The Origin of the Genus Flavivirus and the Ecology of Tick-Borne Pathogens

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1100

JOHN H.-O. PETTERSSON

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-554-8814-7 UPPSALA urn:nbn:se:uu:diva-211090 2013 Dissertation presented at Uppsala University to be publicly examined in Zootissalen, EBC, Villavägen 9, Uppsala, Friday, 10 January 2014 at 13:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Per- Eric Lindgren (Linköpings universitet, Institutionen för klinisk och experimentell medicin).

Abstract Pettersson, J. H. -O. 2013. The Origin of the Genus Flavivirus and the Ecology of Tick- Borne Pathogens. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1100. 60 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-8814-7.

The present thesis examines questions related to the temporal origin of the Flavivirus genus and the ecology of tick-borne pathogens. In the first study, we date the origin and divergence time of the Flavivirus genus. It has been argued that the first flaviviruses originated after the last glacial maximum. This has been contradicted by recent analyses estimating that the tick-borne flaviviruses emerged at least before 16,000 years ago. It has also been argued that the Powassan virus was introduced into North America at the time between the opening and splitting of the Beringian land bridge. Supported by tip date and biogeographical calibration, our results suggest that this genus originated circa 120,000 (156,100–322,700) years ago if the Tamana bat virus is included in the genus, or circa 85,000 (63,700–109,600) years ago excluding the Tamana bat virus. In the second study we estimate the prevalence of tick-borne encephalitis virus (TBEV) in host-seeking Ixodes ricinus from 29 localities in Sweden and compare our data with those of neighbouring countries. Nymphs and adult ticks were screened for TBEV using a real-time PCR assay. The mean TBEV prevalence for all tick stages combined was 0.26% for Sweden and 0.28% for all Scandinavian countries, excluding Iceland. The low prevalence of TBEV in nature may partly be explained by the fact that TBEV occurs in spatially small foci and that the inclusion of ticks from non-infected foci will reduce the prevalence estimate. In the third and fourth study, we conducted the first large-scale investigations to estimate the prevalence and geographical distribution of Anaplasma spp. and Rickettsia spp. in host-seeking larvae, nymphs and adults of I. ricinus ticks in Sweden. Ticks were collected from several localities in central and southern Sweden and were subsequently screened for the presence of Anaplasma spp. and Rickettsia spp. using a real-time PCR assay. For all active tick stages combined, the mean prevalence of Anaplasma spp. and Rickettsia spp. in I. ricinus in Sweden was estimated to 1.1% and 4.8%, respectively. It was also shown that A. phagocytophilum and R. helvetica are the main Anaplasma and Rickettsia species occurring in Sweden.

Keywords: Flavivirus, Virus dating, Molecular dating, Biogeography, Ixodes ricinus, Minimum infection rate, TBE, Tick-borne encephalitis virus, Rickettsia helvetica, Anaplasma phagocytophilum, Infection prevalence, RT-PCR

John H.-O. Pettersson, Department of Organismal Biology, Systematic Biology, Norbyv. 18 D, Uppsala University, SE-75236 Uppsala, Sweden.

ISSN 1651-6214 ISBN 978-91-554-8814-7 urn:nbn:se:uu:diva-211090 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-211090)

Till Élise, Julian, Helen, mor och far

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List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals:

I Pettersson, J.H.-O. & Fiz-Palacios, O. (2013) Dating the origin of the genus Flavivirus in the light of Beringian biogeography. Submit- ted.

II Pettersson, J.H.-O., Golovljova, I., Vene, S., and Jaenson, T.G.T. (2013) Prevalence of tick-borne encephalitis virus in Ixodes ricinus ticks in Northern Europe with particular reference to Southern Swe- den. Submitted.

III Severinsson, K., Jaenson, T.G., Pettersson, J., Falk, K., and Nilsson, K. (2010) Detection and prevalence of Anaplasma phagocytophilum and Rickettsia helvetica in Ixodes ricinus ticks in seven study areas in Sweden. Parasites & Vectors. 3:66. Doi: 10.1186/1756-3305-3-66

IV Wallménius*, K., Pettersson*, J.H.-O., Jaenson, T.G.T., and Nilsson, K. (2012) Prevalence of Rickettsia spp., Anaplasma phagocytophilum, and Coxiella burnetii in adult Ixodes ricinus ticks from 29 study areas in central and southern Sweden. Ticks and Tick-borne Diseas- es. 3(2):100-106. Doi: 10.1016/j.ttbdis.2011.11.003

Reprints were made with permission from the respective publishers. * Both authors contributed equally. Cover image by Élise and Julian Orest Petters- son.

Contents

Introduction ...... 9 What are zoonoses and how do they emerge? ...... 9 Vector-borne zoonoses ...... 11 The origin of the genus Flavivirus and virus dating ...... 12 Ticks and tick-borne zoonoses ...... 17 Tick-borne encephalitis virus ...... 19 Tick-borne anaplasmoses and rickettsioses ...... 23 Tick-borne anaplasmoses ...... 24 Tick-borne rickettsioses ...... 27 Research aims ...... 29 Summary paper I ...... 30 Summary paper II ...... 32 Summary paper III and IV ...... 34 Conclusions ...... 36 Svensk sammanfattning ...... 37 Acknowledgements ...... 41 References ...... 44

Abbreviations

16S rRNA gene Encodes small subunit of the ribosome 95% HPD Highest posterior density interval that contains 95% of the posterior probability BEAST Bayesian analysis by sampling trees GGEV Greek goat encephalitis virus gltA gene Citrate synthase-encoding gene in Rickettsia spp. HGA Human granulocytic anaplasmosis HGE Human granulocytic erhlichiosis ISF Insect-specific flaviviruses kDa Kilo Dalton LIV Louping ill virus MIR Minimum infection rate MBF Mosquito-borne flaviviruses MMLV Montana myotis leukoencephalitis virus MrBayes Software for Bayesian phylogenetic inference NKVF No-known vector flaviviruses ompB gene Outer membrane protein gene OHFV Omsk haemorrhagic fever virus POWV Powassan virus RAxML Randomized axelerated maximum likelihood RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid SFG Spotted-fever group SSEV Spanish sheep encephalitis virus TABV Tamana bat virus TBE Tick-borne encephalitis TBEV Tick-borne encephalitis virus TBF Tick-borne flaviviruses TG Typhus group tMRCA Time to most recent common ancestor TSEV Turkish sheep encephalitis virus YFV Yellow fever virus

Introduction

What are zoonoses and how do they emerge? Zoonoses, as defined in the present thesis, are infectious diseases, caused by various organisms (including protozoa, fungi, helminths and bacteria) and viruses, that can be transmitted between humans and other animals (WHO 1959; Palmer, Soulsby & Simpson 1998; Taylor, Latham & Woolhouse 2001). Of all diseases known to humans (>1400), more than 60% are zoonoses. Zoonoses account for approximately 75% of all diseases that are considered to be emerging, i.e. that has recently appeared or has been present earlier but are now expanding their geographical ranges and/or incidences in humans. Of all emerging zoonotic diseases, more than 40% and 30% are caused by viruses and bacteria, respectively (Cleaveland, Laurenson & Tay- lor 2001; Taylor et al. 2001). It is also known that the majority (68%) of all zoonotic pathogens are able to infect multiple host species of several taxonomic families or even different orders (Pedersen et al. 2005).

How then does a microorganism or virus evolve to become a zoonotic pathogen? The short answer is contact. Human contact and exposure to other animals is the fundamental basis by which zoonotic pathogens evolve and become established (Kruse, Kirkemo & Handeland 2004; Woolhouse, Haydon & Antia 2005). Throughout history and evolution, human population migration, expansion and increase in density, changes in human behaviour, development of farming and livestock, and exploitation of new areas through logging and habitat, destruction, changes in climate and ecology, and phenotypic change have provided ample opportunities for new pathogens to appear and establish themselves in human populations (Morse 1995; Woolhouse et al. 2005; Nunn 2006; Wolfe, Dunavan & Diamond 2007).

Furthermore, it is known that the best overall predictor of whether two species share the same pathogens is their evolutionary relationship (Davies & Pedersen 2008). Humans are therefore more likely to share pathogens with chimpanzees and gorillas than with mandrills or baboons or other more distantly related animals (Figure 1). This is supported by the fact that non- human primates have contributed to about 20% of all major human infectious diseases, for example hepatitis B, vivax malaria, dengue fever, yellow fever, HIV-1 and HIV-2 (Weiss 2001; Wolfe et al. 2007), although primates

9 only comprise 0.5% of all vertebrate species (Wolfe et al., 2007). This is also supported by the fact that the majority of all human diseases, established, emerging, and re-emerging, have their origin in the Old World rather than in the New World. This is partly explained by differences in shared evolutionary history and phylogenetic distance. For instance, humans share a much longer evolutionary history with Old World animals during which pathogen transfer could have occurred, than with New World animals.

Phylogenetic relationship Frequency of contact

Figure 1. Comparison of the relative risk of a pathogen of animal origin to infect and being established in humans. The risk is illustrated as dependent on phylogenetic relatedness and frequency of contact with other animals. Phylogenetic relationship is a good predictor of risk for cross-species transmission. However, abundance and frequency of contact, here represented by difference in thickness of the lines, can compensate for lack of phylogenetic relatedness. The thicker the line, the greater the chance of a pathogen crossing species boundaries. After Holmes (2009).

However, what also needs to be pointed out is that high abundance and frequent contact can compensate for lack of phylogenetic relationship. For example the role of rodents and the transmission of plague; poultry in the transmission of different influenza viruses; and bats as reservoirs of Corona virus (Figure 1) (Olsen 2006; Wolfe et al. 2007; Drexler et al. 2010; Corman et al. 2013). Similarly, for viruses alone, geographical range overlap is a better predictor for host range of pathogens than phylogenetic relatedness. High mutation rate, short generation time and high population diversity allow viruses to rapidly adapt to other taxonomic families or orders (Davies & Pedersen 2008). Likewise, it has been pointed out that a zoonotic pathogen might be shared between two species due to similarities in habitat type, food preferences, geographical ranges and ecological characteristics (Nunn 2006). Two main factors regulate the capability of a pathogen to infect multiple

10 hosts: (i) having numerous opportunities of transmission between host species and/or (ii) high genetic variation and short generation time of the pathogen allowing for rapid evolution and adaptation to novel host species (Woolhouse 2001).

The perhaps most well-known pathogen in humans with an animal origin is the causative agent of AIDS, i.e. the human immunodeficiency viruses HIV- 1 and HIV-2. Evidence suggests that the HIV-strains in humans are the result of multiple cross-species transmission events of simian immunodeficiency viruses (SIVs). HIV-1 evolved from the transmission of SIV-strains in chimpanzees (Pan troglodytes troglodytes) to humans, following cross-species transmission on at least three separate occasions during the early 20th centu- ry (Gao et al. 1999; Hahn 2000; Korber 2000). HIV-2 originated from SIV transmitted from sooty mangabeys (Cercocebus atys) (Hirsch et al. 1989; Santiago et al. 2005).

Vector-borne zoonoses The transmission dynamic of several zoonoses have evolved in the face of a vector, i.e. usually an arthropod species that can carry and transmit the disease-causing organism or virus to a new host. Diseases caused by such pathogens are defined as vector-borne zoonoses.

Arthropods and disease-causing organisms and viruses have co-evolved for millions of years before mammals, let alone humans, originated (Lovisolo, Hull & Rösler 2003). Blood-feeding arthropod parasites, of which mosquitoes and ticks are the two most important groups, can increase transmission of pathogens to new and between hosts. However it is less clear how some pathogens originally became established in a vector population, partly because of the multifactorial nature and complexity of pathogen-vector-host species jumps. As such, a virus or a microorganism faces several challenges when adapting to a vector-host system. The challenges are mostly related to the genetics and physiology (i.e. phylogenetic relatedness, barriers for entry into host cells and tissues, different types of immune defence systems and resistance, etc.) and the ecology and behaviour (i.e. population size, density and social construction, natality and mortality rates, longevity, feeding behaviour, local and global migration, etc.) of the host and vector (Edman 2003; Kuno & Chang 2005; Woolhouse et al. 2005; Parrish et al. 2008).

Two main ideas exist regarding the origin of vector-borne zoonotic pathogens. (i) Some zoonotic pathogens might have originated directly in blood- feeding arthropods from which they subsequently were transferred to vertebrates where they could cause disease. This may have been the case with the

11 different species Rickettsia. (ii) Other zoonotic pathogens are likely to first have been established in vertebrates’ whereafter the pathogens were transmitted and became established in the vector population (Edman 2003). Once a potential pathogen becomes established in a certain vector, the vector may allow the pathogen to be spread across larger distances, transmitted more efficiently and more frequently (Edman 2003; Kuno & Chang 2005). Fur- thermore, it is unclear why only some groups of blood-feeding arthropods have evolved virus-vector-capabilities. For example, tabanid flies (Tab- anidae) have no or only very limited function as vectors of zoonotic viruses, even though they are just as species-rich as blood-feeding mosquitoes (Cu- licidae), which are vectors of multiple zoonotic pathogens (Kuno & Chang 2005). What is clear is that even though blood-feeding arthropods only con- stitute about 1.5% of the total species diversity of Arthropoda (Ribeiro 1995; Kuno & Chang 2005), the haematophagus arthropods – by transmission of zoonotic pathogens – have had and still have a significant impact on human health and life (Edman 2003).

The origin of the genus Flavivirus and virus dating One virus genus that includes several of the most important human zoonotic viral pathogens is Flavivirus (Flaviviridae). This genus has a worldwide distribution and comprise close to 80 unique viral species, including some that are tentatively assigned to this genus (Gould et al. 2003; Gubler, Kuno & Markoff 2007; Lindenbach, Thiel & Rice 2007; Gould & Solomon 2008). Members of Flavivirus are characterized by a set of morphological characters of the virus particles and how the viral genome is organised. These viruses are enveloped, 50nm in diameter and spherical in shape with a genome consisting of an approximately 11kbp long single-stranded positive sense RNA molecule. The genome is made up of ten genes, three structural and seven non-structural, which are flanked by a 5´and 3´- non-coding terminal (Figure 2) (Lindenbach et al. 2007).

One single open reading frame

5´-NCRR Structural Non-Structural 3´-NCR

C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5

Figure 2. Genome organisation of the Flavivirus genus. Structural genes; capsid (C), pre-membrane (prM) and envelope (E). Non-structural genes; NS1–NS5. After Lin- denbach et al. (2007).

