate heterogeneity was applied using discrete gamma distribution with eight rate categories, and the shape parameter alpha was estimated from the data sets. Phylogenetic trees were visualized with the TreeView program distributed with the PHYLIP program package and edited with commersial graphics software packages. S segment sequences of different hantavirus strains obtained from sequence databases and used for phylogenetic analyses are listed in the Appendix I. Results and Discussion 53

RESULTS AND DISCUSSION

Isolation of the Saaremaa virus from lung samples of A. agrarius

The lung tissues of all three antigen-positive A. agrarius were homogenized and inoculated onto confluent monolayers of Vero E6 cells. Every three weeks cells were collected and passaged onto fresh uninfected Vero E6 cells. Monitoring of hantavirus antigen performed by IFA demonstrated that after 54 days post-infection most cells were positive. The cell culture supernatants were collected 59 days post-infection and used to infect fresh Vero E6 cultures, which all became infected in eight days. One of the three isolates (originating from A. agrarius no. 160) was passaged further and selected as the reference strain DOBV/Saaremaa/160v (or SAAV, for short) (I).

Antigenic characterization of the new isolate by FRNT and MAbs

To analyze serological relationships between SAAV, DOBV and HTNV by FRNT, antisera to DOBV and SAAV were raised in rabbits by intranasal immunization. Cross-FRNT comparison of the DOBV and SAAV demonstrated moderate cross-reactivity between them and very little or no cross reactivity of either virus with HTNV (I). The end-point titers to the homologous virus were 2- or 4-fold higher than to heterologous virus indicating significant antigenic differences between the two isolates. Analysis of four convalescent human sera from hantavirus-infected people (two from Estonia and two from Slovenia) further indicated antigenic differences: both sera from Slovenia reacted with DOBV at 4-fold higher titer than with SAAV. On the other hand, two sera from Estonia showed a more divergent reactivity: one of them reacted with a 4-fold higher titer to SAAV, while the other one reacted with 2-fold higher titer to DOBV (I). In order to compare the antigenic features of the two genotypes to each other and to three other hantaviruses (PUUV, HTNV and SEOV) slides containing acetone-fixed Vero E6 cells (141), infected with each of the five hantaviruses, were analyzed with a panel of 25 MAbs. The panel included MAbs raised against glycoproteins of HTNV (4) and PUUV (142) and N proteins of HTNV, PUUV, and TULV (141, 146, 271). The analysis revealed prominent similarity of the gross antigenic structures of SAAV and DOBV, as no differences in reactivity of 23 MAbs were found. A small difference in the reactivity was noted with two MAbs: PUUV-N specific MAb 4E5 (3-fold higher reactivity with DOBV) and HTNV-G1 specific MAb 8B6 (4-fold higher reactivity with SAAV). The cross-reactivity between SAAV and the other three Results and Discussion 54 hantaviruses was lower: four MAbs reacted differently with SAAV and SEOV, five MAbs showed different reactivity to SAAV and HTNV, and nine MAbs reacted differently with SAAV and PUUV (I). Taken together, the data suggested that the antigenic structure of SAAV is closer to that of DOBV than to HTNV or other hantaviruses. Cross-FRNT comparison of DOBV and SAAV demonstrated 2- to 4-fold higher end-point titers to the homologous virus compared with the heterologous virus, suggesting that SAAV and DOBV have a similar antigenic structure, although they are clearly distinguishable by FRNT (I).

Characterization of nt and deduced aa sequences of SAAV

The S segment sequence of SAAV is 1671 nucleotides long and contains a single ORF of 1287 nt encoding a nucleocapsid protein of 429 aa. The N-ORF is flanked by 5’ and 3’ NCRs of 35 and 349 nt in length, respectively. Comparison of the S segment/N protein sequences of SAAV with those of other hantaviruses showed that it is most similar to hantaviruses carried by Murinae rodent family: DOBV, HTNV and SEOV (less than 28%/21% of nt/aa divergence in the sequences) (I, VI). The level of divergence between SAAV and hantaviruses carried by Arvicolinae and Sigmodontinae rodent families was substantially higher: up to 38%/39% and up to 36%/37%, respectively. The primary structure of N proteins of all Murinae-borne hantaviruses is similar: all of them are 429 aa long (four aa shorter than N proteins of all Arvicolinae- borne viruses and one aa longer than that of all Sigmodontinae-borne hantaviruses). Alignment of the N protein sequences of all known hantaviruses demonstrated that N proteins of Murinae-borne hantaviruses do not contain the four aa present at positions 260-263 of all Arvicolinae- and Sigmodontinae- borne hantaviruses. At the same time, another stretch of five aa located at positions 248-252 of all Arvicolinae- and Murinae-borne hantaviruses was not found in the sequences of Sigmodontinae-borne viruses. No sequencing homology could be identified between Sigmodontinae- and Arvicolinae-borne hantaviruses in the first region or between Murinae- and Arvicolinae-borne viruses in the second region. The M segment sequence of SAAV is 3645 nt long and contains a coding region of 3405 nt encoding the GPS of 1135 aa. The 5’ and 3’ NCRs (cRNA- sense) are 40 and 190 nt, respectively. The highest nt and aa homology was observed between SAAV and other Murinae-borne viruses (up to 81%/94%), while homology to the other two hantavirus families was substantially lower: 59%/54% with the Sigmodontinae-borne SNV and 59%/55% with the Arvicolinae-borne TULV (I, VI). Results and Discussion 55

