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Emerging Microbes & Infections
ISSN: (Print) 2222-1751 (Online) Journal homepage: https://www.tandfonline.com/loi/temi20
Recent establishment of tick-borne encephalitis
foci with distinct viral lineages in the Helsinki area,
Finland
Teemu Smura, Elina Tonteri, Anu Jääskeläinen, Gabriel von Troil, Suvi
Kuivanen, Otso Huitu, Lauri Kareinen, Joni Uusitalo, Ruut Uusitalo, Tuula
Hannila-Handelberg, Liina Voutilainen, Simo Nikkari, Tarja Sironen, Jussi
Sane, Janne Castrén & Olli Vapalahti
To cite this article: Teemu Smura, Elina Tonteri, Anu Jääskeläinen, Gabriel von Troil, Suvi
Kuivanen, Otso Huitu, Lauri Kareinen, Joni Uusitalo, Ruut Uusitalo, Tuula Hannila-Handelberg,
Liina Voutilainen, Simo Nikkari, Tarja Sironen, Jussi Sane, Janne Castrén & Olli Vapalahti (2019)
Recent establishment of tick-borne encephalitis foci with distinct viral lineages in the Helsinki area,
Finland, Emerging Microbes & Infections, 8:1, 675-683, DOI: 10.1080/22221751.2019.1612279
To link to this article:  https://doi.org/10.1080/22221751.2019.1612279
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group on behalf of Shanghai Shangyixun
Cultural Communication Co., Ltd
Published online: 14 May 2019.
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Recent establishment of tick-borne encephalitis foci with distinct viral lineages
in the Helsinki area, Finland
Teemu Smuraa,b, Elina Tonteric, Anu Jääskeläinenb, Gabriel von Troild, Suvi Kuivanena, Otso Huitue,
Lauri Kareinena, Joni Uusitaloa, Ruut Uusitaloa,f,g, Tuula Hannila-Handelbergc, Liina Voutilainenc,
Simo Nikkaric, Tarja Sironena, Jussi Saneh, Janne Castrénd and Olli Vapalahtia,b,g
aDepartment of Virology, University of Helsinki, Helsinki, Finland; bDivision of Clinical Microbiology, Helsinki University Hospital Laboratory
Services (HUSLAB), Helsinki, Finland; cCenters for Military Medicine and Biothreat Preparedness, Helsinki, Finland; dArchipelago Doctors Ltd,
Parainen, Finland; eNatural Resources Institute Finland (Luke), Helsinki, Finland; fDepartment of Geosciences and Geography, University of
Helsinki, Helsinki, Finland; gDepartment of Veterinary Biosciences, University of Helsinki, Helsinki, Finland; hDepartment of Health Security,
Infectious Disease Control and Vaccinations Unit, National Institute for Health and Welfare, Helsinki, Finland
ABSTRACT
Number of tick-borne encephalitis (TBE) cases has increased and new foci have emerged in Finland during the last decade.
We evaluated risk for locally acquired TBE in the capital region inhabited by 1.2 million people. We screened ticks and
small mammals from probable places of TBE virus (TBEV) transmission and places without reported circulation. The
TBEV positive samples were sequenced and subjected to phylogenetic analysis. Within the study period 2007–2017,
there was a clear increase of both all TBE cases and locally acquired cases in the Helsinki area. The surveillance of ticks
and small mammals for TBEV confirmed four distinct TBEV foci in the Helsinki area. All detected TBEV strains were of
the European subtype. TBEV genome sequences indicated that distinct TBEV lineages circulate in each focus.
Molecular clock analysis suggested that the virus lineages were introduced to these foci decades ago. In conclusion,
TBE has emerged in the mainland of Helsinki area during the last decade, with at least four distinct virus lineages
independently introduced into the region previously. Although the overall annual TBE incidence is below the
threshold for recommending general vaccinations, the situation requires further surveillance to detect and prevent
possible further emergence of local TBE clusters.