12 Flaviviruses are transmitted by four different modes, three of which are dependent on arthropods, for their transmission. These include (i) mosquito- borne flaviviruses (MBF), such as the Dengue virus (DENV) and the Yellow fever virus (YFV) (Grard et al. 2010), (ii) tick-borne flaviviruses (TBF), such as the Tick-borne encephalitis virus (TBEV) and the Omsk haemorrhagic fever virus (OHFV) (Gritsun, Nuttall & Gould 2003b; Grard et al. 2007). It also includes flaviviruses that are transmitted without an apparent vector, (iii) the no-known arthropod vector flaviviruses (NKVF) (Porterfield 1980). NKVFs include such members as the Montana myotis leukoencephalitis virus (MMLV) and the Tamana bat virus (TABV). Finally, within Fla- vivirus there are also viruses that are non-zoonotic. These (iiii) insect- specific flaviviruses (ISF) are only capable of replicating in insects cells and are not known to cause any disease in humans (Kuno 2007; Cook et al. 2012). In general, phylogenetic data support the clustering by vector-host association within Flavivirus (Figure 3) (Gould et al. 2001, 2003; Gaunt et al. 2001).

It has been proposed that Flavivirus originated from a non-vectored mamma- lian virus ancestor somewhere in Africa since Flavivirus, compared to He- pacivirus and Pestivirus, is the only genus within Flaviviridae that contains arboviruses (viruses vectored by arthropods) (Gould et al. 2003). In the present thesis we showed that Flavivirus is relatively old (I). Instead of having originated sometime after the last glacial maximum, i.e. less than 10,000 years ago (Zanotto et al. 1996; Gould et al. 2003), Flavivirus is estimated to have emerged circa 120,000 (95% HPD 87,100–158,900) to circa 85,000 (95% HPD 63,700–109,600) years ago (I). The age of the genus depends on if TABV should be considered a member of Flavivirus or not. If TABV is considered separate from Flavivirus and the split from TABV occurred around 85,000 years ago, it leaves about 75,000 years of Flavivirus evolution that previously were not taken into consideration. This begs the question, “How did the flaviviruses become distributed around the world?” After the split from the earliest ancestor, most members of Flavivirus started to diverge about 50,000 years ago into the host-vector associations that we currently see in Flavivirus (I). In view of these ages, it is very likely that the spread of several Flavivirus members accompanied the spread and migration of humans out of Africa (I), that took place sometime between 80,000 and 40,000 years ago (Macaulay 2005; Rasmussen et al. 2011; Blome et al. 2012; Henn, Cavalli-Sforza & Feldman 2012). Likewise, it is very likely that birds and other animals, both vertebrates and invertebrates, contributed to the distribution of some viruses (Mackenzie, Gubler & Petersen 2004).

13 SSEV LIV TBEV-Euu TBEV-Euu GGEV TSEV TBEV-FEE TBEV-FEE TBEV-X TBEV-X TBEV-Sibb TBEV-Sibb Tick-borne OHFV OHFV flaviviruses LGTV AHFV KFDV POWV DTV GGYV KSIV RFV KADV SREV MEAV TYUV MMLV No-known vector RBV MODV flaviviruses APOIV KUNV WNV WNV WNV KOUV YAOV MVEV ALFV JEV USUV SLEV ROCV ILHV TMUV Mosquito-borne BYDV STWV flaviviruses BAGV BAGV (Culex) NTAV IGUV BSQV NMV KOKV DENV-1 DENV-3 DENV-2 DENV-4 ZIKV SPOV KEDV NOUV CHAOV Insect-specific LAMV DGV flaviviruses SABV POTV JUGV BANV Mosquito-borne UGSV BOUV flaviviruses EHV WESSV (Aedes) SEPV YFV YOKV No-known vector ENTV CxFV flaviviruses QBV NAKV PCV Insect specific KRV AEFV flaviviruses CFAV HANKV No-known vector TABV flaviviruses

Figure 3. Phylogenetic relationships and vector-host associations within Flavivirus. Aedes virus (AEFV), Alfuy virus (ALFV), Alkhurma virus (AHFV), Apoi virus (APOIV), Bagaza virus (BAGV), Bagaza virus (BAGV), Baiyangdian virus (BYDV), Banzi virus (BANV), Bouboui virus (BOUV), Bussuquara virus (BSQV), Cell fusing agent virus (CFAV), Chaoyang virus (CHAOV), Culex flavivirus (CxFV), Deer tick virus (DTV), Dengue virus type 1 (DENV-1), Dengue virus type 2 (DENV-2), Dengue virus type 3 (DENV-3), Dengue virus type 4 (DENV-4), Donggang virus (DGV), Edge Hill virus (EHV), Entebbe bat virus (ENTV), Gadgets Gully virus (GGYV), Greek goat encephalitis virus (GGEV), Hanko virus (HANKV), Iguape virus (IGUV), Ilheus virus (ILHV), Japanese encephalitis virus (JEV), Jugra virus (JUGV), Kadam virus (KADV), Kamiti River virus (KRV), Karshi virus (KSIV), Kedougou virus (KEDV), Kokobera virus (KOKV), Koutango virus (KOUV), Kunjin virus (KUNV), Kyasanur forest disease virus (KFDV), Lammi virus (LAMV), Langat virus (LGTV), Louping ill virus (LIV), Meaban virus

14 (MEAV), Modoc virus (MODV), Montana myotis leukoencephalitis virus (MMLV), Murray Valley encephalitis virus (MVEV), Nakiwogo virus (NAKV), New Mapoon Virus (NMV), Nounane virus (NOUV), Ntaya virus (NTAV), Omsk hemorrhagic fever virus (OHFV), Palm Creek Virus (PCV), Potiskum virus (POTV), Powassan virus (POWV), Quang Binh virus (QBV), Rio Bravo virus (RBV), Rocio virus (ROCV), Royal Farm virus (RFV), Saboya virus (SABV), Saumarez Reef virus (SREV), Sepik virus (SEPV), Sitiawan virus (STWV), Spanish sheep encephalitis virus (SSEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), Ta- mana bat virus (TABV), Tick-borne encephalitis virus - Siberian subtype (TBEV- Sib), Tick-borne encephalitis virus Far-Eastern subtype (TBEV-FE), Tick-borne encephalitis virus - European subtype (TBEV-Eu), Tick-borne encephalitis virus - intermediate subtype (TBEV-X), Tembusu virus (TMUV), Turkish sheep encephalitis virus (TSEV), Tyuleniy virus (TYUV), Uganda S virus (UGSV), Usutu virus (USUV), Wesselsbron virus (WESSV), West Nile virus (WNV), Yaounde virus (YAOV), Yellow fever virus (YFV), Yokose virus (YOKV), Zika virus (ZIKV). After (I).

This new temporal scale during which Flavivirus has evolved will require a revision and renewed discussion about how some Flavivirus members were distributed throughout the globe. For example, it has been argued that DENV emerged within the last 2,000 years following virus crossover events from monkeys to humans. And that thereafter DENV became globally distributed by humans (Twiddy, Holmes & Rambaut 2003; Dunham & Holmes 2007). However in (I), DENV is estimated to have emerged more than 14,000 (95% HPD 10,645–18,210) years ago. Therefore, provided that the dating in (I) is correct, humans probably came into contact with DENV subtypes much earlier than previously suggested. However, the older estimated origin of DENV (I) does not exclude the spread of DENV at a later stage when the human population density had increased (Twiddy et al. 2003; Dunham & Holmes 2007). Likewise, it is indicated that WNV is more than 5,000 (95% HPD 3,866–7,134) years old (I) compared to the 200 years estimated by May and co-workers (May et al. 2011). The discrepancies between these two estimates are difficult to reconcile. It is clear that the occurrence of WNV in a certain region is due to a recent introduction of the virus from a different region (Garmendia, Van Kruiningen & French 2001; Nicholas Komar 2003; LaDeau, Kilpatrick & Marra 2007). At the same time, it is possible that prior to the introduction into one area, the virus has been circulating in a different area for a longer time. Hence the virus is "re-emerging" if being introduced into a new area.

Many of the incongruities found between different dating studies relate to the problem of accurately dating deep viral origins. In order to date the divergence of viruses in time it is necessary to have a type of temporal calibration. The temporal calibration in combination with a molecular clock, the idea that the accumulation of nucleotide substitutions occur at a certain rate,

15 allows time to be inferred in the phylogenetic tree. In virological dating studies, isolation dates, or tip dates, is the type of calibration used in the majority of all dating studies. The problems associated with inferring deep evolutionary history in viruses by tip date calibration are also caused by the very same trait that makes viruses so useful for inferring recent evolutionary history: their high substitution rate (Holmes 2003; Drummond et al. 2003). What follows from the high substitution rate of several viruses, which is due to their error prone repair and replication machinery (Bromham & Penny 2003; Duffy, Shackelton & Holmes 2008), is that the repeated substitution changes in the genome at the same position will weaken the phylogenetic signal with time (Holmes 2003). Added also by the fact that different lineages may have different substitution rates (Holmes 2003; Duffy et al. 2008; Sanjuán 2012), dating becomes more and more problematic and uncertain the deeper the node you are trying to date.

A way to improve the dating is to include an internally calibrated node, which is calibrated by a fossil or a biogeographical event (Ho & Phillips 2009). For viruses, fossils are unavailable. However biogeographical calibration has been used successfully to infer that the Simian immunodeficiency virus has been present for at least 32,000 years (Worobey et al. 2010). Bio- geographical calibration was also used by us to infer an older than previously assumed age for Flavivirus (I). A different type of internal calibration comes from the field of paleovirology, i.e. the study of extinct viruses and/or variance of activity among anti-viral genes between host species. Paleoviro- logical studies have shown that the dating of the interaction between virus and host can lead to estimates as far back as to prehistorical and geological timescales (Emerman & Malik 2010; Gilbert & Feschotte 2010; Patel, Emerman & Malik 2011; Katzourakis 2013). When using isolation date calibration only, the uncertainty increases with node depth, since there is no information from this part of the tree. The internal calibration helps the analysis by adding more information deeper into the tree by specifying the age for a certain node, and thereby indirectly limiting the age-range that the other non-calibrated nodes may have. This will allow for rate of substitution to be more accurately determined (Ho & Phillips 2009). Even though it might be difficult to exclude other explanations, the calibration of internal nodes by biogeographical events is probably the best method to infer deep evolutionary history for viruses, and will likely help to date deep ancestry of virus clades other than the Flavivirus and the Simian immunodeficiency virus clades.

16 Ticks and tick-borne zoonoses With a distributional range from northern Africa to northern Scandinavia, the tick Ixodes ricinus tick (Acari: Ixodidae) is considered to be one of the most important vectors of pathogens for human diseases.

I. ricinus has a life-cycle that consists of three active life-stages, larva, nymph and adult, involving multiple vertebrate hosts. Tick larvae feed mostly on smaller vertebrates moving on, under or close to the ground, such as rodents, whereas hosts of tick nymphs and adults also include larger vertebrates, such as hares, cervids and humans (Jaenson et al. 1994; Tälleklint & Jaenson 1997). Cervids, especially roe deer (Capreolus capreolus), are considered to be the main maintenance hosts of I. ricinus, and it is not uncom- mon to find one individual cervid to be infested with more than 100 adult female ticks (Tälleklint & Jaenson 1997). The seasonal activity patterns of the different life stages of I. ricinus can be either unimodal, with a single peak in spring-summer, or bimodal, with peaks in May to June and August to September (Hendrick, Moore & Morison 1938; Nilsson 1988; Mejlon & Jaenson 1993; Tälleklint & Jaenson 1996).

The abundance and activity pattern of ticks in the season is dependent on several inter-seasonal related factors, such as temperature and humidity, host abundance and dynamics, and tick behaviour (Nilsson 1988; Randolph et al. 2002; Medlock et al. 2013). I. ricinus occurs most abundantly in moist, de- cidious or mixed coniferous/deciduous woodlands and forests and are less abundant in open and dry habitats (Mejlon & Jaenson 1993; Lindström & Jaenson 2003). Furthermore, climate is the main determinant for the altitudinal and latitudinal geographical distribution of I. ricinus (Lindgren, Tälleklint & Polfeldt 1999; Jaenson & Lindgren 2011). For example, it has been shown that the expansion of I. ricinus to higher altitudes in the Krko- nose mountains was due to climate change (Materna, Daniel & Danielová 2005). However, climate and extended vegetation periods alone cannot explain the increased abundance of ticks. Within its potential distributional range, the abundance and occurrence of I. ricinus is strongly dependent on the availability and movement of tick amplification hosts, i.e. larger mammals such as cervids (Jaenson et al. 2012b). It has been suggested that the expansion of I. ricinus northwards in Sweden from the early 1980s to 2008 was due to both climate change, in particular milder winter season and an extended autumn and earlier spring period of activity, and, mainly, due to changes in the abundance and distribution of roe deer (Jaenson et al. 2012b). It is also known that the microclimatic conditions, in particular a high relative humidity at ground level, are significant determinants for I. ricinus sur- vival and distribution (Gray 1991).

17 I. ricinus is a vector of several pathogens (Table 1), where the nymphs of I. ricinus are considered to be the main vectors. For example, densities of I. ricinus nymphs are correlated with densities of Borrelia-infected nymphs (Tälleklint & Jaenson 1996; Nicholson & Mather 1996) and most people who became infected with the TBEV in Europe had been bitten by a TBEV- infected nymph (Korenberg 1994). However, in Russia, the TBEV-infection is usually transferred to humans by adult I. persulcatus (Korenberg 1994; Korenberg et al. 2001). The recent and continued increase of different high- throughput sequencing techniques has allowed for the discovery of previously unknown and undetected pathogens as well as a more detailed view of the flora of potential pathogens present in I. ricinus populations (Carpi et al. 2011).

Table 1. Notable potential human pathogens detected in I. ricinus ticks in Europe. After Pluta et al. (2011); Paduraru et al. (2012); Coipan et al. (2013).

Pathogen Disease Anaplasma phagocytophilum Human granulocytic anaplasmosis Babesia divergens Babesiosis Babesia microti Babesiosis Borrelia afzelii Lyme borreliosis / Lyme disease Borrelia burgdorferi sensu stricto Lyme borreliosis / Lyme disease Borrelia garinii Lyme borreliosis / Lyme disease Borrelia miyamotoi Relapsing fever Borrelia valaisiana Borreliosis / Lyme disease Candidatus Neoehrlichia mikurensis Diffuse symptoms, Serious disease in immuno- compromized people Coxiella burnetii Q-fever Crimean-Congo haemorrhagic fever virus Crimean-Congo haemorrhagic fever Eyach virus Colorado tick fever-like Francisella tularensis Tularemia Louping ill virus Menigoencephalitis Rickettsia conorii “Spotted fever” Rickettsia helvetica “Spotted fever” Rickettsia monacensis “Spotted fever” Rickettsia sibirica Siberian tick typhus Tick-borne encephalitis virus, European subtype Tick-borne encephalitis

18 Tick-borne encephalitis virus The tick-borne encephalitis virus (TBEV), the causative agent of tick-borne encephalitis (TBE), was first discovered in 1937 by Lev Zilber during an expedition in Far-Eastern Russia. TBE is now considered to be one of the most important arthropod-borne viral diseases in Europe and Asia (Granström 1997; Gritsun, Lashkevich & Gould 2003a; Charrel et al. 2004; Mansfield et al. 2009).