The primary structure of hantaviral G1-G2 proteins shows more variation than the N protein structure. The N-terminus of G1 contains a signal peptide rich in non-polar, non-charged aa residues (Leu, Ile, Met, Trp, Phe and Ala) and is highly variable in length between different hantaviruses. The N-terminus of the mature G1 protein of HTNV has been shown to be Thr (position 18) (220), but the first site conserved in all hantaviral sequences (Arg/Lys) is located at position 20 of the deduced GPS sequence of HTNV. The variable region is slightly shorter in Murinae-borne hantaviruses (17 aa in SEOV and THAIV, 19 aa in HTNV, DOBV, and SAAV), followed by Sigmodontinae-borne viruses (all have 20 aa) and Arvicolinae-borne viruses (19 aa in TULV, 21 aa in TOPV, KHAV and PHV, and 24 aa in PUUV). The other major differences in the primary structure of G1 and G2 include: (i) three aa missing in the G1 sequences (positions 94-96 of total alignment) of all Murinae-borne viruses (Leu/Thr-Ala-Glu/Asp/Tyr in Arvicolinae-borne hantaviruses and Thr-Thr- Asp in Sigmodontinae-borne hantaviruses); (ii) two/three aa residues present in the G1 sequences of all Arvicolinae-borne hantaviruses and missing from the sequences of Murinae- and Sigmodontinae-borne (positions 229-231 of total alignment, no apparent homology between the sequences of different hantaviruses); (iii) Ala/Ser residue present at position 244 of G1 protein in all Murinae-borne hantaviruses and missing from the sequences of other hantaviruses; (iv) Pro in G2 protein of all Murinae-borne hantaviruses at position 893 is missing from the sequences of other hantaviruses. The C- terminal region of the G2 protein (aa 1144-1154 of total alignment) is rich in positively charged aa residues (Arg, Lys, and His) and varies in length between different hantaviruses species. Murinae-borne hantaviruses have the shortest C-terminal domain (all have 6 aa residues), followed by that of Sigmodontinae- borne hantaviruses (6-8 aa residues) and Arvicolinae-borne hantaviruses (9-11 aa residues). Important features recognized in the primary structure of hantavirus G1-G2 proteins include conserved cysteine residues and glycosylation sites (222). Cys residues may be involved in the folding of G1-G2 heterodimer and therefore residues conserved between all hantaviruses or hantaviruses carried by a particular rodent sub-family deserve a special attention. The G1 protein of SAAV contains 33 Cys residues. Twenty-seven of them are conserved between all hantaviral sequences, four aa are conserved between the vast majority of hantaviruses and two residues are shared by all Murinae-borne hantaviruses. All 27 Cys residues present in the G2 protein of SAAV are conserved in all hantavirus sequences known to date. The predicted G1 protein of SAAV has five putative sites for N-linked glycosylation in the ectodomain. Two of them are shared by all hantaviruses (aa 347-349 and 399-401); one is present in all, except KBRV (aa 134-136); one is Results and Discussion 56 shared by all hantaviruses carried by Murinae rodents (aa 235-237); and one site is present only in DOBV and SAAV (aa 252-254) (12, VI). The L segment sequence of SAAV is 6532 nucleotides long and contains a single long ORF (nt 38-6493), encoding the L protein of 2151 aa. The nt and aa sequences of SAAV L segment are very similar to the sequences of other Murinae-borne viruses (up to 86%/97%), and less similar to the hantaviruses carried by two other rodent sub-families: up to 62%/67% with the Sigmodontinae-borne hantaviruses and up to 66%/68% with the Arvicolinae- borne hantaviruses (VI). Important regions recognized in the primary structure of hantaviral L proteins included six conserved aa motifs which were found in all RNA- dependent RNA-polymerases and presumably responsible for nucleoside triphosphate binding and catalysis (motifs A, C and D) and for the positioning of the template and primer relative to the active site (motifs B and E and premotif A) (174). Other conserved regions were found in the polymerases of viruses with segmented negative-stranded RNA genome (Arenaviridae, Bunyaviridae and Orthomyxoviridae), namely: (i) two conserved amino acid residues, Glu and Lys, which might be involved in the promoter recognition; and (ii) motif E with the conserved tetrapeptide Glu-(Phe/Tyr)-X-Ser that could play a role in the initiation of transcription, also present in the L protein of SAAV. Other important features of hantavirus L proteins include two N- terminal conserved regions found previously in polymerases of bunyaviruses and arenaviruses and C-terminal region containing mostly acidic aa described in polymerases of Sin Nombre virus, Puumala virus, Tula virus and tomato spotted wilt virus (genus Tospovirus) (27, 118). All these domains were conserved in the sequence of SAAV L protein.

Genetic diversity and geographic distribution of hantaviruses carried by A. flavicollis and A. agrarius in Europe

A. agrarius-derived SAAV is currently represented by strains originating from 6 European countries: Estonia, Russia, Slovenia, Slovakia, Denmark, and Hungary (I, II, III, 217, 232, VII) (Fig. 7). While the complete sequences of all three genes are available only for the prototype SAAV strain from Estonia, complete sequences of the S segment and partial M segment sequences were recovered for strains from Estonia, Russia, Slovakia and Denmark. On the phylogenetic tree based on the partial S segment sequences (nt 720-1040), strains from different countries form 6 distinct lineages (Fig. 8A)1. The

1The Slovenian strain could not be included in the phylogenetic analysis, because partial sequences available for the strains from Slovenia and Hungary have a short overlap. However, separate phylogenetic analysis that included this strain (III) showed that it forms independent lineage on the phylogenetic tree. Results and Discussion 57

Fig. 7. Geographic distribution of DOBV and SAAV in Europe. Geographic region shaded in light-gray color indicate the area inhabited only by A. flavicollis. The region shaded in dark-gray color mark the area co-inhabited by A. flavicollis and A. agrarius. Black and white mouse figures indicate sites where hantavirus sequences were recovered from A. flavicollis and A. agrarius, respectively. Black and white circles mark countries with confirmed or suspected cases of HFRS, respectively, caused by DOBV or SAAV.

Hungarian lineage is represented by two strains characterized only by partial sequences of the S segment; the Slovenian lineage is represented by a single strain for which partial sequences of the S and M segments are available. Three Results and Discussion 58

A. SAAV TOPV 63 SAAV/Loll-1403 DENMARK

100 SAAV/Saaremaa-160v ESTONIA SAAV/Saaremaa-90

99 SAAV/Kurkino 53 RUSSIA SAAV/Kurkino 44 91 100 SAAV/Tazar-2 SAAV/Tazar-8 HUNGARY 85 SAAV/esl255Aa 81 SAAV/esl862Aa 100 SAAV/esl856Aa SLOVAKIA 57 SAAV/esl175Aa

SAAV/esl374Aa

B. TOPV DOBV 100 P-s1223/Krasnodar-2000* RUSSIA As-1/Goryachiy Klyuch-2000 BOSNIA-HERZEGOVINA 71 DOBV/Tuzla-43 94 DOBV-prototype

74 DOBV-Dobrava/Af-1/90 SLOVENIA DOBV-Kocevje/Af-3/93 DOBV-Prekmurje/Af-10/96 DOBV-esl400Af SLOVAKIA 60 DOBV-AP 100 56 DOB-TD* 58 DOBV/Greece13 DOBV-PR* DOBV-PA* 99 DOBV-TI* GREECE DOBV-NF*