ARTICLE HISTORY Received 6 March 2019; Revised 11 April 2019; Accepted 20 April 2019
KEYWORDS Tick-borne encephalitis; flavivirus; incidence; phylogeny; Finland
Introduction
Tick-borne encephalitis virus (TBEV) is a flavivirus
(genus Flavivirus, family Flaviviridae) that causes
severe encephalitis across large parts of Europe and
Northern Asia. Globally, TBEV has been estimated to
annually cause 13,000 cases of central nervous system
infection [1]. In nature, TBEV circulates in a fragile
cycle involving ticks and their vertebrate hosts, mainly
small mammals. Humans are accidental hosts and do
not contribute to the circulation of TBEV.
There are three subtypes of TBEV: European (Eur),
Siberian (Sib) and Far-Eastern (FE). TBEV-Eur is carried
mainly by Ixodes ricinus ticks in central and north-east-
ern Europe, whereas TBEV-Sib and -FE are foundmainly
in Ixodes persulcatus ticks in an area reaching from
north-eastern Europe to the Russian Far East, China
and Japan [1]. Recently, new subtypes of TBEV (Himala-
yan and Baikalian) have been characterized [2,3].
The number of tick-borne encephalitis (TBE) cases
has increased during the last decade in Finland. Fin-
land lies at the northernmost limit of the TBE endemic
area in Europe, and the occurrence of TBE in Finland is
currently restricted to geographically separate endemic
foci [4–7]. In the 1960s, screening of cattle for anti-
TBEV antibodies demonstrated the circulation of
TBEV in a few individual locations: the archipelagos
of Åland and Turku, and the Lappeenranta and Kok-
kola regions [8]. TBEV transmission was restricted to
these locations for decades until Isosaari Island in the
outer archipelago of Helsinki was recognized as a
TBEV focus in the 1990s (Figure 1) [9]. Since 2008,
new TBE foci have been detected annually in Finland
[4,10–12].
Apart from Isosaari Island, TBEV was not con-
sidered to circulate in the Helsinki area prior to 2011,
when the first human case with a confirmed local trans-
mission in Helsinki was reported. Since then, new
locally acquired human TBE cases have occurred
every year in the Helsinki region. The distribution of
TBEV is considered to be affected by climate, mainly
through its impact on the vectors and their hosts.
The apparent increase in the locally acquired TBE
© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
CONTACT Teemu Smura teemu.smura@helsinki.fi Department of Virology, University of Helsinki, FI-00014 Helsinki, Finland; Division of Clinical
Microbiology, Helsinki University Hospital Laboratory Services (HUSLAB), FI-00014 Helsinki, Finland
Emerging Microbes & Infections
2019, VOL. 8, NO. 1, 675–683
https://doi.org/10.1080/22221751.2019.1612279
case numbers is compatible with the establishment of
new TBEV foci in southern Norway [13–17] and in
the lake district of Sweden [18–20].
The recent increase of TBE cases in the densely
populated and rapidly growing capital area, home to
approximately 1.2 million inhabitants, together with
the estimations of increased microclimatic suitability
for the establishment of TBEV foci in the southern
coast of Finland [21], are of concern and lead us to
conduct the current study of TBEV in the area. We
investigated the number and origin of TBE cases
reported in the Helsinki area to detect locally acquired
TBEV infections. To affirm local transmission, we col-
lected ticks and small mammals within the Helsinki
area, and screened them for TBEV. Virus genomes
were sequenced and subjected to phylogenetic analysis
that revealed a pattern of multiple introductions of dis-
tinct TBEV lineages to the Helsinki region.
Figure 1. Map of collection sites.
Table 1. Tick and mammal sampling sites, years and TBEV-RT-PCR and TBEV-IFA results.