Taxonomically, TBEV belongs to the genus Flavivirus within the family Flaviviridae and is closely related to Louping ill virus (LIV), Spanish sheep encephalitis virus (SSEV), Greek goat encephalitis virus (GGEV) and Turk- ish sheep encephalitis virus (TSEV) (Figure 4) (Gritsun et al. 2003a; Interna- tional Committee on Taxonomy of Viruses 2012). TBE viruses originated about 5,000 years ago, splitting up into two clades, a European and a Rus- sian/Asian clade, a little more than 3,000 years ago. These clades have continued to diverge since 2,600 years ago (I). TBEV is considered to consist of three virus subtypes with relatively distinct vector- and geographical association: the European (TBEV-Eu), the Far-Eastern (TBEV-FE), and the Siberi- an (TBEV-Sib) TBEV subtype (Ecker et al. 1999; Gaunt et al. 2001; Gritsun et al. 2003a).

The European subtype is found in a patchwork all over Europe and in western Russia. This subtype is mainly vectored by I. ricinus (Süss 2011), but has also been detected in I. persulcatus (Jääskeläinen et al. 2011). TBEV-Eu has also been recorded in South Korea, where it is vectored by Haemphysalis spp. rather than by Ixodes spp. (Kim et al. 2008; Yun et al. 2009; Ko et al. 2010). The Far-Eastern subtype is vectored by I. persulcatus and occurs mainly from eastern Russia to Japan (Ecker et al. 1999; Kovalev, Kokorev & Belyaeva 2010). Like the Far-Eastern subtype, the Siberian subtype is vectored by I. persulcatus and is found in Eastern Europe, including Finland, western Russia and Siberia (Ecker et al. 1999; Jääskeläinen et al. 2006; Ko- valev et al. 2009). In general, all TBEV subtypes follow the geographical distribution of their primary vectors. As such, the subtypes also overlap and co-circulate in regions where I. ricinus and I. persulcatus overlap (Lundkvist et al. 2001; Jääskeläinen et al. 2011).

19 Tick-borne encephalitis virus - Eu

Louping ill virus

Spanish sheep encephalitis virus

Greek goat encephalitis virus

Turkish sheep encephalitis virus

Tick-borne encephalitis virus - FE

Tick-borne encephalitis virus - Sib

Omsk haemorrhagic fever virus

Langat virus

Figure 4. Phylogenetic relationships of TBEV subtypes, TBEV-Eu, TBEV-FE and TBEV-Sib, and more distantly related flaviviruses. After (I).

On average annually, about 3,000 and 6,000 persons are diagnosed with TBE in Europe and Russia, respectively (Süss 2011). Following TBEV transmission after a tick bite, disease symptoms of TBE are usually presented after an incubation period of on average 8 days. Between 30–95% of all TBE infected persons do not present any disease symptoms, whereas people who do develop symptoms usually experience TBE as a monophasic (>70%) or biphasic (<30%) course (Kaiser 1999, 2008; Mickiene et al. 2002; Gritsun et al. 2003a). During the first phase, most symptoms are influenza-like, i.e. fever, general malaise, headache and body pain, lasting on average five days. This is followed by a symptom-free period lasting on average seven days. People who also experience the second phase, may develop disease symptoms ranging from a mild meningitis to a severe and life-threatening encephalitis with paralytic long-lasting sequelae (Kaiser 1999, 2008; Mickiene et al. 2002; Gritsun et al. 2003a). Mortality ranges from 1% for TBEV-Eu to as high as 20–40% for the Far-Eastern TBEV-subtype (Lindquist & Vapalahti 2008; Kaiser 2008). When TBEV-infected children fall ill with TBE they usually children experience mild and vague symptoms (Grubbauer et al. 1992; Iff et al. 2005; Hansson et al. 2011). However, it was recently shown that about 60% of all children that experienced TBE-infection suffer from long-lasting neurological problems including headache, fatigue, and irritabil-

20 ity. Most notably, a third of all children suffered from cognitive problems with significantly impaired cognitive functions, especially a reduced working memory capability (Fowler et al. 2013).

TBEV is maintained in nature between ticks and their vertebrate host by four modes of host-vector transmission (Figure 5): (1) Viraemic transmission: TBEV can be transmitted between ticks and tick-hosts when tick larvae or nymphs feed on a vertebrate host, typically rodents belonging to the genera Apodemus spp. and Myodes spp. These rodents, especially the young ones, may become viraemic following TBEV infection. However, this mode of viraemic transmission is considered not to account solely for the maintenance of TBEV in nature since the viraemia in the rodents usually lasts less than a week. Then, due to a reduced concentrations of virions, the potential for TBEV transmission to feeding ticks is limited or impossible (Nuttall & Labuda 2003; Achazi et al. 2011). However, TBEV can be detected in infected rodents during several months as well as during winter. Thus, rodents should still be regarded as TBEV reservoirs (Tonteri et al. 2011). (2) Transovarial transmission occurs when the TBEV-infection is passed along from the adult female tick to her eggs. This usually occurs at a frequency of less than 1% (Danielová & Holubová 1991; Gould et al. 2003). Since an adult female may lay up to 3,000 eggs, transovarial TBEV transmission might be significant for the long term maintenance of TBEV in the tick population, because ticks, once infected, usually will remain infected throughout the remainder of their life, i.e. by (3) transstadial transmission. Non-viraemic co-feeding transmission: TBEV transmission can also occur when (4) a group of congregated ticks are co-feeding on the same host, a rodent, which may or may not experience viraemia, where the TBEV is transferred from TBEV-infected ticks to susceptible ticks via the host’s leucocytes (Labuda et al. 1993, 1997; Randolph, Gern & Nuttall 1996; HavlíKová, LičKová & Klempa 2013). For this mode of transmission to occur, it is important that there is synchrony in activity between the infective (typically nymphs) and the susceptible (typically larvae) tick stages (Randolph et al. 2000), which usually occurs in the spring if temperatures quickly rise to ≥7°C (Knap et al. 2009).

21 Transovarial transmission

Infected tick larvae Transstadial transmission

TransstadialTransstadial trantransmissionsmission

Figure 5. Tick-borne encephalitis virus transmission cycle between ticks and tick hosts.

In Sweden, and Europe in general, the prevalence of TBEV in all tick stages combined is usually less than 1% (II) and between 150–300 cases of TBE have been recorded each year since 2006 (Jaenson et al. 2012a). In Sweden, the first human TBE case was diagnosed in 1954 (Holmgren & Forsgren 1990). Since then, the number of TBE cases has increased significantly during the last decade. This has been explained mainly by the increased abundance of tick maintenance hosts, particularly roe deer, but also by changes in climate (Jaenson et al. 2012b; a). The relatively low prevalence of TBEV in the vector population has led researchers to question how TBEV can be maintained in nature. It has been shown that TBEV circulates in patchily distributed and spatially small foci (Dobler et al. 2011). Therefore, the inclusion of ticks from outside the actual TBEV foci will bias the virus prevalence estimate towards a lower prevalence. This is partly seen when compar- ing TBEV positive localities with TBEV negative localities (II). Another explanation is perhaps that the TBEV prevalence in ticks is actually an un- derestimate due to TBEV being present in unfed ticks in very low concentrations, which are presumably undetectable by commonly used real-time PCR assays (II).

22 The incidence of TBE and the prevalence of TBEV in ticks in Sweden will likely increase in the future. A changing climate, leading to earlier springs and later arrival of winters, in combination with a high or even increasing number of cervids will lead to an increased abundance of ticks. An increased abundance of ticks will lead to more opportunities for TBEV to be transmitted to and among ticks and TBEV reservoir hosts (Jaenson et al. 2012a). Therefore, it is important to regularly monitor the TBEV prevalence in the tick and tick-host populations, as well as to further explain what habitat characteristics are optimal for TBEV to be maintained in nature.

Tick-borne anaplasmoses and rickettsioses The order Rickettsiales includes two families with particular significance to human health. The family Anaplasmataceae includes four well-known genera, Anaplasma, Ehrlichia, Neorickettsia and Wolbachia (Dumler et al. 2001). The family Rickettsiaceae includes two genera, Orientia and Rickettsia (Fig- ure 6) (Weiss & Dasch 1991; Tamura et al. 1995; Fournier & Raoult 2009). Both Anaplasma and Rickettsia are obligate intracellular gram-negative al- phaproteobacteria. The genome size of Anaplasma is approximately 1.5 M bases long, encoding more than 1300 proteins, whereas the genome of Rick- ettsia is in general smaller and more conserved with a genome size of about 1.1M bases that encode a bit more than 800 proteins (Andersson et al. 1998; Dunning Hotopp et al. 2006). Approximately 150 million years ago, Rickett- sia diverged into two clades; one that mainly infects arthropods, which include most human pathogens, and one that infects various arthropods, pro- tists and other eukaryotes (Weinert et al. 2009). Anaplasma is estimated to have started to diverge between 78–43 million years ago, possibly co- diverging with its main vector and reservoir, the ixodid ticks (Foley et al. 2008).

23 Order Family Genus Species

A. phagocytophilum

A. platys

A. bovis Anaplasma

A. marginale

Ehrlichia

Wolbachia Anaplasmataceae

SFG Neorickettsia R. helvetica, sibirica Rickettsiales Rickettsia

TG Rickettsiaceae R. typhi, prowazekii Orientia

Figure 6. Schematic overview of Rickettsiales taxonomy. After Dumler et al. (2005); Gillespie et al. (2010).

Tick-borne anaplasmoses The genus Anaplasma is a geographically widely distributed taxonomic group. Members of Anaplasma have for several decades been known to cause infections in cattle (Foggie 1951). Prior to 2000, A. phagocytophilum was known as Ehrlichia phagocytophila, i.e. the etiological agent of human granulocytic erhlichiosis (HGE). In 1990 it was for the first time discovered in the United States as a disease-causing agent in humans (Chen et al. 1994). Then, studies were initiated which subsequently led to its taxonomical re- classification as belonging to the Anaplasma genus (Figure 6). The corre- sponding disease was thus changed to human granulocytic anaplasmosis (HGA) (Dumler et al. 2001).

In nature, A. phagocytophilum is maintained in a cycle between ixodid ticks and tick-hosts, i.e. rodents and cervids (Burkot et al. 2001; Hildebrandt et al. 2003; Hartelt et al. 2004; Ohashi et al. 2005). The cycle may be depicted as beginning when (1) an uninfected tick larva becomes infected after feeding on an infected vertebrate, e.g. a rodent. (2) The infected larva then moults to become an infected nymph, since the infection is transstadially transferred. (3) The infected nymphs feed on a second vertebrate host, e.g. a rodent or a cervid, which may get infected with A. phagocytophilum from the tick bite or the host may already be infected with the bacterium. (4) The infection is then transstadially transferred by the infected nymph to the adult tick, either

24 male or female. The infected tick female – and presumably occasionally also the infected tick male – may transmit the Anaplasma infection to another vertebrate host (Rikihisa 2011). Compared to Rickettsia spp., Anaplasma spp. are not transovarially transferred (from the adult female to her eggs) (Figure 7) (Long et al. 2003). The importance of cervids, in particular roe deer (Capreolus capreolus) and red deer (Cervus elaphus), for the maintenance of A. phagocytophilum is explained by their high abundance in several countries as well as the high prevalence of A. phagocytophilum in the cervid population. Apart from humans and wild animals, also domesticated animals, such as cats, dogs, cattle, goats, sheep and horses can become infected with A. phagocytophilum and exhibit symptoms of disease (Stuen, Granquist & Silaghi 2013).

Unlike transmission of TBEV, co-feeding may not be important for the maintenance of Anaplasma spp. (Kocan & de la Fuente 2003). Similarly to rickettsial diseases, the common symptoms of HGA are in general nonspe- cific ones, such as fever, headache, general malaise, myalgia and nausea (Dumler 2012). A. phagocytophilum mostly infect cells originating from blood stem cells, e.g. leucocytes (type of white blood cell), neutrophils (type of white blood cell), thrombocytes (platelets), erythrocytes (red blood cells) and endothelial cells, for its propagation while simultaneously evading the host immune defence system (Rikihisa 2010). Therefore, HGA is usually characterized by blood disorders, such as leucocytopenia, lymphocytopenia, and thrombocytopenia, (Dumler 2005; Dumler et al. 2007). Since the time when the first human HGA case was diagnosed in the US, A. phagocytophilum has been confirmed all over Europe and Asia. In Europe, A. phagocytophilum is mainly vectored by I. ricinus. The prevalence in the tick population, reviewed by Stuen and co-workers (Stuen et al. 2013), ranges from 0.4–34% depending on tick stage and geographical region. In Sweden, two large-scale investigations found that the mean MIR of A. phagocytophilum in all blood-feeding tick stages in central and southern Sweden is approximately 1% (III; IV). It has been suggested that the presence of different A. phagocytophilum-variants may explain the large differences in prevalence found in different studies (IV). The low prevalence found in adult ticks in IV can possibly be explained by (i) an inefficient transstadial transmission of A. phagocytophilum (variants). The low prevalence might also be due to that (ii) when the nymphs are feeding on a second host, there are antibacterial factors present in the blood of the host that eliminate the bacteria (IV). The annual number of persons experiencing A. phagocytophilum-related diseases in Sweden is not known, mainly because HGA is not a notifiable disease in Sweden as well as in most other countries.

25 Transstadial TransovarialTransovarial transmission transmissiontransmission

TransstadialTransstadial TransstadialTransstadial transmissiontransmission transmissiontransmission

Rickettsia spp. transmission cycle

Anaplasma spp. transmission cycle

TransstadialTr transmissionsion

Non-infectedNon-in Transstadial tick eggseg transmission

Non-infected tick larvae

Figure 7. Transmissions cycles of Anaplasma spp. and SFG Rickettsia species. After Long et al. (2003); Parola, Paddock & Raoult (2005); Socolovschi et al. (2009); Rikihisa (2011).