70 DOBV-CG* 94 DOBV-SZ* DOBV-EA* 87 DOBV-HA* DOBV-SI* DOBV-GA*

Fig. 8. Phylogenetic trees based on the partial S segment sequences of SAAV (nt 720- 1040) (A) and DOBV (nt 393-753) (B). Both trees were estimated based on the pairwise distance matrix calculated using the Kimura two-parameter evolutionary model. Tree topology was estimated using the Neighbor-Joining method (500 bootstrap replicates). Bootstrap support values of greater than 50% are shown at the appropriate branch points. Genetic lineages that received bootstrap support >70% are shown in frames. Sequences marked by asterisks were recovered from HFRS patients. Results and Discussion 60 strains of the Estonian lineage were recovered from Haeska and Loode Tammik localities in Saaremaa island (I). The prototype SAAV strain originated from Haeska in 1996 along with two other strains, Saaremaa 111 and Saaremaa 171. Partial S and M segment sequences recovered for the two latter strains were identical to the sequences of the prototype strain, and, therefore they were not characterized further. In 1997, during another trapping expedition, strains Saaremaa/90/97 (SAAV-90) and Saaremaa/91/97 (SAAV-91) were recovered from Loode Tammik locality. For both strains complete sequences of the S segment and partial M segment sequences (nt 2008-2584 for SAAV-90 and nt 2898-3630 for both strains) were recovered. The S segment sequences of the two strains were distinguished by a single nt difference. The partial sequences of the M segment recovered from both strains were identical. Strains originated from Loode Tammik and Haeska showed 1.7% divergence in the coding sequences of the S segment and 1.9% in the M segment sequences. Both strains that represent the Russian genetic lineage originated from Tula region (approximately 300-400 km south of Moscow), where a large outbreak of HFRS occurred in 1991-1992 (148). In 1998 Apodemus agrarius were trapped near Kurkino village and seven of them were found positive for hantaviral antigen in the ELISA test (II). Three specimens with the highest antigen titer were analyzed by RT-PCR and complete nucleotide sequences of the S segment as well as partial sequences of the M segment (nt 1704-2584 and nt 2913-3618) were recovered from two of them. Corresponding wt strains, that were not isolated in cell culture, were designated as Kurkino/Aa44/1998 and Kurkino /Aa53/1998 (or Kurkino 44 and Kurkino 53, for short). The sequences of the two strains appeared to be very similar, with less than 1% of nt divergence in both the coding region of the S segment and sequenced regions of the M segment. Genetic lineages from Estonia and Russia showed up to 12.8% of nt divergence in the coding sequences of the S segment and up to 14.6% in the M segment sequences (III, Table 2). The genetic lineage from Slovakia (232) includes five strains recovered from A. agrarius trapped in the eastern part of Slovakia (Kosice region). Complete S segment sequences were recovered from two rodents (East Slovakia-856-Aa and East Slovakia-862-Aa), while the other three strains (East Slovakia-374-Aa, East Slovakia-175-Aa, and East Slovakia-255-Aa) were characterized only by partial S segments sequences (nt 381-935). Similarly to the low intra-lineage divergence observed in the lineages from Estonia and Russia, the nt divergence between two complete S sequences of Slovakian strains was low, only 0.3%. The divergence between the lineage from Slovakia and lineages from Estonia and Russia was up to 13.1% and 10.2% (Table 2), respectively, thus suggesting

Results and Discussion 60

g n . i 2 3 d 3 2 o 2 4 0 7 8 2 8 2 2 3 6 1 c ...... 0 . . 140 - - 2. 3 2 2 1 0 0 2 l 0. 7 e 11 1 1 l 1 1 10 10 1 1 1 1 t 3 o e t L l n p n m 90 o 6 1 7 7 3 8 6 4 7 - i o . . 7 0 . . . c 2 2 4. 4. 2. 2 2. 2. A 1 0. 1 1 1 1 1 1 12 1 1 eg A e r . S e th d h l t n o i b or 0V e f c 0 3 6 7 3 8 1 4 0 in . . . . 0 . . . . 16 d n - 2. 4 4 2 2 3 0. e e n 13 1 1 1 1 12 13 1 1 A n g A w r S o e mi h v i er s us i d 3 e et V r 5 t r - 2 9 3 6 7 d a 6 . . . 5 0 7 . a n k . A . . r 2. 3 3 2 2 e r 9 0 0. f 1 1 1 1 1 u A er o us ag K S e w . ir g v e A 4 c ta 4 h n - 1 5 1 5 9 29 0 . 0 . c n e k 0. 2 4. 3. 2. 0 3. e r a g 0. 1 1 1 1 1 1 s e r er f Ku e v o i p s d e f t in 862 4 5 3 8 3 o 4 . 01 - a . . a 3 k c s 0 0 r i 14. 1 14. 14. 14. v e t l d s u S l t n i a n v s e r r e e e f b 856 4 4 3 7 2 Th f 0 . - . i 4. 4. 4. 4. k 0 m e. 1 13 1 1 1 d v l l u b n S a N e l i . e a s w v n t i a e a b ot tr s e n c s i V v 5 5 0 0 7 en 0 o . A . . l 0 s g 0 4 3 11. 11. S i r 0 A l e 2 S l - v r o V i 3 d c 1 7 7 d n - B . 9 0 . . e a f 1. 1 oda 1 0 r 1 1 O o n avi G V s l f a D B es r P . u O 0 0 K A l . 0 - A / D a 2. e 0. 1 12 3 r v n 2 G t e 2 s e 1 e s - w h t P g e i n b h i 3* a e us e r 2 0 c t c . . h i s 122 0 0 n s T V - e e . at P t h g B t r v l f e en o y v i 1 s m e DO - 0 d . . c s g 0V 0 n e c A A e 16 90 s ti P 4 3 u 3 4 5 e S A 1 a aa- aa- a a i - 03 n o o eq e e e n 23 m m s n n c c n h 14 e i i e e - e e t 1 e Ge l v k k 12 56A 62A - e e l ai s 8 8 o s r r - r et o l aar aar ur ur of l t 2. sl sl P S A G G S S L s e e K K / / / / / - / / / - - - e. s l V V V V V V V V V V V V on u i B B B B B omp b A A A A A A A r g i a O O O O C A A A A A e D V DO D D D S S SA SA S S S T r *