Site, year
Human
cases
Ticks TBEV RNA
pos/total Mammal species (n)
Brains TBEV RNA
pos/total
Spleens TBEV RNA
pos/total
Serology TBEV Ab
pos/total
Sipoo archipelago, 2013 1 in 2011
2 in 2016
5/90 Myodes glareolus (21) 0/21 0/20 1/21
Microtus agrestis (1) 0/1 0/1 0/1
Apodemus flavicollis
(3)
0/3 0/3 0/3
Sorex sp (2) 0/1 0/2 0/2
Helsinki Karhusaari, 2013 1 in 2016 0/9 Myodes glareolus (1) N/A N/A 0/1
Apodemus flavicollis
(1)
0/1 0/1 0/1
Helsinki Jollas, 2016–18 2 in 2014
1 in 2017
0/62 Myodes glareolus (26) 1/26 0/26 6/26
Microtus agrestis (4) 0/4 0/4 0/4
Apodemus flavicollis
(3)
0/2 0/3 0/3
Sorex araneus (2) 0/2 0/2 0/2
Helsinki Santahamina,
2015–2017
no cases 0/18 Myodes glareolus (30) 0/27 0/29 0/30
Microtus agrestis (7) 0/7 0/7 0/7
Apodemus flavicollis
(8)
0/8 0/8 0/8
Sorex sp (3) 0/2 0/3 0/3
Helsinki Vallisaari, 2016 no cases 0/34 No samples
Helsinki Lauttasaari 2016–
2017
no cases 0/80 No samples
Espoo Tontunmäki 2014,
2016
1 in 2013 0/6 Myodes glareolus (6) N/A N/A 0/6
Microtus agrestis (2) N/A N/A 0/2
Apodemus flavicollis
(8)
N/A N/A 0/8
Espoo and Kauniainen
2016–2017
see Figure
1
14/679
676 T. SMURA ET AL.
Material and methods
Human cases
TBE is a notifiable disease in Finland and the cases are
reported in the National Infectious Diseases Register
(NIDR) of the National Institute for Health and
Welfare (THL) (https://thl.fi/ttr/gen/rpt/tilastot.html).
The register data of each acute case is studied and, if
possible, the patients are interviewed to determine
the most likely site of infection. The method has been
described earlier [10]. Only cases with the known
most likely site of infection were included in the
primary epidemiological analyses.
Study area
TheHelsinkiMetropolitan area covers the cities of Espoo,
Helsinki, Kauniainen and Vantaa. In addition, the Hel-
sinki area covers also the municipalities of Kirkkonummi,
Vihti, Nurmijärvi, Tuusula, Kerava, Järvenpää, Hyvinkää
and Sipoo. The population of the Helsinki area was
1,214,447 inhabitants at the end of 2017 (https://www.
tilastokeskus.fi/tup/tilastotietokannat/index.html). All
TBE cases during the years 2007–2017, in which any
one of the municipalities of the Helsinki area was regis-
tered as the place of infection, place of diagnosis or as
the home municipality of the patient, were included in
this study. The TBE incidence trends were studied
using joinpoint regression model implemented in Join-
point Trend Analysis Software [22].
Sample collection
Ticks, rodents and shrews were collected from various
locations in Helsinki, Espoo and the Sipoo archipelago
between 2013 and 2017 (Figure 1 and Table 1). The col-
lection was focused on the probable sites of infection of
the TBE cases. Tick collections were conducted by
flagging questing ticks. Small mammals were collected
using snap traps set over night and either dissected
immediately or stored at −80 °C until dissected. Tick
samples were also acquired from a crowdsourced collec-
tion initiative organized by a private medical clinic that
provides TBEV vaccination in the area. This collection
was not targeted towards suspected TBEV endemic foci.
Nucleic acid extraction and TBEV-RT-PCR
The ticks and tissue samples (brain and spleen) were
homogenized in Dulbecco’s PBS + 0,2% BSA and ster-
ile sand using a MagnaPure homogeniser at 7000 rpm
for 2 × 30 s. RNA was extracted using TriPure reagent
according to the manufacturer’s instructions (Roche)
or AllPrep DNA/RNA Mini Kit (Qiagen) and eluted
in dH2O. TBEV RT-PCR was performed as described
previously [23], using Taqman fast virus 1-step
mastermix (Thermo Fischer Scientific), 500 nM pri-
mers and 200 nM probe.