26 Tick-borne rickettsioses Rickettsia is a genetically diverse genus and a geographically widely distributed taxonomic group (Parola et al. 2013). It includes 25 validated Rickett- sia-species, of which 16 are acknowledged human pathogens. Several other species are yet to be described (Weiss & Dasch 1991; Fournier & Raoult 2009). The human pathogens of the genus are classically divided into two groups: the typhus group (TG) and spotted-fever group (SFG. This division is based on how their phenotypic characters, clinical features, vector association and genealogical relationships are viewed (Fournier & Raoult 2009) (Figure 6). However, it should be noted that there are discrepancies between categorizing rickettsial species by phenotypic characters and by molecular phylogenies, which is why the naming and delimitation are still debated (Roux & Raoult 2000; Parola et al. 2005; Vitorino et al. 2007; Walker 2007; Gillespie et al. 2010). Also, throughout the evolution of the Rickettsia genus, there has been frequent host switching and host associations are in general considered to be short-lived (Weinert et al. 2009). The TG rickettsiae are mainly vectored by insects, such as fleas (Xenopsylla cheopis) and lice (Pe- diculus humanus). This permits for rapid transmission of the rickettsiae since both fleas and lice are capable to feed on multiple hosts during a short time (Azad & Beard 1998). The SFG rickettsiae are mainly vectored by ixodid ticks, but are also vectored by fleas and mites. The mites and ticks allows for a slower transmission; ticks usually only feed on one host per active life stage the duration of which may last for more than a year (Raoult & Roux 1997; Azad & Beard 1998; Parola et al. 2005; Socolovschi et al. 2009).

In Europe, several Rickettsia-species are transmitted between different ixodid ticks (Oteo & Portillo 2012). The main maintenance host of the SFG rickettsiae, R. helvetica, is the common tick, I. ricinus. SFG rickettsiae are transmitted and maintained by three modes of host-vector transmission (Fig- ure 7): (1) Transovarial transmission, i.e. from the adult female to her eggs, (2) transstadial transmission, i.e. the infection is preserved between one tick life stage and the next stage. In this way, ticks act as both reservoir and vector for the bacteria. SFG rickettsiae are also maintained when (3) ticks ac- quire rickettsiae from infected small or large vertebrates, such as a rodent or possibly a cervid (Azad & Beard 1998; Parola et al. 2005; Stefanidesova et al. 2007; Schex et al. 2011). Also, birds may act as reservoirs for Rickettsia spp. (Elfving et al. 2010). Co-feeding has been suggested to play a role in the maintenance of Rickettsia spp. by various ixodid ticks (Macaluso et al. 2001; Zemtsova et al. 2010). As with Anaplasma-infections, humans are dead-end hosts and do not contribute to the maintenance of SFG rickettsiae in nature. R. helvetica is considered to be the cause of acute febrile illness, headache, myalgia, and rash following a tick bite (Fournier 2000; Parola et al. 2005; Nilsson 2009; Nilsson, Elfving & Påhlson 2010). R. helvetica has

27 also been suggested to be associated with perimyocarditis and sudden death in humans (Nilsson, Lindquist & Påhlson 1999). This species was recently also reported to be of importance as a cause of facial paralysis and sudden deafness (Nilsson et al. 2013). As with A. phagocytophilum-infections, Rick- ettsia-infections are in most countries not notifiable. Therefore, the number of people falling ill due to R. helvetica infections in e.g. Sweden each year is largely unknown.

In Sweden, in 1997 R. helvetica was the first SFG Rickettsia species to be detected (Nilsson et al. 1997). Since then, only R. helvetica and R. sibirica, the causative agent of Siberian tick typhus, in the SFG of Rickettsia have been found in I. ricinus ticks in Sweden (III; IV). Prevalence (MIR) in Swe- den ranges between 1 and 11% depending on tick stage and locality, with an overall prevalence of 4.8% for southern and central Sweden (III; IV). In a Eurasian perspective, the prevalence range of R. helvetica in I. ricinus is 0.6–47% (Socolovschi et al. 2009). As such, it follows that the abundance and distribution of I. ricinus and tick-hosts are also important for the geographical distribution of SFG rickettsiae. The abundance and geographical distribution of I. ricinus ticks in Sweden have increased and expanded during the last decade, most likely due to an increase of tick maintenance hosts and a changing climate (Jaenson et al. 2012b). With an increasing abundance of ticks, it is also likely that an increase in the prevalence of SFG rickettsiae will follow. In the future, it is likely that “novel” Rickettsia-species will become established in regions where they have not previously been found (Parola 2004). In view of the rapidly changing climate and environment, the increase in travel of people accompanied by pets, the increasing transport of livestock across country borders, and vast numbers of tick-infested birds migrating to and from Europe each year, the potential for new species of Rickettsia and other tick-borne pathogens to become established in Europe is significant (Parola et al. 2005; Elfving et al. 2010; Conraths & Mettenleiter 2011).

28 Research aims

This thesis aims to address and answer questions related to the origin of the Flavivirus genus and the ecology and tick-borne pathogens.

In paper I we test the hypothesis of a Flavivirus origin within the last 10,000 years in view of the Powassan virus-Beringian land bridge biogeography.

In paper II we attempt to estimate the prevalence of TBEV in host-seeking I. ricinus ticks in Sweden and to compare our estimates with neighbouring Scandinavian countries. We also try to explain how TBEV can be maintained in nature even though the TBEV prevalence in ticks is relatively low.

In paper III we seek to estimate the prevalence and distribution of Anaplas- ma spp. and Rickettsia spp. mainly in host-seeking I. ricinus tick larvae and nymphs in Sweden.

In paper IV we seek to estimate the prevalence and distribution of Anaplas- ma spp. and Rickettsia spp. in host-seeking adult I. ricinus ticks in Sweden.

29 Summary paper I

In paper I, we attempt to date the origin of the entire Flavivirus clade and its major groups. The deep ancestry of Flavivirus has for long been discussed. Previous studies have estimated and argued, based on single gene molecular clock extrapolations, that the Flavivirus clade originated approximately 10,000 years ago and that its major groups have been diverging since 5,000 years ago.

It is well known that dating deep divergence times in viruses is particularly difficult as viruses do not leave any fossils, which could otherwise have been used to calibrate internal nodes for more accurate dating. Virtually all virus- dating studies rely on isolation date (or tip date) calibration. However this type of calibration becomes problematic when it comes to dating deeper nodes. As the variation in substitution rate among lineages differs strongly, the age of the deep internal nodes will also be uncertain with very wide con- fidence intervals as a consequence. Recent molecular clock analysis of the tick-borne flaviviruses (TBF) clade has indicated that TBF originated more than 16,000 years ago, which suggests that the Flavivirus clade should be significantly older than 10,000 years. Also, it has been argued and shown that the emergence of the Powassan virus (POWV) into North America was linked with the time of the opening and closing of the Beringian land bridge, the landmass that connected Asia with North America. The Beringian land bridge was open to mammal land migration between 15,000–11,000 years ago until the land bridge was over-flooded and the Bering Strait was formed. Thereafter, migration from Asia to North America by mammals other than humans was strongly reduced/almost completely restricted. In view of this knowledge, and in combination with the availability of complete genomes from most Flavivirus members, we again examine the temporal origin of the genus.

The POWV-Beringian biogeographical event allowed us to calibrate the split of POWV from its sister clade of closely related TBF to have occurred between 15,000–11,000 years ago. First, we gathered complete coding genome sequences with known isolation dates of all Flavivirus members available at public databases. All sequences were aligned as amino acids to keep codon positioning while being aligned, and then back-translated to nucleotides. Phylogenetic trees were then inferred using MrBayes and RAxML. The

30 same topology was subsequently used as a starting tree in the BEAST analysis. The BEAST analysis was performed using three modes of calibration under a relaxed molecular clock and Bayesian skyline coalescence; (i) using only tip dates, (ii) using tip date and secondary internal calibration from a previous molecular clock study on tick-borne flaviviruses, and (iii) using tip dates and primary internal calibration based on Beringian biogeography of POWV.

The estimated time to most resent common ancestor (tMRCA) from the different calibration schemes used in our study were highly congruent, the main difference being the range of their 95% HPD-intervals. We show that the inclusion of internal calibration can improve dating to a high existent and therefore focus on the combined tip date and Beringian calibration analysis. Here we show that, depending on if Tamana bat virus (TABV) is to be considered to be a part of the genus or not, Flavivirus originated circa 120,000 (95% HPD 156,100–322,700) years ago including TABV or circa 85,000 (95% HPD 63,700–109,600) years ago excluding TABV (Table 2).

Table 2. Estimated tMRCA and 95% HPD intervals for Flavivirus and its major groups using tip date and biogeographical calibration. A dash denotes a split between groups.

Node tMRCA 95% HPD Root 230.5 156.1 – 322.7

TABV/ISF-MBF-TBF-NKVF 119.8 87.1 – 158.9

ISF /MBF -TBF-NKVF 84.7 63.7 – 109.6 α dom α MBF /TBF-NKVF 47.2 37.3 – 58.7 dom α

MBFdom 41.5 32.6 – 51.6 ISF 40.7 30.8 – 52.3 α TBF/NKVF 39.3 30.7 – 49.2 α TBF 27.5 21.7 – 34.2

NKVF 24.1 18.0 – 30.9 α POWV/sister clade of TBF 12.8 11.0 – 14.8

In any case, our estimates indicate that the genus is much (8 to 12 times) older than previously estimated. This has implications for how we should view the emergence of Flavivirus. Instead of a relatively recent origin, with an emergence after the last glacial maximum, we show that Flavivirus has evolved and diversified during a much longer time scale with a temporal origin coinciding with the migration and spread of humans out of Africa.

31 Summary paper II

Paper II investigates the prevalence of tick-borne encephalitis virus (TBEV) in Sweden and compares the prevalence in Sweden with that of other Scan- dinavian countries. We also hypothesise and try to explain how TBEV can be maintained in nature despite such a relatively low prevalence of TBEV in ticks.

During 2008, we collected close to 3,000 (2,074 nymphs and 906 adults) I. ricinus ticks from several localities mainly in southern Sweden. RNA was extracted and analysed for TBEV by real-time reverse-transcriptase PCR assay targeting a variable section in the non-coding 3´-region of the TBEV genome. In total, we detected TBEV in seven of our tick samples. Two nymphal pools (one from Herrhamra and one from Kolarvik) and in five adult tick specimens (one from Jönköping, three from Herrhamra, and one from Skutskär) were positive for TBEV.

TBEV prevalence was estimated as Minimum Infection Rate (MIRTBEV). For Sweden, the MIRTBEV was estimated to be approximately 0.23% (0.10% for nymphs and 0.55% for adult ticks). Thus, the TBEV prevalence is generally higher in adults than in nymphs. This is because the potential for a tick to become infected with TBEV increases with the number of blood meals con- sumed: in general, a nymph has ingested blood once whereas an adult tick has ingested blood twice. This fact was also supported by the data from Her- rhamra where the mean MIRTBEV for nymphal ticks was estimated to 0.5% and for adult ticks to 4.5%. Characterisation, by phylogenetic analysis of full genome sequences of TBEV, detected in the nymphal pools, confirmed that the TBEV-infected ticks harboured the European TBE virus subtype. The European TBE virus subtype is the only subtype detected in Sweden so far.

Compared to neighbouring Scandinavian countries, Sweden has a similar TBEV prevalence in the I. ricinus tick populations (Table 3). On average for Scandinavia, nearly 1 in every 360 ticks collected was infected with TBEV. This should be seen as a crude estimate, since the prevalence can fluctuate widely among different TBEV “hot spot areas” and areas where TBEV is present at a very low prevalence or is completely absent.

32 Table 3. Mean minimum infection rates of TBEV expressed as percentages of ticks (including all active tick life stages) collected and screened for TBEV in Scandina- via. Number of collected Number of TBEV- ticks positive ticks Collection Number of studies Pools Pools Country years and localities Total collected positive MIR %

Denmark 1999–2011 3 studies, ≥ 18 sites 6865 ≥ 70 11 0.16

Finland 1957–2008 4 studies, ≥ 27 sites 14320 2490 42 0.29

Norway 2003–2009 2 studies, 9 sites 6440 ≥ 563 26 0.40

Sweden 1958–2008 4 studies, 45 sites 11733 1510 30 0.26

Scandinavia 1958–2011 All sites (≥ 99) 39358 ≥ 4633 109 0.28

We believe that the low TBEV prevalence in I. ricinus ticks has three possible explanations that are not mutually exclusive. (i) The TBEV occurs in spatially very small foci. Therefore, the inclusion of ticks from outside of these foci is likely to bias (reduce) the estimate of the prevalence within foci where TBEV actually occurs. (ii) The potential of a tick being infected with TBEV increases with its developmental stage (fed larva

33 Summary paper III and IV

Papers III and IV are the first large-scale studies investigations on the prevalence and geographical occurrence of the gram-negative Anaplasma spp. and Rickettsia spp. bacteria in host-seeking Ixodes ricinus larvae, nymphs and adults in Sweden. A total of 2,132 I. ricinus ticks (204 larvae, 963 nymphs, 509 adult females and 456 adult males) were collected and analysed for presence of these bacteria. We used a real-time PCR, targeting the citrate synthase-encoding gltA gene in Rickettsia spp. and the 16S gene in Ana- plasma spp. Based on all active tick stages we estimated the prevalence to about 1% and 5% for A. phagocytophilum and R. helvetica, respectively (Table 4). In paper IV, we also screened for Coxiella burnetii, the causative agent of Q-fever in humans. However, C. burnetii was not detected in any of our samples.

Table 4. Mean minimum infection rate of A. phagocytophilum and R. helvetica in ticks collected in Ixodes ricinus collected in Sweden (results from paper III and IV). Larvae Nymphs Adult Adult Total males females A. phagocytoph- 0 1.7 0.2 1.1 1.1 ilum (0 / 204) (16 / 963) (1 / 427) (5 / 466) (22 / 2060)

R. helvetica 1.0 1.7 6.8 10.6 4.8 (2 / 204) (16 / 963) (31 / 456) (54 / 509) (103 / 2132)

Sequencing of the ompB and the 17-kDa antigen genes showed that the majority of all Rickettsia spp. positive ticks in Sweden are infected with R. helvetica. However, in paper IV the sequencing of one Rickettsia-positive tick specimen indicated that it was infected with a species closely related to Rickettsia species not previously detected in Sweden, i.e. R. sibirica, the etiological agent of Siberian tick typhus. It is likely that this species had been brought to Sweden by migrating birds and/or pet owners travelling with their animals across country borders. Likewise, partial sequencing of the 16S rRNA gene indicated that A. phagocytophilum is the predominant Anaplas- ma species occurring in Sweden.

A. phagocytophilum has been linked to febrile disease in dogs, horses and ruminants, and is the causative agent of human granulocytic anaplasmosis,

34 HGA. The medical importance of R. helvetica, the predominant Rickettsia species occurring in Sweden, is less well understood. However, it has recently been shown to be a pathogen of humans. In paper III and IV we show that this rickettisa is present in most areas of Sweden investigated. It is relatively prevalent in some areas of Sweden, and should therefore be considered as a possible cause of human disease following tick bites.