Results and Discussion 61 that lineages from Russia and Slovakia are more closely related to each other than to the Estonian lineage. Two sequences that form the Hungarian lineage, Tazar-2 and Tazar-8, were recovered from eastern Hungary (Tazar NATO Airbase) between 1995 and 1996 (217). Both strains were characterized by sequencing of a 321 nt-long fragment of the S segment (nt 720-1040), and had a nt divergence of 1%. This lineage demonstrates up to 13.8% nt divergence from the Estonian lineage, 7.5% from the Russian lineage and 7.2% from the Slovakian lineage, and thus appears to be substantially closer to the lineages from Russia and Slovakia than to the Estonian one. The single strain that represents the genetic lineage from Denmark, (Saaremaa/Lolland/Aa1403/2000, or Loll-1403 for short), was recovered from A. agrarius trapped in Lolland island in year 2000 (VII). This strain was characterized by the complete sequence of the S segment and partial sequence of the M segment (nt 1711-2239). Genetic divergence between this strain and other lineages of SAAV determined from the coding region of the S segment was up to 10.2% with the Estonian lineage, up to 10.8% with the Russian lineage, up to 11.2% with the lineage from Slovakia and up to 9.7% with the Hungarian lineage (Table 2). Divergence determined based on the M segment sequences was up to 13.8% with the Estonian lineage, and 14.0% with the Russian lineage. The last genetic lineage of the A. agrarius-derived European hantavirus is represented by a single strain from Eastern Slovenia (Prekmurje village), recovered close to the Slovenian-Hungarian border. Only partial sequences of the S (nt 376-761) and M (nt 1314-1590) segments of this strain were recovered (III). The nt sequence of the S segment shows up to 14.3% divergence with the sequences from Estonia, 9.4% divergence with the sequences from Russia, and up to 7.6% divergence with the Slovakian strains. The M segment sequence showed 18.1% nt difference to the prototype strain from Estonia and a difference of 11.1% to the strains from Russia (III). The values of genetic divergence calculated between different genetic lineages demonstrated that strains from Russia, Slovakia, Slovenia and Hungary appear to form a group of more closely related strains with the inter- strain divergence ranging from 7.2% to 10.2%. The Estonian lineage is more distantly related to this group with the least divergence, 12.8%, with the lineage from Russia, and the highest divergence, 18.1%, with the Slovenian strain. The origin of the Danish strain is less clear as the genetic distances between this strain and representatives of other lineages are nearly equal. The strain from Denmark clusters with the Estonian lineage on the phylogenetic trees although Results and Discussion 62 bootstrap support for this clustering is moderate and varies depending on the set of analyzed sequences (Fig. 3, Fig. 8A, VII). DOBV, carried by A. flavicollis, is represented by at least 25 strains: nine strains originating from Slovenia (III); one strain from Bosnia-Herzegovina (147); 16 strains from Greece (183; IV); one strain from Albania (183); and one strain from Slovakia (232) (Fig 8). Nearly half of the known sequences (13) were recovered directly from A. flavicollis. Sequences of other 12 strains were obtained from clinical samples of HFRS patients and thus the precise origin of the infecting hantavirus is unknown. However, close genetic relationships between patient-derived sequences and those recovered from A. flavicollis suggest that this rodent species is the most likely source of infection. From all the Slovenian strains only the prototype strain, Dobrava, has been characterized by complete sequencing of the S and M segments (12) and a partial sequencing of the L segment (I). For the other eight strains only partial sequences of the M segment (nt 1314-1590), and partial S segment sequences (nt 377-761) (for three of them) are available (III). Slovenian strains were recovered from 5 geographic localities all over the country: Dobrava, Kocevie, Tenetise, Gorjanci and Prekmurje. Genetic divergence observed between the strains from one locality estimated from the S and M segment sequences was up to 1% and 0.4%, respectively. The nt divergence between strains from the different localities ranged from 2.5% to 4.9% for the S segment sequences and from 0.4% to 5.8% for the M sequences (III). Phylogenetic analysis based on the M segment sequences showed at least three genetic lineages: (i) formed by strains from Southern Slovenia (Dobrava, Gorjanci and Kocevje); (ii) formed by strains originated from Central Slovenia (Tenetise); and (iii) formed by a single strain from Eastern Slovenia (Prekmurje). Such a grouping indicated geographic clustering of DOBV genetic variants observed previously for other hantaviral species. Phylogenetic analysis based on the S segment sequences placed all sequences from Slovenia (two from Dobrava and one from Tenetise) within a single lineage (III, Fig 8B). Partial S segment sequence originating from neighboring Bosnia-Herzegovina was grouped together with the Slovenian strains forming a distinctive phylogenetic lineage with them (III, Fig 8B). Sequences of 16 DOBV strains originating from Greece were recovered during three different studies. First, partial M segment sequence was recovered from the serum sample of an Albanian patient with HFRS (3). Two years later, partial S segment sequences of 11 strains (and also partial M sequences for four of them) were recovered from sera of 30 Greek HFRS patients admitted to the hospitals over the last 17 years from all over the country (183). In the same study partial sequences of the M segment were recovered from a patient with Results and Discussion 63