Virus isolation
Homogenized ticks were suspended in PBS, inoculated
to SK-N-SH human neuroblastoma cells and incubated
for 1 h at +37°C. Tick suspension was replaced by Dul-
becco’s Modified Eagle’s Media (DMEM) sup-
plemented with glutamine, 100 units/ml penicillin,
100 μg/ml streptomycin and 2% foetal bovine serum
(FBS). Samples were collected immediately after infec-
tion and at day 6 for RNA quantification and the cells
were prepared for immunostaining on day of collection
(6 dpi). RNA was extracted from the cell culture super-
natant using the Viral RNA Mini Kit (Qiagen) accord-
ing to the manufacturer’s instructions.
Sequencing
The primers for complete TBEV coding region sequen-
cing were designed using PrimalScheme tool http://
primal.zibraproject.org/ [24] using 500 bp target
amplicon size and 50 bp overlap. cDNA was sythesized
from RT-PCR positive RNA samples with SuperScript
III enzyme (Invitrogen) using random hexamers, and
PCR was conducted using Q5 PCR kit (New England
Biolabs) under previously published conditions [24].
The PCR products were purified using AmpureXP
magnetic beads (Beckman Coulter) and sequencing
libraries prepared using Nextera XT kit (Illumina)
according to the manufacturer’s instructions.
The TBEV RT-PCR positive vole brain was hom-
ogenized at 3000 rpm for 60 s. Prior to RNA extraction,
the homogenate was treated with a cocktail of micro-
coccal nuclease (New England BioLabs, Ipswich,
USA) and benzonase (Millipore) for 1 h at +37°C.
Ribosomal RNA was removed using a NEBNext
rRNA depletion kit (New England BioLabs), according
to the manufacturer’s protocol. The sequencing library
was prepared using a NEBNext Ultra II RNA library
prep kit (New England BioLabs).
The library fragment sizes were measured using
agarose gel electrophoresis and the concentrations
using Qubit Broad-Range dsDNA Assay Kit (Life
Technologies) and NEBNext Library Quant kit for Illu-
mina (New England BioLabs). Sequencing was con-
ducted using MiSeq V2 reagent kit with 150 bp reads.
Raw sequence reads were trimmed and low quality
(quality score <30) and short (<50 nt) sequences removed
using Trimmomatic [25]. The trimmed sequence reads
were de-novo assembled using Velvet de novo assembler
[26] followed by re-assembly against the de-novo
assembled consensus sequences using BWA-MEM algor-
ithm [27] implemented in SAMTools version 1.8 [28].
In case no products were obtained using complete
coding sequence PCR, amplification of smaller
EMERGING MICROBES AND INFECTIONS 677
fragments of E-gene and NS5 were attempted using
previously described protocols [29].
Phylogenetic analysis
Complete genome and E gene sequences of all available
European subtype TBEV were downloaded from Gen-
Bank (accessed June 2018). The sequences were aligned
using the ClustalW algorithm followed by manual
refinement. Substitution model was estimated using
MEGA7 [30].
Phylogenetic tree based on the complete coding
sequences was constructed using Bayesian Monte
Carlo Markov Chain (MCMC) method implemented
in BEAST version 1.8.0 [31]. Two different clock
models (strict clock and log normal relaxed clock)
and two demographic models (constant and Bayesian
skyline models) were compared by calculation of
Bayes Factors (ratio of marginal likelihoods of the
models). The marginal likelihoods of different model
combinations were estimated using path sampling
and stepping stone methods [32,33]. Bayes factors
(BF) were calculated for each pair of models. The
final analysis was conducted using General Time
Reversible (GTR) substitution model with 4-category
gamma-distributed variation among sites and a pro-
portion of invariant sites, strict clock model and Baye-
sian skyline demographic model. The analyses were
run in duplicates for 50 million states and sampled
every 5000 steps. Posterior probabilities were calcu-
lated with a burn-in of 10% and checked for conver-
gence using the Tracer version 1.7. [34].