35 Conclusions

We are more and more moving towards an integrated approach to understand the origin, evolution and ecology of human pathogens, a One Health perspective, by taking into account knowledge and information from different fields of research, as also shown by the contribution from the present thesis and the manuscripts and papers therein:

To understand the origin and evolution of pathogens, it is fundamental to know during what temporal scale they have been evolving. We showed that the genus Flavivirus is 85,000–120,000 years old, which is significantly older than previous estimates (I).

In order to understand the relationship between pathogens, vectors and vector hosts, we need to study, analyse and explain the patterns that certain pathogens exhibit in relation to their ecology. In manuscript II we argued that the low prevalence of TBEV in ticks in Sweden and elsewhere might be due to the inclusion of ticks collected outside of the patchily distributed and spatially restricted foci by which TBEV occurs in nature.

In order to make the general public and health care officials aware of the potentially diseases-causing organisms and viruses that is transmitted around them, we need to conduct large-scale field investigation to find out where and at what prevalence a certain pathogen occurs. In manuscript and papers II, III and IV, the prevalences of three medically important pathogens, A. phagocytophilum, R. helvetica and TBEV, were estimated to occur at 1.1, 4.8 and 0.26%, respectively, as overall mean values including all active tick stages in Sweden.

In many ways, we are only just beginning to understand the evolution, ecology and diversity of pathogens that humans are or may potentially be ex- posed to. Therefore, we need to continue to conduct basic research and col- laborate across different scientific disciplines.

36 Svensk sammanfattning

Zoonoser, det vill säga infektionssjukdomar orsakade av mikroorganismer (bakterier, maskar, protozoer, svampar) och virus som kan överföras mellan andra djur och människor, upptar över 60% av alla för människan kända infektionssjukdomar. De flesta zoonoser är antingen orsakade av bakterier eller virus. Zoonoser uppkommer när människan kommer i kontakt med andra djur, vilda eller domesticerade. Ju närmare besläktade människan är med det djur som bär på en smittsam mikroorganism eller virus, desto större chans är det att samma mikroorganism eller virus skall kunna etablera sig som en zoonotisk patogen och orsaka sjukdom hos människan. Till exempel är det större risk att människan delar zoonotiska patogener med människoa- por än med andra primater. Samtidigt kan avsaknad av genetiskt släktskap kompenseras med frekvent kontakt där det finns återkommande möjligheter för zoonotiska patogener att överföras från eller till människan. Till exempel i fallet med uppkomsten av digerdöden (pesten) på medeltiden och männi- skans kontakt med gnagare, eller uppkomsten av olika influensor som beror på människans nära och frekventa kontakt med främst fåglar och grisar.

Flera zoonotiska virus och mikroorganismer överförs till människan med hjälp av en vektor, en bärare och överförare av den sjukdomsframkallande mikroorganismen eller viruset. Vektorn är oftast en blodsugande ektoparasi- tisk artropod, till exempel en fästing eller stickmygga. Hur vektorburna zoonotiska patogener uppkommer är fortfarande till stor del oklart, beroende på att så många olika faktorer är inblandade. Bland annat måste en blivande potentiell zoonotisk patogen anpassa sig till både vektorn och värdens im- munförsvar och alla fysiska barriärer som är ämnade att förhindra den att orsaka infektion. Likaså är både populationsstorlek och -densitet samt social konstruktion hos värdpopulationen viktiga för en blivande zoonotisk patogen. I till exempel en liten population, där individerna sällan har kontakt med varandra, riskerar patogenen att inte kunna spridas vidare och riskerar på så sätt dö ut. Det finns två grundläggande idéer om hur zoonotiska patogener uppkommer. Den ena är att vissa patogener har uppkommit direkt ur vektorn från vilka de sedermera har även anpassats till att överföras till och replikeras i ryggradsdjur. Den andra är att vissa patogener först uppstod hos ryggradsdjur varefter dessa togs upp och anpassade sig till en vektor.

37 Inom Flaviviridae finns släktet Flavivirus, som består av närmare 80 virusar- ter spridda över hela världen. Släktet inkluderar flera mycket viktiga humanpatogener, såsom DENV (Dengue feber-virus) och TBEV (fästingburen encefalit-virus). Inom släktet har flera olika virustransmissionsvägar utveck- lats. De flesta medlemmarna av Flavivirus överförs med hjälp av en vektor. Dessa inkluderar fästing- och myggburna flavivirus. Vissa överförs utan en vektor, dvs. de är icke-vektorburna flavivirus, och några är helt specifika till insekter, dvs. insektsspecifika flavivirus. Enligt en hypotes uppstod Flavivi- rus i Afrika från ett icke-vektorburet virus. Tidigare uppskattningar indike- rade att Flavivirus uppstod för mindre än 10 000 år sedan. I denna avhand- ling indikeras snarare att Flavivirus sannolikt uppstod för ca 100 000 år sedan och att flera flavivirus kom att spridas över stora delar av världen i samband med att den moderna människan började emigrera ut ur Afrika.

Fästingen Ixodes ricinus är Sveriges och Europas viktigaste vektor av flerta- let humanpatogener, så som Anaplasma-, Borrelia- och Rickettsia-bakterier samt TBE-viruset. Fästingens livscykel består av tre aktiva stadier: larv, nymf och adult, vilka alla biter människan. I naturen är det främst hjortdjur och gnagare som fästingen suger blod av. Hjortdjur främst fungerar som amplifikationsvärdar för fästingen, medan gnagare främst är viktiga reservoarer för olika zoonotiska virus och mikroorganismer. Om det blir ett fästin- går eller inte är beroende av flera faktorer som är kopplade mellan åren. Till exempel om det fanns gott om värddjur under föregående år, om föregående höst var mild, om det fanns ett snötäcke som isolerade mot den värsta kylan, när våren startar, hur mycket det regnar under sommaren, och så vidare. Fästingens utbredningsområde begränsas främst av förekomsten av medel- stora och stora däggdjur, framförallt rådjur, samt av klimatet och då framför allt av längden på vegetationsperioden.

En av de för människan viktigaste av alla zoonotiska patogener som I. ricinus bär på är TBEV som orsakar fästingburen encefalit, TBE. TBEV delas vanligen in i tre olika subtyper, en europeisk, en östlig och en sibirisk. Den Europeiska TBEV-subtypen hittar man främst i just Europa där den årligen orsakar ca 3000 sjukdomsfall av TBE. De östliga och sibiriska TBEV- subtyperna förekommer hela vägen från östra Europa till Japan och orsakar årligen ca 6000 sjukdomsfall av TBE. Utöver epidemiologi skiljer sig de tre subtyperna skiljer sig även en del i sjukdomsförlopp, där den europeiska subtypen har ett relativt milt förlopp jämfört med den östliga och sibiriska subtypen. Den senaste gemensamma förfadern till alla TBEV-subtyper uppstod för ca 5000 år sedan och började divergera för ca 2600 år sedan till de olika subtyper vi nu kan identifiera.

I naturen vidmaktshålls TBE-viruset genom att kontinuerligt överföras mellan fästingar och dess värddjur, där främst gnagare fungerar som TBEV-

38 reservoarer eftersom de kan förbli infekterade under långa perioder. TBEV kan överföras från fästinghonan till hennes ägg, och när väl en fästing har blivit infekterad förblir den TBEV-infekterad under hela sitt liv. Vidare kan fästingen bli infekterad genom att inta en blodmåltid från en värd som har viremi. Alternativt kan TBEV överföras från fästing till fästing när ett flertal fästingar sitter tätt tillsammans och suger blod från samma värd, där TBEV överförs när en infekterad fästing injicerar viruspartiklar i värden varefter en icke-infekterad fästing i direkt anslutning suger upp samma viruspartiklar. I Sverige har antalet sjukdomsfall av TBE bland människor tydligt ökat under det senaste decenniet till mellan 150–300 fall årligen, vilket förklaras av en ökning av antalet värddjur samt en för fästingen gynnsam förändring i klimatet. Samtidigt ligger TBEV-prevalensen på mindre än en procent jämfört över alla fästingstadier. Detta förklaras av att TBEV bara finns inom geografiskt mycket begränsade områden. Om man då mäter prevalensen genom att inkludera fästingar utanför dessa små TBEV-områden kommer man att få ett för lågt prevalensvärde.

Inom bakteriefamiljerna Anaplasmataceae och Rickettsiaceae (ordningen Rickettsiales) finns två släkten av gram-negativa intracellulära bakterier, Anaplasma och Rickettsia, vilka inkluderar flera kända och potentiella fäs- tingburna humanpatogener. Tidigare känd inom veterinärmedicinen som patogen för tamdjur, visade det sig under 1990-talet att Anaplasma phagocytophilum orsakar human granulocytär anaplasmos (HGA). Det beror på att bakterien infekterar olika typer av röda och vita blodkroppar. HGA yttrar sig med diffusa symptom såsom feber, huvudvärk, illamående och kräkningar. I Europa vidmakthålls A. phagocytophilum i naturen, såsom TBEV, genom att överföras mellan främst I. ricinus och dess värddjur, där hjortdjur anses vara de viktigaste värdarna. Dock inte genom transovariell överföring i fästingar- na. Prevalensen av A. phagocytophilum i mellersta och södra Sverige visade sig vara omkring 1%, sett över alla fästingstadier, men beroende på fästing- stadium och geografiskt område kan prevalensen i Europa ligga på mellan 0.4–34%. Antalet människor som blir sjuka med HGA i Sverige under ett år är okänt eftersom sjukdomen inte är anmälningspliktig samt för att sympto- men har likheter med de som uppträder i samband med flera andra vanliga sjukdomar.

Släktet Rickettsia inkluderar fler än tio kända humanpatogener som sprids av olika fästingar inom Ixodidae. Av dessa har R. helvetica och R. sibirica nu påvisats i I. ricinus i Sverige. Jämfört med A. phagocytophilum är symptom- atologin som orsakas av dessa Rickettsia-arter i Sverige inte lika väl kända. Men man har relaterat flera sjukdomssymptom, såsom feber, huvudvärk, hudutslag och muskelvärk, till infektion med i Sverige förekommande Rick- ettsia-art(er). Precis som för Anaplasma-infektionerna, så är antalet årliga sjukdomsfall inte känd då infektioner orsakade av de svenska Rickettsia-

39 bakterierna inte är anmälningspliktiga samt på grund av de diffusa sympto- men. Men i takt med att den kliniska erfarenheten har ökat, så har också en mer specifika symptombild kunnat beskrivas. I naturen vidmakthålls Rickett- sia-bakterierna i en fästing-värd-cykel liknande den för TBEV och brukar, till skillnad från Anaplasma-bakterierna, överföras transovariellt. Prevalens- en i fästingar i Europa varierar mellan 0.6–47%. I södra och mellersta Sve- rige har prevalensen uppskattats till ca 4.8% utslaget över alla fästingstadier. Med ett förändrat klimat, med tidigare vårar och senare höstar, samt en ök- ning av fästingens värddjur kommer vi sannolikt i framtiden att få uppleva att både fästingarna och de fästingburna infektionerna kommer att öka.

40 Acknowledgements

Throughout my PhD I have come across so many people that have helped me, motivated me, inspired me, or in any way made my life as a PhD student much easier and more rewarding. Here are a few:

Afsaneh Ahmadzadeh (Ingår i SystBios fantastiska forskaringenjörsduo. Alltid glad och positiv. Många tack för all hjälp i labbet!), Agneta Brandt- berg-Falkman (Avdelningens poet, det är svårt att inte bli på bra humör med dig omkring Agneta), Alex Nater (Good luck with your postdoc here at EBC!), Alexander Eiler (Hopefully we’ll get that 454-paper done soon..;), Allison Perrigo (Little miss multi-tasking and globetrotter. Good luck with your postdoc!), Anders Larsson (Jag är ju lite orolig nu när ”min” bioinfor- matiker inte kommer att vara inom en armlängds avstånd, blir nog tvungen att köpa en mobil åt dig..;), Anders Rydberg (Skärmflygande tidigare kontorskamrat med intresse för lingvistik), Andreas Wallberg (Numera vid BMC där han räddar Sveriges honungsbin från undergång), Anette Roth (Många tack för all hjälp i labbet med mina fästingar), Anja Rautenberg (Rysslandskännare som numera jobbar som Linné-lobbyist på Länsstyrel- sen), Ann-Sofi Tylö (Numera pensionerad, men tidigare labbmeister på avd för klinisk virologi, Sahlgrenska. Många tack för alla tips och råd i labbet!), Anna Aspán (Tack för all hjälp på SVA), Anna Kopps (Dr Dolphin, I hope to see you in Zürich soon!), Anneleen Kool (Übermamma med ett smittsamt positivt humör. Rensar numera rabatten på botan i Oslo), Astrid Schuster (Good luck with your PhD at SystBio!), Baset Ghorbani (Good luck with your postdoc at SystBio!), Björn Herrman (Många tack för all hjälp med screeningen av mina orangutangprover!), Carel van Schaik (Many thanks to you and the rest of the AIM crew for giving me the opportunity to work with the orangutan-material), Catarina Rydin (Entusiastisk och driven systemati- ker, numera vid SU), Chanda Vongsombath (Searches for effective tradit- ional botanical insect repellents), Chengjie Fu (Hard-working postdoc at SystBio that just got his position extended for a few more years. Best of luck to you and your family), Ding He (a.k.a. Professor Banana, sushi-buffé champion, I’m still waiting for an invitation to join ”the He lab” in Taiwan :), Elin Nilsson (Tack för all hjälp i labbet uppe i Umeå!), Erik Alestig (He- patit-B-forskande dygnet-runt-arbetande läkare med koll på Göteborgs lunchställen), Gemma Atkinson (Bioinformatics wizard, likes snow and darkness so much that she and the rest of the family moved to Umeå), Helge