HFRS and A. flavicollis trapped in the patient´s home area. Finally, complete sequences of the S segment and partial M segment sequences were recovered for two strains (Ano-Poroia/Af9/1999 and Ano-Poroia/Af13/1999) originating from A. flavicollis trapped in northeastern Greece (III, IV). One of the strains was isolated in Vero E6 cell culture (Ano-Poroia/Af9V/1999, or DOBV-AP for short) and subjected to complete genomic sequencing, which made it the only DOBV strain characterized by complete sequence analysis (VI). On the phylogenetic tree based on the partial S segment sequences, all strains from Greece were placed in three main groups: Northeastern, Northwestern and North Central (183; III). Two strains recovered later from A. flavicollis trapped in Northeastern Greece (Ano-Poroia) were also placed within the Northeastern lineage (III), suggesting a possible link between infected A. flavicollis and HFRS cases in that area (IV). A phylogenetic tree based on the partial M segment sequences demonstrated reliable clustering of two A. flavicollis-derived sequences with an A. flavicollis- and HFRS patient-derived sequences recovered in the second study (183) (IV). The nt diversity within the Northeastern and Northwestern lineages was similar (up to 1.4%). Northwestern and North Central lineages were more closely related to each other (up to 2.9% nt diversity), while Northeastern lineage was more distantly related to them with up to 4.7% and up to 4.5% nt diversity, respectively. Nt diversity between Greek and Slovenian strains in the S segment sequences ranged from 3.2% to 6.2%. The level of diversity observed in partial M segment sequences was slightly higher: up to 9.9% between different Greek strains and up to 11.2% with the sequences from Slovenia and Bosnia-Herzegovina. A single strain originated from Slovakia demonstrated up to 4.7% of divergence with the strains from Slovenia and Bosnia-Herzegovina, and up to 5.2% with sequences from Greece. Most recently S segment sequences of new DOBV variant were recovered from wood mouse (A. sylvaticus) and HFRS patient in Russia (248). The low level of nt divergence between the sequences (0.2%) suggested A. sylvaticus as a likely source of the infection. Both sequences showed similar divergence with other DOBV lineages: 9.3-12% with the strains from Greece, 9.3-11.5% with sequences from Slovenia and 9.7% with the Slovakian sequence (Table 2). Thus, DOBV lineage from A. sylvaticus appears to be the most distantly related to any A. flavicollis-derived lineage, although lineages from A. flavicollis and A. sylvaticus are more closely related to each other than to the sequences of SAAV (Table 2). Phylogenetic analysis including most DOBV strains based on partial sequences of the S segments (nt 393-753) (Fig. 8B) identifies three well- supported genetic lineages: (i) formed by sequences from Slovenia and Bosnia- Results and Discussion 64

Herzegovina; (ii) formed by sequences from Northwestern and North Central Greece; and (iii) A. sylvaticus-derived sequences from Russia. Phylogenetic relationships between other sequences originated from Northeastern Greece, Slovakia and Slovenia were not resolved by this analysis. This can be explained by the short region used for the analysis and low genetic divergence between analyzed DOBV strains. Nevertheless, analysis based on the complete coding sequences of the S segments demonstrates reliable geographic clustering of DOBV sequences (Fig. 3). Altogether the comparison of different DOBV and SAAV strains showed that genetic divergence observed between different genetic lineages of both viruses was comparable. The highest divergence observed between SAAV lineages was 14.3% and 18.1% for the S and M segment sequences, respectively, and for DOBV lineages 12.2% and 11.2%, respectively. These values were also comparable with the inter-lineage divergence of other Murinae-borne hantaviruses (HTNV and SEOV) or Sigmodontinae-borne hantaviruses (SNV), although they were lower than those of PUUV (see Introduction). Generally, as is the case also for other hantaviruses, M segment sequences of SAAV and DOBV strains were slightly more divergent than the S segment sequences. The slightly higher values of inter-lineage divergence observed within SAAV in comparison to DOBV may reflect the fact that SAAV strains originated from far more distant geographic locations than DOBV strains, rather than indicate a genuine difference in the variability. More extensive studies should be conducted to clarify this point.

Evidence that hantaviruses carried by A. agrarius and A. flavicollis represent distinct genotypes

Analysis of phylogenetic relationships (Fig. 3, III, VII) and genetic diversity (Table 2) of the strains derived from A. agrarius and A. flavicollis demonstrated that virus strains carried by the different host species are distinct from each other even when they are found in the same geographic locality. For example, in Slovenia sequences of A. agrarius–derived strain Prekmurje/Aa-9/96 and A. flavicollis-derived strain Prekmurje/Af-10/96 were recovered from rodents trapped close to Prekmurje village (III). However, comparison of partial S and M segment sequences of these two strains with each other and with the sequences of other A. flavicollis- and A. agrarius-derived strains demonstrated that Prekmurje/Aa-9/96 is more closely related to A. agrarius-derived strains from Slovakia and Russia than to Prekmurje/Af-10/96 or any other A. flavicollis-derived strain from Balkans. Since Slovakian and Russian strains were found approximately 400 km and 2100 km away from Slovenia, i.e. in distant Results and Discussion 65 geographic regions, this example demonstrated host-specific rather than geographic clustering of strains originated from A. flavicollis and A. agrarius (III). A second example illustrating the same rule was provided by the phylogenetic analysis of strains from Slovakia (232). The sequence of the single A. flavicollis-derived strain from this country was more closely related to the sequences of A. flavicollis-derived strains from Slovenia and Greece than to the sequences recovered from A. agrarius trapped in Slovakia or other countries. Host-dependent clustering of hantaviral sequences recovered from A. agrarius and A. flavicollis that co-exist in the same geographic area suggests that, currently, transmission of hantaviruses between two rodent species does not occur. All phylogenetic analyses that have been performed so far suggested host dependent clustering of strains derived from A. agrarius and A. flavicollis2. Moreover, FRNT test performed on 37 human serum samples (24 from Estonia and 13 from Balkans) demonstrated that most of the Estonian sera (19), including all sera from Saaremaa island (12) reacted with higher end-point titers to the local SAAV strain (22). The majority of the sera (15) had at least four-fold higher titers to SAAV than to DOBV. In contrast, 10 of 13 human sera from the Balkans had higher titers to the local DOBV strain than to SAAV (9 of 10 with the four-fold or higher titers). Only one serum out of 13 had a higher titer to SAAV, than to DOBV (two-fold). Thus, viruses carried by A. flavicollis and A. agrarius appear to represent distinct hantavirus serotypes. Additional evidence suggesting crucial differences between the viruses carried by A. flavicollis and A. agrarius in Europe has come from the epidemiological data on human hantaviral infections. In the areas where only A. agrarius-carried virus was found, hantavirus infections seem to be milder than in the Balkans, where A. flavicollis-carried virus is predominant and several outbreaks with 9-12% case fatality were registered. In Estonia, where on average 3% of total population have antibodies to a Murinae-borne hantavirus (and only the virus carried by A. agrarius is present) no severe HFRS cases have been encountered (63). Another example is the outbreak of HFRS that took place in Russia in 1991-1992 where no severe cases of HFRS were registered among approximately 130 total cases (148). Altogether these data suggest that the hantaviruses carried by A. agrarius and A. flavicollis represent two different virus genotypes with a possible difference in the pathogenicity. Accordingly, it has been suggested that SAAV carried by A. agrarius in Europe should be regarded as a distinct hantavirus