41 Ax:son Johnsons stiftelse, Henrik Lantz (Konstant många järn i elden. Huse- rar numera på BMC), Henrik Sundberg (Svamp-gourmand och doktorand som letar efter svamp på baggar), Hugo de Boer (Inspirerande och produktiv arbetsmyra av rang, kan ”systemet”, har antagligen tillräckligt med flygmil- poäng för att bjuda hela avdelningen på en jordenrunt-flygresa i 1:a-klass), Ina Severin (Many thanks for all the help in the lab!), Inga Hedberg (Syst- Bios grand lady, arbetar på till synes oförtröttligt), Ingela Nilsson (Tack för all assistans på labbet i Umeå), Irina Golovljova (Many thanks for all the help at SMI!), Janneke van Woerden (Cave woman! Thanks for always ma- king me feel welcome when coming to Zürich. Hope I’ll see you soon!), Jenna Anderson (Previous project student that is now working to save the world [s cattle] from Bluetongue disease), Karin Lundberg (Ekonomiadmi- nistratör med koll på det mesta), Karin Steffen (a.k.a. Knas-Karin/kleine kokosnuss. It will be really quiet not having you around. You’ll be dearly missed!), Katarina Andreasen (Numera på IBG där hon bl.a. försöker göra studenter och lärare till kommunikativa underverk), Katarina Wallménius (Lycka till med doktorerandet!), Katinka Pålsson (Resofil och kattofil), Ken- neth Nilsson (Handledare och Rickettsia-fantast), Kerstin Falk (Driven och kunnig virolog på SMI), Kungliga vetenskapsakademin, Leif Tibell (Grattis till lill-knodden!), Liljewalchs och Sederholms resestipendier genom Upp- sala Universitet, Magnus Lidén (Vetenskaplig allkonstnär och upptäcktsre- sare), Magnus Lindh (Många många tack för all hjälp med mitt orangutang- projekt!), Maria Lindström (Du lämnade oss alla alldeles för fort. Saknar dig så!), Maria Romeralo (Slime molds r’ us), Maria van Noordwijk (Many thanks to you and the rest of the AIM crew for giving me the opportunity to work with the orangutan-material), Mariette Manktelow (Carl von Linné- vetare och fantast. Många tack för all hjälp med Linnékursen!), Martin Ry- berg (Schvamptaxonom och parameterguru), Mats Thulin (En levande data- bas över blommande växter, numera ”utsliten” professor), Mattias Forshage (Min första kontorskamrat som introducerade mig till livet som doktorand), Michael Krützen (Many thanks to you and the rest of the AIM crew for giving me the opportunity to work with the orangutan-material), Mikael Thollesson (Handledare och teknik-fantast uti fingerspetsarna), Nahid Hei- dari (Ingår i SystBios fantastiska forskaringenjörsduo. Stort rättvisepatos. Många tack för all hjälp i labbet!), Olle Engkvist Byggmästares stiftelse, Omar Fiz-Palacios (Mr Molecular Clock. Thanks for all the inspiring di- scussions!), Paco Cardenas (24h-under-the-microscope-looking-at-never- ending-boxes-full-of-sponges-Paco), Petra Korall (En av få som kan arbeta dygnet-runt utan [?] något centralstimulerande ;), Pravech Ajawatanawong (I will miss your thai-cooking! Good luck with your defense!), Sandra Baldauf (Head of Department who is fighting and working hard for SystBio. Strong integrity), Sanea Sheikh (Good luck with your PhD!), Sanja Savic (Grattis till lill-knodden!), Sarina Veldman (Upcoming edible orchid specialist. Good luck with your PhD! Be sure to give Hugo a run for his money), Sirkka Vene

42 (Många tack för all hjälp på SMI!), Stefan Bertilsson (Många tack för möj- ligheten att jobba i erat labb!), Stiftelsen Lars Hierta Minne, Stina Wester- strand (?... Vem skall nu bjuda på spontankladdkakor och vem skall jag nu bråka med om alla mina Illustrator-frågor? ;), Suuniva Aagaard (Go og glad jente som numera ser till att norrbaggarna följer sina CITES-åtaganden), Sven Bergström (Handledare och Borrelia-fantast, tack för all hjälp uppä i Umä), Thomas Jaenson (Sveriges främsta fästingforskare och tillika min huvudhandledare. Har fått genomlida mycket under alla dessa år. Tusen tack för allt!), Thomas Persson Vinnersten (Lycka till på ditt nya jobb! Om det sker ett utbrott av vägglöss på vår gata så vet jag vem jag skall kon- takta/skylla på ;), Tomas Bergström (Virolog som hoppas knäcka nöten om hur TBEV sprids i Sverige), Vichith Lamxay (Does very interesting research aiming to identify effective culturally important medicinal traditions), Wal- lenbergstiftelsen, Åke Lundkvist (Kunnig virolog, numera på BMC) and Åsa Kruys (Tokig träna-före-tuppen-vaknar-löpare. Dubbelfrukost. Vi ses [när jag springer om dig…;] i Slottsbacken!).

However, none of it would have been possible without the strong support from my loving and caring family. Helen, Élise, Julian, mom and dad, I love you all!

43 References

Achazi, K., Růžek, D., Donoso-Mantke, O., Schlegel, M., Ali, H.S., Wenk, M., Schmidt-Chanasit, J., Ohlmeyer, L., Rühe, F., Vor, T., Kiffner, C., Kallies, R., Ulrich, R.G. & Niedrig, M. (2011) Rodents as senti- nels for the prevalence of tick-borne encephalitis virus. Vector- Borne and Zoonotic Diseases, 11, 641–647.

Andersson, S.G., Zomorodipour, A., Andersson, J.O., Sicheritz-Pontén, T., Alsmark, U.C., Podowski, R.M., Näslund, A.K., Eriksson, A.S., Winkler, H.H. & Kurland, C.G. (1998) The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature, 396, 133–140.

Azad, A.F. & Beard, C.B. (1998) Rickettsial pathogens and their arthropod vectors. Emerging Infectious Diseases, 4, 179–186.

Blome, M.W., Cohen, A.S., Tryon, C.A., Brooks, A.S. & Russell, J. (2012) The environmental context for the origins of modern human diversity: A synthesis of regional variability in African climate 150,000– 30,000 years ago. Journal of Human Evolution, 62, 563–592.

Bromham, L. & Penny, D. (2003) The modern molecular clock. Nature Re- views Genetics, 4, 216–224.

Burkot, T.R., Maupin, G.O., Schneider, B.S., Denatale, C., Happ, C.M., Rutherford, J.S. & Zeidner, N.S. (2001) Use of a sentinel host system to study the questing behavior of Ixodes spinipalpis and its role in the transmission of Borrelia bissettii, human granulocytic ehrlichiosis, and Babesia microti. The American Journal of Tropical Medicine and Hygiene, 65, 293–299.

Carpi, G., Cagnacci, F., Wittekindt, N.E., Zhao, F., Qi, J., Tomsho, L.P., Drautz, D.I., Rizzoli, A. & Schuster, S.C. (2011) Metagenomic profile of the bacterial communities associated with Ixodes ricinus ticks. PLoS ONE, 6, e25604.

44 Charrel, R.N., Attoui, H., Butenko, A.M., Clegg, J.C., Deubel, V., Frolova, T.V., Gould, E.A., Gritsun, T.S., Heinz, F.X., Labuda, M., Lash- kevich, V.A., Loktev, V., Lundkvist, A., Lvov, D.V., Mandl, C.W., Niedrig, M., Papa, A., Petrov, V.S., Plyusnin, A., Randolph, S., Süss, J., Zlobin, V.I. & de Lamballerie, X. (2004) Tick-borne virus diseases of human interest in Europe. Clinical Microbiology and In- fection, 10, 1040–1055.

Chen, S.M., Dumler, J.S., Bakken, J.S. & Walker, D.H. (1994) Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. Journal of Clinical Microbiology, 32, 589–595.

Cleaveland, S., Laurenson, M.K. & Taylor, L.H. (2001) Diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence. Philosophical Transactions of the Royal Society B: Biological Sciences, 356, 991–999.

Coipan, E.C., Jahfari, S., Fonville, M., Maassen, C.B., van der Giessen, J., Takken, W., Takumi, K. & Sprong, H. (2013) Spatiotemporal dynamics of emerging pathogens in questing Ixodes ricinus. Frontiers in Cellular and Infection Microbiology, 3.

Conraths, F.J. & Mettenleiter, T.C. (2011) Infectious Diseases Under the Influence of Changing Environmental Factors. Progress in Parasit- ology (ed H. Mehlhorn), pp. 253–258. Springer Berlin Heidelberg, Berlin, Heidelberg.

Cook, S., Moureau, G., Kitchen, A., Gould, E.A., de Lamballerie, X., Holmes, E.C. & Harbach, R.E. (2012) Molecular evolution of the insect-specific flaviviruses. The Journal of General Virology, 93, 223– 234.

Corman, V.M., Rasche, A., Diallo, T.D., Cottontail, V.M., Stocker, A., Sou- za, B.F. d. C.D., Correa, J.I., Carneiro, A.J.B., Franke, C.R., Nagy, M., Metz, M., Knornschild, M., Kalko, E.K.V., Ghanem, S.J., Mo- rales, K.D.S., Salsamendi, E., Spinola, M., Herrler, G., Voigt, C.C., Tschapka, M., Drosten, C. & Drexler, J.F. (2013) Highly diversified coronaviruses in neotropical bats. Journal of General Virology, 94, 1984–1994.

Danielová, V. & Holubová, J. (1991) Transovarial transmission rates of tick- borne encephalitis virus in Ixodes ricinus ticks. Modern Acarology (eds F. Dusbabek & V. Bukva), pp. 7–10. SPB Academic Publish- ing, Prague, Czech Republic.

45 Davies, T.J. & Pedersen, A.B. (2008) Phylogeny and geography predict pathogen community similarity in wild primates and humans. Pro- ceedings of the Royal Society B: Biological Sciences, 275, 1695– 1701.

Dobler, G., Hufert, F., Pfeffer, M. & Essbauer, S. (2011) Tick-borne encephalitis: From microfocus to human disease. Progress in Parasitology (ed H. Mehlhorn), pp. 323–331. Springer Berlin Heidelberg, Berlin, Heidelberg.

Drexler, J.F., Gloza-Rausch, F., Glende, J., Corman, V.M., Muth, D., Goettsche, M., Seebens, A., Niedrig, M., Pfefferle, S., Yordanov, S., Zhelyazkov, L., Hermanns, U., Vallo, P., Lukashev, A., Muller, M.A., Deng, H., Herrler, G. & Drosten, C. (2010) Genomic characterization of severe acute respiratory syndrome-related Coronavirus in European bats and classification of Coronaviruses based on partial RNA-dependent RNA polymerase gene sequences. Journal of Vi- rology, 84, 11336–11349.

Drummond, A.J., Pybus, O.G., Rambaut, A., Forsberg, R. & Rodrigo, A.G. (2003) Measurably evolving populations. Trends in Ecology & Evo- lution, 18, 481–488.

Duffy, S., Shackelton, L.A. & Holmes, E.C. (2008) Rates of evolutionary change in viruses: Patterns and determinants. Nature Reviews Genet- ics, 9, 267–276.

Dumler, J.S. (2005) Anaplasma and Ehrlichia Infection. Annals of the New York Academy of Sciences, 1063, 361–373.

Dumler, J.S. (2012) The biological basis of severe outcomes in Anaplasma phagocytophilum infection. FEMS Immunology & Medical Microbi- ology, 64, 13–20.

Dumler, J.S., Barbet, A.F., Bekker, C., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y. & Rurangirwa, F.R. (2001) Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickett- sia, descriptions of six new species combinations and designation of Ehrlichia equi and “HGE agent” as subjective synonyms of Ehr- lichia phagocytophila. International Journal of Systematic and Evo- lutionary Microbiology, 51, 2145–2165.

46 Dumler, J.S., Choi, K.-S., Garcia-Garcia, J.C., Barat, N.S., Scorpio, D.G., Garyu, J.W., Grab, D.J. & Bakken, J.S. (2005) Human Granulocytic Anaplasmosis and Anaplasma phagocytophilum. Emerging Infec- tious Diseases, 11, 1828–1834.

Dumler, J.S., Madigan, J.E., Pusterla, N. & Bakken, J.S. (2007) Ehrlichioses in humans: Epidemiology, clinical presentation, diagnosis, and treatment. Clinical Infectious Diseases, 45 Suppl 1, S45–51.

Dunham, E.J. & Holmes, E.C. (2007) Inferring the timescale of Dengue virus evolution under realistic models of DNA substitution. Journal of Molecular Evolution, 64, 656–661.

Dunning Hotopp, J.C., Lin, M., Madupu, R., Crabtree, J., Angiuoli, S.V., Eisen, J., Seshadri, R., Ren, Q., Wu, M., Utterback, T.R., Smith, S., Lewis, M., Khouri, H., Zhang, C., Niu, H., Lin, Q., Ohashi, N., Zhi, N., Nelson, W., Brinkac, L.M., Dodson, R.J., Rosovitz, M.J., Sundaram, J., Daugherty, S.C., Davidsen, T., Durkin, A.S., Gwinn, M., Haft, D.H., Selengut, J.D., Sullivan, S.A., Zafar, N., Zhou, L., Benahmed, F., Forberger, H., Halpin, R., Mulligan, S., Robinson, J., White, O., Rikihisa, Y. & Tettelin, H. (2006) Comparative Ge- nomics of Emerging Human Ehrlichiosis Agents. PLoS Genetics, 2, e21.

Ecker, M., Allison, S.L., Meixner, T. & Heinz, F.X. (1999) Sequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and Asia. The Journal of General Virology, 80 (Pt 1), 179– 185.

Edman, J.D. (2003) Arthropod transmission of vetebrate parasites. Medical entomology: A textbook on public health and veterinary problems caused by arthropods, Rev. ed (eds B.F. Eldridge & J.D. Edman), pp. 151–163. Kluwer Academic Publishers, Dordrecht ; Boston.

Elfving, K., Olsen, B., Bergström, S., Waldenström, J., Lundkvist, Å., Sjöstedt, A., Mejlon, H. & Nilsson, K. (2010) Dissemination of spotted fever Rickettsia agents in Europe by migrating birds. PLoS ONE, 5, e8572.

Emerman, M. & Malik, H.S. (2010) Paleovirology–Modern Consequences of Ancient Viruses. PLoS Biology, 8, e1000301.

Foggie, A. (1951) Studies on the infectious agent of tick-borne fever in sheep. The Journal of Pathology and Bacteriology, 63, 1–15.

47 Foley, J., Nieto, N.C., Foley, P. & Teglas, M.B. (2008) Co-phylogenetic analysis of Anaplasma phagocytophilum and its vectors, Ixodes spp. ticks. Experimental and Applied Acarology, 45, 155–170.

Fournier, P.-E. (2000) Evidence of Infection in Humans with Rickettsia helvetica in Eastern France. Emerging Infectious Diseases, 6, 389–392.

Fournier, P.-E. & Raoult, D. (2009) Current Knowledge on Phylogeny and Taxonomy of Rickettsia spp. Annals of the New York Academy of Sciences, 1166, 1–11.

Fowler, Å., Forsman, L., Eriksson, M. & Wickström, R. (2013) Tick-borne encephalitis carries a high risk of incomplete recovery in children. The Journal of Pediatrics, 163, 555–560.

Gao, F., Bailes, E., Robertson, D.L., Chen, Y., Rodenburg, C.M., Michael, S.F., Cummins, L.B., Arthur, L.O., Peeters, M., Shaw, G.M., Sharp, P.M. & Hahn, B.H. (1999) Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature, 397, 436–441.

Garmendia, A.E., Van Kruiningen, H.J. & French, R.A. (2001) The West Nile virus: Its recent emergence in North America. Microbes and In- fection, 3, 223–229.