2 A. agrarius-derived genetic lineage originating from Estonia (Saaremaa) is almost equally divergent from other A. agrarius- and A. flavicollis-derived lineages and may be placed with A. flavicollis-derived strains by some phylogenetic methods, especially when partial sequences are used for the analysis (232). Nevertheless, analysis of a larger set of complete S segment sequences (Fig. 3) and phylogenetic analysis of a large portion of the M segment (not shown) demonstrate host-dependent clustering of the variants from A. agrarius and A. flavicollis. Results and Discussion 66 species (with a prototype strain Saaremaa/160v). The corresponding taxonomic proposal is now under consideration by the International Committee for Virus Taxonomy (A. Plyusnin, pers. comm.). Although phylogenetic analysis of more than 20 wild-type strains does not show cross-species transmission of DOBV and SAAV, the possibility of such transmission cannot be totally ruled out. In a recent study on the host specificity of European hantaviruses, SAAV was shown to infect colonized A. agrarius and A. flavicollis with a similar efficiency after subcutaneous (s.c.) inoculation (115). However, these results should be considered cautiously because s.c. infection does not mimic natural way of viral transmission. Remarkably, the same study has shown that both DOBV and SAAV could also infect laboratory mice (albeit with a lower efficiency than Apodemus mice). Laboratory mice originate from Mus musculus species, which also belong to Murinae rodent family, but has never been shown to carry DOBV or SAAV in nature.

Host switching

The fact that SAAV is genetically closer to DOBV than to HTNV is unusual since both SAAV and HTNV are carried by A. agrarius and it is a widely accepted view that hantaviruses are firmly associated with their rodent hosts (82, 197, 178, 200). The likely explanation for the observed phenomenon is host switching event, i.e. transmission of a hantavirus to a new host, like those previously suggested for MGLV-NYV and TOPV-KHAV (169, 256). In the paper V we described phylogenetic analysis of rodent species and compared phylogenetic trees inferred for rodents and hantaviruses in order to test the hypothesis about host switching in the evolution of SAAV, DOBV, and HTNV. The analysis was based on the fragment of mitochondrial DNA (mtDNA) that surrounds the origin of replication (D-loop region), which was previously successfully used for resolving phylogenetic relationships of P. maniculatus in North America (169). The D-loop sequences were recovered from fifteen rodent tissue samples, including eight A. flavicollis (one from Estonia, two from Poland, one from Slovenia, two from Greece, and two from Italy), four A. agrarius (two from Estonia, one from Poland, and one from Slovenia), and three A. sylvaticus (all from Belgium). The newly recovered sequences were combined with sequences already deposited in the databases and subjected to phylogenetic analysis that revealed a logical grouping of rodents from Murinae, Arvicolinae, and Sigmodontinae subfamilies. All types of phylogenetic methods applied for the reconstruction (DM, MP, and ML) showed the same clustering of the rodent species. Sequences belonging to different rodent sub-families: Sigmodontinae, Arvicolinae, and Murinae, Results and Discussion 67 formed three separate clusters. All Murinae-originated sequences were monophyletic, although the grouping of Rattus sequence with mouse species received only a moderate bootstrap support. Three Apodemus species: A. agrarius, A. flavicollis, and A. sylvaticus, formed a well-supported group further divided into two subgroups, one of which was formed by sequences of A. agrarius and the other by sequences of A. sylvaticus and A. flavicollis. Such a grouping confirmed the morphological division of the genus Apodemus into subgenera Sylvaemus (includes A. flavicollis and A. sylvaticus) and bona fide Apodemus (includes A. agrarius) (156). The A. agrarius subgroup, in turn, included two well-supported clusters comprised of sequences from Europe and the Far East, respectively. Thus, the analysis of the D-loop region of the mtDNA demonstrated the monophyletic origin of all A. agrarius-derived sequences, which were undoubtedly separated from A. flavicollis-derived sequences. Direct alignment of the rodent tree with the tree based on complete coding sequences of the hantavirus S segment revealed discrepant phylogenies of the trio HTNV-DOBV-SAAV and their respective rodent hosts, the eastern subspecies of A. agrarius, A. flavicollis, and the western subspecies of A. agrarius, thus providing phylogenetic evidence for host switching in the evolution of Dobrava and Saaremaa hantaviruses (V). The likely model of the host switching suggests that the common ancestor of DOBV and SAAV, carried by A. flavicollis, was transmitted to the western subspecies of A. agrarius and diversified into a distinct genotype, SAAV. The alternative scenario includes transmission in the opposite direction (i.e. of an ancestral SAAV from A. agrarius to A. flavicollis). Although this scenario may look more parsimonius, it implies that an ancestral hantavirus associated with A. agrarius had diversified into variants as distinct as HTNV and SAAV (divergence up to 29% at the nt level, and up to 23% at the aa level, not to mention that they represent clearly distinct serotypes and show different pathogenicity for humans). So far, this level of diversity has been seen only for hantaviruses carried by distinct rodent species. The host switching seems to be a historically recent event because DOBV and SAAV demonstrate relatively low level of diversity between their S/N sequences, up to 12.9/3.7% only (I, III, 232). This can explain the high cross- reactivity observed in IFA and ELISA directed against the N protein (21, 147). The substantially higher diversity (up to 18/5.9%) in the M/G1G2 sequences of DOBV and SAAV make them behave as distinct serotypes in cross- neutralization tests (I, VI, 22). The different levels of diversity observed between the G1G2 sequences than N sequences probably reflect a slower evolution of hantaviral N protein due to more stringent functional constraints and/or different pressures from host immune response. Results and Discussion 68

In order to estimate the approximate time point of the host switching, viral and rodent sequences were tested for the clock-like mode of evolution. The model of clock-like evolution (which assumes an equal rate of accumulation of mutations in all phylogenetic lineages) would allow estimation of time for the hypothetical DOBV/SAAV host switching, which should mark the beginning of their evolutionary split. It was shown that S segment sequences of Murinae- borne viruses evolve in a clock-like manner and, therefore, can be used for the calculations. M segment sequences did not fit this model, and became clock-like only when Г-distribution of substitution rates was chosen and the third position of every codon excluded. Time estimates were done based on the branch lengths of clock-like ML trees. According for the paleontological data available for rodent species, predecessors of mice and rats had split approximately 10-12.2 millions years ago (MYA) (31 and references therein). Since hantaviruses are thought to have co-evolved with their rodent hosts for a much longer time, SEOV carried by Rattus, and the common ancestor of all hantaviruses associated with Apodemus mice should have been separated at that time. Based on this time-point, the hypothetical split (host switching) between DOBV and SAAV was estimated from the branch lengths of the S segment- based ML tree at 2.7-3.4 MYA. The time of the evolutionary split between HTNV and DOBV (which also should indicate the split between A. agrarius and A. flavicollis) was estimated from the same tree as 8.1-10.8 MYA. When the same evolutionary time-points were estimated from the ML tree based on the M segment sequences, corresponding values appeared to be slightly lower: 2.1-2.6 MYA, for the hypothetical split between DOBV and SAAV, and 7.9-9.6 MYA for the split between DOBV and HTNV. The differences in the estimations based on ML trees inferred from different viral segments can be explained by the fact that M segment sequences passed the test for clock-like evolution only when the third positions of codons were excluded from the analysis, and therefore some phylogenetically important information was not taken into consideration.

Conclusions and Future Prospects 69

CONCLUSIONS AND FUTURE PROSPECTS

This thesis summarizes the current knowledge concerning genetics and geographic distribution of two European hantaviruses: SAAV and DOBV. Both are pathogenic for humans and, therefore, the information about their genetic variability and geographic distribution is vital for the understanding of HFRS epidemiology, diagnostics and vaccine development.

Genetic comparison and phylogenetic analysis of DOBV and SAAV demonstrated that they are genetically related, but ecologically, serologically and phylogenetically distinct, suggesting that they represent two distinct hantavirus genotypes with a possible difference in pathogenicity.

SAAV was shown to be exclusively associated with the western form of striped field mouse, A. agrarius, in a wide geographic area: from Denmark in the west to the central Russia in the east. DOBV has been found only in Southern Europe (Balkan area). Such limited geographic distribution of DOBV is interesting because its natural host, yellow-necked mouse (A. flavicollis), inhabits a much wider geographic area including most of Western Europe and stretched to the Caucasus in the east. Since DOBV is highly pathogenic for humans, it is important to know if its limited geographic distribution is dependent on ecological factors, or simply reflects the lack of knowledge concerning HFRS epidemiology. If latter is the case, the actual geographic range of DOBV and DOBV-caused HFRS should be clarified and health risk associated with DOBV infections in other parts of Europe should be properly assessed.

At present, little is known about the mechanisms that are responsible for hantaviral pathogenesis. This study has reported the first cloning and sequencing of complete genomes of SAAV and DOBV. Since the data on the epidemiology of DOBV and SAAV suggest that these genotypes have different pathogenicity for humans, detailed comparison of DOBV and SAAV sequences could reveal candidate genetic markers for hantavirus pathogenicity and host specificity. Cloned genomic segments can be used for genetic manipulations with the viral genomes in order to identify functionally important regions and molecular determinants of hantaviral pathogenesis.

Another important topic of hantavirus research concerns clarification of evolutionary mechanisms of these viruses, studies of hantaviral maintenance in rodent species and means of rodent-to-rodent/rodent-to-human transmissions. Conclusions and Future Prospects 70

Analysis of SAAV and DOBV demonstrated that the genetic diversity within each of the two genotypes is similar to that observed within other hantavirus genotypes. Phylogenetic analyses of DOBV and SAAV suggested that evolution of both genotypes follows the general principles established for other hantaviruses: host-dependent clustering governs over geographic clustering of the genetic variants. Comparative phylogenetic analysis of hantaviruses and rodent host species provided the evidence for the first known host switching event in the evolution of Murinae-borne hantaviruses: a common ancestor of DOBV and SAAV, carried by A. flavicollis, was transmitted to the western form of A. agrarius, leading to the ecological and most likely reproductive isolation of SAAV.

Acknowledgements 71

ACKNOWLEDGEMENTS

The research described in this thesis was carried out in the Department of Virology, University of Helsinki, during years 1997-2003, with support and contribution of many people, and I will always be grateful to all of them. I would like to express my most sincere gratitude to my supervisors: Alex Plyusnin and Antti Vaheri. I am especially indebted to Alex for inviting me to Finland, teaching me everything about virology in general and viral genetics in particular, but most of all, for always being there for me through all the “ups” and “downs” of my research work. I would like to acknowledge him for his willingness to quickly address any scientific or non-scientific problem that I had during this years, and for doing everything in his power to make my work here safe and enjoyable. Antti is acknowledged for creating friendly and encouraging working environment, for stimulating discussions and his indispensable comments and advices. I also would like to especially thank both Alex and Antti for being such a great partners in our tennis games, which kept me slim and fit for the lab work. My most warm thanks goes to Angelina Plyusnina for teaching me all the methods I have been using during this work, for always being supportive and helpful with a practical advice, technical help, or a kind word. I would also like to thank her for the delicious homemade pies and cakes that kept me going during those long, dark, scary evenings. I would like to thank all the people in our “hantavirus” (and later upgraded to “viral zoonoses”) group for being such a nice bunch of guys, girls, ladies and gentlemen as they were, for sharing the lab and the office space with me, for all the small talks and scientific discussions that we had during the day, and for all those beers we drank together. I especially want to acknowledge Sami Kukkonen and Pasi Kaukinen for helping with the translation of those countless documents in Finnish that I found in my mailbox during these years. I would like to thank Sami for taking care of the computers and for his help in resolving all kinds of technical problems that I experienced from time to time. I am also grateful to Sami and Anu Jääskeläinen for all the nice time and fun we shared during our trip to Amsterdam. I am especially grateful to Leena Kostamovaara for being so helpful in the lab, for taking care of all reagents and equipment and for keeping our “kitchen” in the amazing order, but, most of all, for greeting and talking to me in Russian, that made me fill a bit more like home. Olli Vapalahti is gratefully acknowledged for helpful scientific discussions and useful practical advices during this work and preparation of the thesis. Acknowledgements 72

I would like to thank all the people in the Virology department for creating warm and friendly working climate. I especially want to acknowledge all people from different laboratories in Finland and elsewhere who contributed to this research work. Most of all I want to thank Heikki Henttonen, our rodent expert, for his excellent trapping work, and Åke Lundkvist and his stuff at Karolinska institute for doing all the neutralization tests. All members of Zoonoses group, but first of all Tytti Manni and Raija Leveelahti, are acknowledged for doing all the work on isolation and passaging of the hantaviruses I studied. I thank the members of our “Tennis Club”: Alex, Antti, Henrikki Brummer-Konverkontio, Timo Hyypiä and Tuomo Timonen for the fair play and for being real gentlemen on the tennis court and outside it. Timo and Maria Söderlund-Venermo are also acknowledged for evaluating the progress of this thesis for the Graduate School. Professors Ralf Pettersson and Mika Salminen are gratefully acknowledged for revising this thesis and giving valuable comments and suggestions and also for writing their expert reviews on short notice. Finally, I would like to thank all my friends here and in Russia, who supported me during these years: Ilya Plyusnin and Pauly Helberg for being great tennis partners and very good friends in social life, Olya Samuilova for being helpful and supportive, and Andrey Parfentjev for indispensable support and help to me and my family during good and bad times for nearly two decades of my live. Most of all, I would like to thank my mom and dad and all my family for their love and support.