Gaunt, M.W., Sall, A.A., de Lamballerie, X., Falconar, A.K., Dzhivanian, T.I. & Gould, E.A. (2001) Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography. The Journal of General Virology, 82, 1867–1876.

Gilbert, C. & Feschotte, C. (2010) Genomic fossils calibrate the long-term evolution of Hepadnaviruses. PLoS Biology, 8, e1000495.

Gillespie, J.J., Brayton, K.A., Williams, K.P., Quevedo Diaz, M.A., Brown, W.C., Azad, A.F. & Sobral, B.W. (2010) Phylogenomics Reveals a Diverse Rickettsiales Type IV Secretion System. Infection and Im- munity, 78, 1809–1823.

Gould, E.A., de Lamballerie, X., Zanotto, P.M. & Holmes, E.C. (2001) Evo- lution, epidemiology, and dispersal of flaviviruses revealed by molecular phylogenies. Advances in Virus Research, 57, 71–103.

Gould, E.A., de Lamballerie, X., Zanotto, P.M. & Holmes, E.C. (2003) Ori- gins, evolution, and vector/host coadaptations within the genus Fla- vivirus. Advances in Virus Research, 59, 277–314.

48 Gould, E.A. & Solomon, T. (2008) Pathogenic flaviviruses. Lancet, 371, 500–509.

Granström, M. (1997) Tick-borne zoonoses in Europe. Clinical Microbiolo- gy and Infection, 3, 156–169.

Grard, G., Moureau, G., Charrel, R.N., Holmes, E.C., Gould, E.A. & de Lamballerie, X. (2010) Genomics and evolution of Aedes-borne flaviviruses. The Journal of General Virology, 91, 87–94.

Grard, G., Moureau, G., Charrel, R.N., Lemasson, J.-J., Gonzalez, J.-P., Gal- lian, P., Gritsun, T.S., Holmes, E.C., Gould, E.A. & de Lamballerie, X. (2007) Genetic characterization of tick-borne flaviviruses: New insights into evolution, pathogenetic determinants and taxonomy. Virology, 361, 80–92.

Gray, J.S. (1991) The development and seasonal activity of the tick Ixodes ricinus: A vector of Lyme borreliosis. Review of Medical and Veter- inary Entomology, 79, 323–333.

Gritsun, T.S., Lashkevich, V.A. & Gould, E.A. (2003a) Tick-borne encephalitis. Antiviral Research, 57, 129–146.

Gritsun, T.S., Nuttall, P.A. & Gould, E.A. (2003b) Tick-borne flaviviruses. Advances in Virus Research, 61, 317–371.

Grubbauer, H.M., Dornbusch, H.J., Spork, D., Zobel, G., Trop, M. & Zenz, W. (1992) Tick-borne encephalitis in a 3-month-old child. European Journal of Pediatrics, 151, 743–744.

Gubler, D., Kuno, G. & Markoff, L. (2007) Flaviviruses. Fields Virology 5th ed vol 1, 5th ed (eds D. Knipe, P. Howley, D. Griffin, R. Lamb, M. Martin, B. Roizman & S. Straus), pp. 1153–1253. Lippincott-Raven Publishers, Philadelphia, PA.

Hahn, B.H. (2000) AIDS as a zoonosis: Scientific and public health implications. Science, 287, 607–614.

Hansson, M.E.A., Örvell, C., Engman, M.-L., Wide, K., Lindquist, L., Lide- felt, K.-J. & Sundin, M. (2011) Tick-borne encephalitis in child- hood: Rare or missed? The Pediatric Infectious Disease Journal, 30, 355–357.

49 Hartelt, K., Oehme, R., Frank, H., Brockmann, S.O., Hassler, D. & Kimmig, P. (2004) Pathogens and symbionts in ticks: prevalence of Anaplas- ma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and Babesia sp. in Southern Germany. International Journal of Medical Microbiology Supplements, 293, 86–92.

HavlíKová, S., LičKová, M. & Klempa, B. (2013) Non-viraemic transmission of tick-borne viruses. Acta Virologica, 57, 123–129.

Hendrick, J., Moore, W. & Morison, G.D. (1938) Activity of the Sheep tick. Nature, 141, 648–648.

Henn, B.M., Cavalli-Sforza, L.L. & Feldman, M.W. (2012) The great human expansion. Proceedings of the National Academy of Sciences, 109, 17758–17764.

Hildebrandt, A., Schmidt, K.H., Wilske, B., Dorn, W., Straube, E. & Fin- gerle, V. (2003) Prevalence of Four Species of Borrelia burgdorferi Sensu Lato and Coinfection with Anaplasma phagocytophila in Ix- odes ricinus Ticks in Central Germany. European Journal of Clini- cal Microbiology & Infectious Diseases, 22, 364–367.

Hirsch, V.M., Olmsted, R.A., Murphey-Corb, M., Purcell, R.H. & Johnson, P.R. (1989) An African primate lentivirus (SIVsm) closely related to HIV-2. Nature, 339, 389–392.

Ho, S.Y.W. & Phillips, M.J. (2009) Accounting for calibration uncertainty in phylogenetic estimation of evolutionary divergence times. Systemat- ic Biology, 58, 367–380.

Holmes, E.C. (2003) Molecular clocks and the puzzle of RNA virus origins. Journal of Virology, 77, 3893–3897.

Holmes, E.C. (2009) The Evolution and Emergence of RNA Viruses. Oxford University Press, Oxford; New York.

Holmgren, E.B. & Forsgren, M. (1990) Epidemiology of tick-borne encephalitis in Sweden 1956-1989: A study of 1116 cases. Scandina- vian Journal of Infectious Diseases, 22, 287–295.

50 Iff, T., Meier, R., Olah, E., Schneider, J.F.L., Tibussek, D. & Berger, C. (2005) Tick-borne meningo-encephalitis in a 6-week-old infant. Eu- ropean Journal of Pediatrics, 164, 787–788.

International Committee on Taxonomy of Viruses. (2012) Virus Taxonomy: 2012 Release.

Jääskeläinen, A.E., Tikkakoski, T., Nathalie Y. Uzcategui, Alekseev, A.N., Vaheri, A. & Vapalahti, O. (2006) Siberian subtype tick-borne encephalitis virus, Finland. Emerging Infectious Diseases, 12, 1568– 1571.

Jääskeläinen, A.E., Tonteri, E., Sironen, T., Pakarinen, L., Vaheri, A. & Vapalahti, O. (2011) European subtype tick-borne encephalitis virus in Ixodes persulcatus ticks. Emerging Infectious Diseases, 17, 323– 325.

Jaenson, T.G., Hjertqvist, M., Bergstrom, T. & Lundkvist, A. (2012a) Why is tick-borne encephalitis increasing? A review of the key factors causing the increasing incidence of human TBE in Sweden. Para- sites & Vectors, 5, 184.

Jaenson, T.G.T., Jaenson, D.G.E., Eisen, L., Petersson, E. & Lindgren, E. (2012b) Changes in the geographical distribution and abundance of the tick Ixodes ricinus during the past 30 years in Sweden. Parasites & Vectors, 5, 8.

Jaenson, T.G.T. & Lindgren, E. (2011) The range of Ixodes ricinus and the risk of contracting Lyme borreliosis will increase northwards when the vegetation period becomes longer. Ticks and Tick-borne Diseas- es, 2, 44–49.

Jaenson, T.G., Tälleklint, L., Lundqvist, L., Olsen, B., Chirico, J. & Mejlon, H. (1994) Geographical distribution, host associations, and vector roles of ticks (Acari: Ixodidae, Argasidae) in Sweden. Journal of Medical Entomology, 31, 240–256.

Kaiser, R. (1999) The clinical and epidemiological profile of tick-borne encephalitis in southern Germany 1994-98: A prospective study of 656 patients. Brain, 122, 2067–2078.

Kaiser, R. (2008) Tick-borne encephalitis. Infectious Disease Clinics of North America, 22, 561–575.

51 Katzourakis, A. (2013) Paleovirology: Inferring viral evolution from host genome sequence data. Philosophical Transactions of the Royal So- ciety B: Biological Sciences, 368, 20120493–20120493.

Kim, S.-Y., Yun, S.-M., Han, M.G., Lee, I.Y., Lee, N.Y., Jeong, Y.E., Lee, B.C. & Ju, Y.R. (2008) Isolation of tick-borne encephalitis viruses from wild rodents, South Korea. Vector-Borne and Zoonotic Diseas- es, 8, 7–14.

Knap, N., Durmiši, E., Saksida, A., Korva, M., Petrovec, M. & Avšič- Županc, T. (2009) Influence of climatic factors on dynamics of questing Ixodes ricinus ticks in Slovenia. Veterinary Parasitology, 164, 275–281.

Ko, S., Kang, J.-G., Kim, S.Y., Kim, H.-C., Klein, T.A., Chong, S.-T., Sames, W.J., Yun, S.-M., Ju, Y.-R. & Chae, J.-S. (2010) Prevalence of tick-borne encephalitis virus in ticks from southern Korea. Jour- nal of Veterinary Science, 11, 197.

Kocan, K.M. & de la Fuente, J. (2003) Co-feeding studies of ticks infected with Anaplasma marginale. Veterinary Parasitology, 112, 295–305.

Korber, B. (2000) Timing the Ancestor of the HIV-1 Pandemic Strains. Sci- ence, 288, 1789–1796.

Korenberg, E.I. (1994) Comparative ecology and epidemiology of lyme disease and tick-borne encephalitis in the former Soviet Union. Para- sitology Today (Personal ed.), 10, 157–160.

Korenberg, E.I., Gorban’, L.Y., Kovalevskii, Y.V., Frizen, V.I. & Kara- vanov, A.S. (2001) Risk for human tick-borne encephalitis, borrelio- ses, and double infection in the Pre-Ural region of Russia. Emerging Infectious Diseases, 7, 459–462.

Kovalev, S.Y., Chernykh, D.N., Kokorev, V.S., Snitkovskaya, T.E. & Rom- anenko, V.V. (2009) Origin and distribution of tick-borne encephalitis virus strains of the Siberian subtype in the Middle Urals, the north-west of Russia and the Baltic countries. The Journal of Gen- eral Virology, 90, 2884–2892.

Kovalev, S.Y., Kokorev, V.S. & Belyaeva, I.V. (2010) Distribution of Far- Eastern tick-borne encephalitis virus subtype strains in the former Soviet Union. Journal of General Virology, 91, 2941–2946.

52 Kruse, H., Kirkemo, A.-M. & Handeland, K. (2004) Wildlife as source of zoonotic infections. Emerging Infectious Diseases, 10, 2067–2072.

Kuno, G. (2007) Host range specificity of flaviviruses: Correlation with in vitro replication. Journal of Medical Entomology, 44, 93–101.

Kuno, G. & Chang, G.-J.J. (2005) Biological transmission of arboviruses: Reexamination of and new insights into components, mechanisms, and unique traits as well as their evolutionary trends. Clinical Mi- crobiology Reviews, 18, 608–637.

Labuda, M., Jones, L.D., Williams, T., Danielova, V. & Nuttall, P.A. (1993) Efficient transmission of tick-borne encephalitis virus between cofeeding ticks. Journal of Medical Entomology, 30, 295–299.

Labuda, M., Kozuch, O., Zuffová, E., Elecková, E., Hails, R.S. & Nuttall, P.A. (1997) Tick-borne encephalitis virus transmission between ticks cofeeding on specific immune natural rodent hosts. Virology, 235, 138–143.

LaDeau, S.L., Kilpatrick, A.M. & Marra, P.P. (2007) West Nile virus emergence and large-scale declines of North American bird populations. Nature, 447, 710–713.

Lindenbach, B.D., Thiel, H.-J. & Rice, C.M. (2007) Flaviviridae: the viruses and their replication. Fields Virology 5th ed vol 1, 5th ed (eds D. Knipe, P. Howley, D. Griffin, R. Lamb, M. Martin, B. Roizman & S. Straus), pp. 1101–1152. Lippincott-Raven Publishers, Philadelphia, PA.

Lindgren, E., Tälleklint, L. & Polfeldt, T. (1999) Impact of climatic change on the northern latitude limit and population density of the disease- transmitting European tick Ixodes ricinus. Environmental Health Perspectives, 108, 119–123.

Lindquist, L. & Vapalahti, O. (2008) Tick-borne encephalitis. Lancet, 371, 1861–1871.

Lindström, A. & Jaenson, T.G.T. (2003) Distribution of the common tick, Ixodes ricinus (Acari: Ixodidae), in different vegetation types in southern Sweden. Journal of Medical Entomology, 40, 375–378.

53 Long, S.W., Zhang, X., Zhang, J., Ruble, R.P., Teel, P. & Yu, X.-J. (2003) Evaluation of transovarial transmission and transmissibility of Ehr- lichia chaffeensis (Rickettsiales: Anaplasmataceae) in Amblyomma americanum (Acari: Ixodidae). Journal of Medical Entomology, 40, 1000–1004.

Lovisolo, O., Hull, R. & Rösler, O. (2003) Coevolution of viruses with hosts and vectors and possible paleontology. Advances in Virus Research, 62, 325–379.

Lundkvist, Å., Vene, S., Golovljova, I., Mavtchoutko, V., Forsgren, M., Kalnina, V. & Plyusnin, A. (2001) Characterization of tick-borne encephalitis virus from Latvia: Evidence for co-circulation of three distinct subtypes. Journal of Medical Virology, 65, 730–735.

Macaluso, K.R., Sonenshine, D.E., Ceraul, S.M. & Azad, A.F. (2001) Infec- tion and Transovarial Transmission of Rickettsiae in Dermacentor variabilis Ticks Acquired by Artificial Feeding. Vector-Borne and Zoonotic Diseases, 1, 45–53.

Macaulay, V. (2005) Single, rapid coastal settlement of Asia revealed by analysis of complete mitochondrial genomes. Science, 308, 1034– 1036.

Mackenzie, J.S., Gubler, D.J. & Petersen, L.R. (2004) Emerging flaviviruses: The spread and resurgence of Japanese encephalitis, West nile and Dengue viruses. Nature Medicine, 10, S98–109.

Mansfield, K.L., Johnson, N., Phipps, L.P., Stephenson, J.R., Fooks, A.R. & Solomon, T. (2009) Tick-borne encephalitis virus - a review of an emerging zoonosis. The Journal of General Virology, 90, 1781– 1794.

Materna, J., Daniel, M. & Danielová, V. (2005) Altitudinal distribution limit of the tick Ixodes ricinus shifted considerably towards higher altitudes in central Europe: Results of three years monitoring in the Krkonose Mts. (Czech Republic). Central European Journal of Pub- lic Health, 13, 24–28.