This research work was supported by the CIMO foundation, Sigrid Juselius foundation and Helsinki Biomedical Graduate School.

Kirill Nemirov Helsinki, 13.2.2003

References 73

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Appendix 1. Accession numbers of hantaviral strains used for the phylogenetic analysis:

Genotype Strain Accession

HTNV 76-118 M14626 Z10 AF159370 Maaji-1 AF321094 LR1 AF288294 CUMC-B11 U37768 A9 AF329390 S85-46 AF288659 Maaji-2 AF321095 Q32 AB027097 HTN261 AF252259 Hu AB027111 AA2499 AF427320 AA1719 AF427319 AA1028 AF427318 A16 AF288646 SN7 AF288657 84Fli AY017064 E142 AF288644 Chen4 AB027101 RG9 AF288296 CFC94-2 X95077 Da Bie Shan AH211 AF288647 AH09 AF285264 NC167 AB027523 Amur AP1371 AF427324 AP1168 AF427323 AP708 AF427322 AP681 AF427321 Solovey/AP61/1999 AB071183 Solovey/AP63/1999 AB071184 Liu AF288649 SEOV K24-e7 AF288653 K24-v2 AF288655 R22 AF288295 L99 AF488708 Hb8610 AF288643 Z37 AF187082 Sapporo Rat M34881 Tchoupitoulas (TCH) AF329389 Pf26 AY006465 zy27 AF406965 IR461 AF329388 Gou3 AF184988 DOBV Dobrava L41916 As-1/Goryachiy Klyuch-2000 AF442622 Ano-Poroia/Afl9/1999(AP) AJ410615 Ano-Poroia/13Af/99 AJ410619 SAAV Saaremaa 160v AJ009773 Saar/90Aa/97 AJ009775 Kurkino/44Aa/98 AJ131672 Appendix 1 93

Genotype Strain Accession

Kurkino/53Aa/98 AJ131673 East Slovakia-862-Aa AJ269550 East Slovakia-856-Aa AJ269549 Lolland/Aa1403/2000 - PUUV Sotkamo X61035 Fyn AJ238791 Puu/Mellansel/Cg47/94 AJ223374 Puu/Mellansel/Cg49/94 AJ223375 Puu/Solleftea/Cg6/95 AJ223377 Puu/Tavelsjo/Cg81/94 AJ223380 Puu/Eidsvoll/Cg1138/87 AJ223369 Puu/Eidsvoll/1124v AJ223368 Puu/Virrat/25Cg/95 Z69985 Vranica U14137 Puu/Solleftea/Cg3/95 AJ223376 Puu/Hundberget/Cg36/94 AJ223371 Udmurtia/444Cg/88 Z30706 Puu/Vindeln/L20Cg/83 Z48586 Kazan Z84204 Udmurtia/458Cg/88 Z30707 Udmurtia/338Cg/92 Z30708 CG1820 M32750 Udmurtia/894Cg/91 Z21497 Kolodozero AJ238789 Puu/Puu/1324Cg/79 Z46942 Evo/13Cg/93 Z30703 Evo/14Cg/93 Z30704 Evo/12Cg/93 Z30702 Evo/15Cg/93 Z30705 Thuin/33Cg/96 AJ277030 Momignies/47Cg/96 AJ277032 Momignies/55Cg/96 AJ277033 Montbliart/23Cg/96 AJ277031 Couvin/59Cg/97 AJ277034 CG 13891 U22423 Karhumaki AJ238788 Gomselga AJ238790 HOKV Tobetsu-60Cr-93 AB010731 Kamiiso-8Cr-95 AB010730 TULV Tula/53Ma/87 Z30942 Tula/23Ma/87 Z30945 Tula/175Ma/87 Z30943 Tula/76Ma/87 Z30941 Tula/249Mr/87 Z30944 Tula/Kosice144/Ma/95 Y13979 Tula/Kosice667/Ma/95 Y13980 Serbia AF017659 Tula/Koziky/5276Ma/94 AJ223601 D5-98 AF289819 Malacky 32/94 Z48235 Malacky 370/94 Z68191 D17-98 AF289820 Tula/Koziky/5247Ma/94 AJ223600 Lodz-1 AF063892 Appendix 1 94

Genotype Strain Accession

D63-98 AF289821 Tula/Moravia/5302Ma/94 Z49915 Lodz-2 AF063897 Tula/Moravia/5294Ma/94 Z48741 Tula/Moravia/5293Ma/94 Z48574 Tula/Moravia/5286Ma/94 Z48573 Tula/Moravia/5302v/95 Z69991 c109-s AF164094 g20-s AF164093 BLLV MO46 U19303 PHV PH-1 Z49098 ISLAV MC-SB-47 U19302 TOPV Ls136V AJ011646 KHAV MF-43 U35255 SNV NM H10 L25784 NM R11 L37904 Convict Creek 74 L33816 NYV H-NY1 U47135 Convict Creek 107 U29210 RI-1 U09488 MGLV Monongahela-1 U32591 BCCV - L39949 BAYV Louisiana L36929 MULV SH-Tx-339 U54575 CADV VHV-574 AF000140 LANV 510B AF005727 MACV 13796 AF482716 ANDV Chile-9717869 AF291702 AH-1 AF324902 9718133 AF482712 ELMCV RM-97 U11427 NM-164 U11429 LCV 68273 AF307322 LECV 22819 AF482714 BERV Oc22531 AF482713 Hu39694 - AF482711 PERGV 14403 AF482717 RIOMV OM-556 U52136 Oran 22996 AF482715 AND Nort - AF325966