May, F.J., Davis, C.T., Tesh, R.B. & Barrett, A.D.T. (2011) Phylogeography of West Nile virus: from the cradle of evolution in Africa to Eurasia, Australia, and the Americas. Journal of Virology, 85, 2964–2974.

54 Medlock, J.M., Hansford, K.M., Bormane, A., Derdakova, M., Estrada-Peña, A., George, J.-C., Golovljova, I., Jaenson, T.G.T., Jensen, J.-K., Jen- sen, P.M., Kazimirova, M., Oteo, J.A., Papa, A., Pfister, K., Plantard, O., Randolph, S.E., Rizzoli, A., Santos-Silva, M.M., Sprong, H., Vial, L., Hendrickx, G., Zeller, H. & Van Bortel, W. (2013) Driving forces for changes in geographical distribution of Ix- odes ricinus ticks in Europe. Parasites & Vectors, 6, 1.

Mejlon, H.A. & Jaenson, T.G. (1993) Seasonal prevalence of Borrelia burgdorferi in Ixodes ricinus in different vegetation types in Swe- den. Scandinavian Journal of Infectious Diseases, 25, 449–456.

Mickiene, A., Laiskonis, A., Günther, G., Vene, S., Lundkvist, Å. & Lind- quist, L. (2002) Tickborne encephalitis in an area of high endemicity in Lithuania: Disease severity and long-term prognosis. Clinical In- fectious Diseases, 35, 650–658.

Morse, S.S. (1995) Factors in the emergence of infectious diseases. Emerg- ing Infectious Diseases, 1, 7–15.

Nicholas Komar. (2003) West Nile Virus: Epidemiology and ecology in North America. Advances in Virus Research pp. 185–234. Elsevier.

Nicholson, M.C. & Mather, T.N. (1996) Methods for evaluating lyme disease risks using geographic information systems and geospatial analysis. Journal of Medical Entomology, 33, 711–720.

Nilsson, A. (1988) Seasonal occurrence of Ixodes ricinus (Acari) in vegetation and on small mammals in southern Sweden. Ecography, 11, 161–165.

Nilsson, K. (2009) Septicaemia with Rickettsia helvetica in a patient with acute febrile illness, rash and myasthenia. Journal of Infection, 58, 79–82.

Nilsson, K., Elfving, K. & Påhlson, C. (2010) Rickettsia helvetica in Patient with Meningitis, Sweden, 2006. Emerging Infectious Diseases, 16, 490–492.

Nilsson, K., Jaenson, T.G., Uhnoo, I., Lindquist, O., Pettersson, B., Uhlén, M., Friman, G. & Påhlson, C. (1997) Characterization of a spotted fever group Rickettsia from Ixodes ricinus ticks in Sweden. Journal of Clinical Microbiology, 35, 243–247.

55 Nilsson, K., Lindquist, O. & Påhlson, C. (1999) Association of Rickettsia helvetica with chronic perimyocarditis in sudden cardiac death. The Lancet, 354, 1169–1173.

Nilsson, K., Wallménius, K., Hartwig, S., Norlander, T. & Påhlson, C. (2013) Bell’s palsy and sudden deafness associated with Rickettsia spp. infection in Sweden. A retrospective and prospective serologi- cal survey including PCR findings. European Journal of Neurology, Doi: 10.1111/ene.12218. Ahead of print.

Nunn, C.L. (2006) Infectious Diseases in Primates: Behavior, Ecology and Evolution. Oxford University Press, Oxford; New York.

Nuttall, P.A. & Labuda, M. (2003) Dynamics of infection in tick vectors and at the tick-host interface. Advances in Virus Research, 60, 233–272.

Ohashi, N., Inayoshi, M., Kitamura, K., Kawamori, F., Kawaguchi, D., Nishimura, Y., Naitou, H., Hiroi, M. & Masuzawa, T. (2005) Ana- plasma phagocytophilum-infected ticks, Japan. Emerging Infectious Diseases, 11, 1780–1783.

Olsen, B. (2006) Global patterns of influenza A virus in wild birds. Science, 312, 384–388.

Oteo, J.A. & Portillo, A. (2012) Tick-borne rickettsioses in Europe. Ticks and Tick-borne Diseases, 3, 271–278.

Paduraru, O.A., Buffet, J.P., Cote, M., Bonnet, S., Moutailler, S., Paduraru, V., Femenia, F., Eloit, M., Savuta, G. & Vayssier-Taussat, M. (2012) Zoonotic transmission of pathogens by Ixodes ricinus ticks, Romania. Emerging Infectious Diseases, 18, 2089–2090.

Palmer, S.R., Soulsby, E.J.L. & Simpson, D.I.H. (1998) Zoonoses: Biology, Clinical Practice, and Public Health Control. Oxford University Press, Oxford; New York.

Parola, P. (2004) Tick-borne rickettsial diseases: Emerging risks in Europe. Comparative Immunology, Microbiology and Infectious Diseases, 27, 297–304.

Parola, P., Paddock, C.D. & Raoult, D. (2005) Tick-borne rickettsioses around the world: Emerging diseases challenging old concepts. Clin- ical Microbiology Reviews, 18, 719–756.

56 Parola, P., Paddock, C.D., Socolovschi, C., Labruna, M.B., Mediannikov, O., Kernif, T., Abdad, M.Y., Stenos, J., Bitam, I., Fournier, P.-E. & Raoult, D. (2013) Update on tick-borne rickettsioses around the world: A geographic approach. Clinical Microbiology Reviews, 26, 657–702.

Parrish, C.R., Holmes, E.C., Morens, D.M., Park, E.-C., Burke, D.S., Calish- er, C.H., Laughlin, C.A., Saif, L.J. & Daszak, P. (2008) Cross- species virus transmission and the emergence of new epidemic diseases. Microbiology and Molecular Biology Reviews, 72, 457–470.

Patel, M.R., Emerman, M. & Malik, H.S. (2011) Paleovirology–Ghosts and gifts of viruses past. Current Opinion in Virology, 1, 304–309.

Pedersen, A.B., Altizer, S., Poss, M., Cunningham, A.A. & Nunn, C.L. (2005) Patterns of host specificity and transmission among parasites of wild primates. International Journal for Parasitology, 35, 647– 657.

Pluta, S., Hartelt, K., Oehme, R., Kimmig, P. & Mackenstedt, U. (2011) What else besides TBE and borreliosis? Tick-transmitted pathogens in Germany and beyond. Progress in Parasitology (ed H. Mehlhorn), pp. 297–322. Springer Berlin Heidelberg, Berlin, Hei- delberg.

Porterfield, J.S. (1980) Antigenic characteristics and classification of To- gaviridae. Schlesinger, RW. (Ed.), The Togaviruses: Biology, Struc- ture, Replication pp. 13–46. Academic Press, New York.

Randolph, S.E., Gern, L. & Nuttall, P.A. (1996) Co-feeding ticks: Epidemio- logical significance for tick-borne pathogen transmission. Parasitol- ogy today (Personal ed.), 12, 472–479.

Randolph, S.E., Green, R.M., Hoodless, A.N. & Peacey, M.F. (2002) An empirical quantitative framework for the seasonal population dynamics of the tick Ixodes ricinus. International Journal for Parasit- ology, 32, 979–989.

Randolph, S.E., Green, R.M., Peacey, M.F. & Rogers, D.J. (2000) Seasonal synchrony: The key to tick-borne encephalitis foci identified by sat- ellite data. Parasitology, 121 (Pt 1), 15–23.

Raoult, D. & Roux, V. (1997) Rickettsioses as paradigms of new or emerging infectious diseases. Clinical Microbiology Reviews, 10, 694– 719.

57 Rasmussen, M., Guo, X., Wang, Y., Lohmueller, K.E., Rasmussen, S., Al- brechtsen, A., et al. (2011) An Aboriginal Australian genome reveals separate human dispersals into Asia. Science, 334, 94–98.

Ribeiro, J.M. (1995) Blood-feeding arthropods: Live syringes or invertebrate pharmacologists? Infectious Agents and Disease, 4, 143–152.

Rikihisa, Y. (2010) Anaplasma phagocytophilum and Ehrlichia chaffeensis: Subversive manipulators of host cells. Nature Reviews Microbiolo- gy, 8, 328–339.

Rikihisa, Y. (2011) Mechanisms of obligatory intracellular infection with Anaplasma phagocytophilum. Clinical Microbiology Reviews, 24, 469–489.

Roux, V. & Raoult, D. (2000) Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer-membrane protein rOmpB (ompB). International Journal of Systematic and Evolution- ary Microbiology, 50 Pt 4, 1449–1455.

Sanjuán, R. (2012) From molecular genetics to phylodynamics: Evolutionary relevance of mutation rates across viruses (ed B Sherry). PLoS Pathogens, 8, e1002685.

Santiago, M.L., Range, F., Keele, B.F., Li, Y., Bailes, E., Bibollet-Ruche, F., Fruteau, C., Noe, R., Peeters, M., Brookfield, J.F.Y., Shaw, G.M., Sharp, P.M. & Hahn, B.H. (2005) Simian immunodeficiency virus infection in free-ranging sooty mangabeys (Cercocebus atys atys) from the Tai Forest, Cote d’Ivoire: Implications for the origin of epidemic human immunodeficiency virus type 2. Journal of Virology, 79, 12515–12527.

Schex, S., Dobler, G., Riehm, J., Müller, J. & Essbauer, S. (2011) Rickettsia spp. in Wild Small Mammals in Lower Bavaria, South-Eastern Germany. Vector-Borne and Zoonotic Diseases, 11, 493–502.

Socolovschi, C., Mediannikov, O., Raoult, D. & Parola, P. (2009) The relationship between spotted fever group Rickettsiae and Ixodid ticks. Veterinary Research, 40, 34.

Stefanidesova, K., Kocianova, E., Boldis, V., Kostanova, Z., Kanka, P., Nemethova, D. & Spitalska, E. (2007) Evidence of Anaplasma phagocytophilum and Rickettsia helvetica infection in free-ranging ungulates in central Slovakia. European Journal of Wildlife Re- search, 54, 519–524.

58 Stuen, S., Granquist, E.G. & Silaghi, C. (2013) Anaplasma phagocytophilum—a widespread multi-host pathogen with highly adaptive strate- gies. Frontiers in Cellular and Infection Microbiology, 3.

Süss, J. (2011) Tick-borne encephalitis 2010: Epidemiology, risk areas, and virus strains in Europe and Asia-an overview. Ticks and Tick-borne Diseases, 2, 2–15.

Tälleklint, L. & Jaenson, T.G. (1996) Seasonal variations in density of questing Ixodes ricinus (Acari: Ixodidae) nymphs and prevalence of infection with B. burgdorferi s.l. in south central Sweden. Journal of Medical Entomology, 33, 592–597.

Tälleklint, L. & Jaenson, T.G. (1997) Infestation of mammals by Ixodes ricinus ticks (Acari: Ixodidae) in south-central Sweden. Experi- mental and Applied Acarology, 21, 755–771.

Tamura, A., Ohashi, N., Urakami, H. & Miyamura, S. (1995) Classification of Rickettsia tsutsugamushi in a New Genus, Orientia gen. nov., as Orientia tsutsugamushi comb. nov. International Journal of System- atic Bacteriology, 45, 589–591.

Taylor, L.H., Latham, S.M. & Woolhouse, M.E.J. (2001) Risk factors for human disease emergence. Philosophical Transactions of the Royal Society B: Biological Sciences, 356, 983–989.

Tonteri, E., Jääskeläinen, A.E., Tikkakoski, T., Voutilainen, L., Niemimaa, J., Henttonen, H., Vaheri, A. & Vapalahti, O. (2011) Tick-borne encephalitis virus in wild rodents in winter, Finland, 2008–2009. Emerging Infectious Diseases, 17, 72–75.

Twiddy, S.S., Holmes, E.C. & Rambaut, A. (2003) Inferring the rate and time-scale of Dengue virus evolution. Molecular Biology and Evolu- tion, 20, 122–129.

Vitorino, L., Chelo, I.M., Bacellar, F. & Ze-Ze, L. (2007) Rickettsiae phylogeny: A multigenic approach. Microbiology, 153, 160–168.

Walker, D.H. (2007) Rickettsiae and Rickettsial Infections: The Current State of Knowledge. Clinical Infectious Diseases, 45, S39–S44.

Weinert, L.A., Werren, J.H., Aebi, A., Stone, G.N. & Jiggins, F.M. (2009) Evolution and diversity of Rickettsia bacteria. BMC Biology, 7, 6.

59 Weiss, R.A. (2001) The Leeuwenhoek Lecture 2001. Animal origins of human infectious disease. Philosophical Transactions of the Royal So- ciety B: Biological Sciences, 356, 957–977.

Weiss, E. & Dasch, G.A. (1991) Introduction to Rickettsiales and other para- sitic or mutualistic prokaryptes. The Prokaryotes, 2nd ed (eds A. Balows, H.G. Trüper, M. Dworking, W. Harder & K.H. Schleifer), pp. 2402–2406. Springer, New York.

WHO. (1959) Joint WHO/FAO Expert Committee on Zoonoses: Second Report. World Health Organization, Geneva; Switzerland.

Wolfe, N.D., Dunavan, C.P. & Diamond, J. (2007) Origins of major human infectious diseases. Nature, 447, 279–283.

Woolhouse, M.E.J. (2001) Population biology of multihost pathogens. Sci- ence, 292, 1109–1112.

Woolhouse, M.E.J., Haydon, D.T. & Antia, R. (2005) Emerging pathogens: The epidemiology and evolution of species jumps. Trends in Ecolo- gy & Evolution, 20, 238–244.

Worobey, M., Telfer, P., Souquiere, S., Hunter, M., Coleman, C.A., Metz- ger, M.J., Reed, P., Makuwa, M., Hearn, G., Honarvar, S., Roques, P., Apetrei, C., Kazanji, M. & Marx, P.A. (2010) Island biogeography reveals the deep history of SIV. Science, 329, 1487–1487.

Yun, S.-M., Kim, S.-Y., Han, M.G., Jeong, Y.E., Yong, T.-S., Lee, C.-H. & Ju, Y.R. (2009) Analysis of the envelope (E) protein gene of tick- borne encephalitis viruses isolated in South Korea. Vector-Borne and Zoonotic Diseases, 9, 287–293.

Zanotto, P.M., Gould, E.A., Gao, G.F., Harvey, P.H. & Holmes, E.C. (1996) Population dynamics of flaviviruses revealed by molecular phylogenies. Proceedings of the National Academy of Sciences of the United States of America, 93, 548–553.

Zemtsova, G., Killmaster, L.F., Mumcuoglu, K.Y. & Levin, M.L. (2010) Co- feeding as a route for transmission of Rickettsia conorii israelensis between Rhipicephalus sanguineus ticks. Experimental and Applied Acarology, 52, 383–392.

Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1100 Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science and Technology, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology.

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