Phylogenetic tree based on E gene was inferred
using the Bayesian method implemented in MrBayes
v3.1.2 [35], using the GTR model, a 4-category
gamma-distribution model of among-site rate hetero-
geneity and a proportion of invariant sites. MrBayes
was run for 5 million generations, with final standard
deviations between 2 runs of 0.011.
The analyses were carried out on the CSC server (IT
Center for Science Ltd., Espoo, Finland).
Immunofluorescence assay
The small mammal samples were screened for the pres-
ence of TBEV antibodies using an immunofluores-
cence assay on TBEV-infected cells on 10-well micro-
titer well slides and detected using FITC-conjugated
mouse immunoglobulins secondary antibody (Agi-
lent), as described previously [36].
Results
TBE cases
During the years 2007–2017, altogether 488 TBE cases
were registered in the National Infectious Diseases
Register (NIDR) of Finland. Out of these, 125 cases
were diagnosed from the residents of the Helsinki
area (Figure 2). The most likely location at which the
tick bite was acquired was determined from an inter-
view of each TBE patient by National Institute for
Health and Welfare (NIHW), as described previously
[10]. In 35 cases, the reported site of infection was in
the Helsinki area. Thirty of these were residents of
the Helsinki area. Eighty-five infections had most likely
been acquired during visits to other known endemic
foci in Finland (N = 72) or abroad (N = 13). In ten
cases the probable location of infection could not be
determined. Within the study period of 2007–2017,
there was a clear increase in both the annual total num-
ber of TBE cases (from 4 cases in 2007 to 22 cases in
2017) and locally acquired TBE cases (from 0 to 13
locally acquired cases in 2016 and 7 locally acquired
cases in 2017) in the Helsinki area (Figure 2).
The incidence of all TBEV cases reported in
the Helsinki area increased from 0.31 to 1.52/year/
100,000 inhabitants [average annual percentage
change (AAPC) 20.6% (95% confidence interval
13.6–28.0; P < .05)], and the incidence of locally
acquired TBEV infections increased from zero to
0.48/year/100,000 inhabitants (AAPC 35.6% [CI
0.6–82.8; P < .05]).
Screening of ticks and small mammals
Five out of 90 ticks collected in 2013 from an island in
Sipoo archipelago were TBEV RNA positive, and one
Figure 2. The number of TBE cases (A) and the TBE incidence
(B) in the Helsinki area.
678 T. SMURA ET AL.
bank vole (Myodes glareolus) was TBEV antibody
positive (Table 1). One patient had contracted TBEV
most likely on this island in 2011. From the Jollas
peninsula in Helsinki, where two patients in 2014
and one patient in 2017 had likely contracted the dis-
ease, one out of 35 bank voles was TBEV RNA positive,
and six had antibodies against TBEV.
Altogether 679 ticks were collected from humans,
pets or directly from the vegetation by volunteer citi-
zens from various locations in Espoo and Helsinki in
2016–2017. Of these ticks, 15.3% were engorged.
Fourteen ticks (2.1%) were TBEV RNA positive.
Twelve of the TBEV positive ticks were collected
from two pet cats (five and seven positive ticks,
respectively) in Espoonkartano/Kauklahti, which
was also a likely site of infection for one TBE patient
in the year 2017.
The other two locations where TBEV positive ticks
were detected were Tontunmäki, where three cases of
TBE have been detected previously, and Laurinlahti,
where no human cases have been detected.
Molecular epidemiology
Sequence data were obtained from twelve RT-PCR
positive ticks and one bank vole brain. In addition,
the complete genome of a previously detected positive
tick pool from the Isosaari island (year 2005) [37] was
sequenced. All TBEV strains were of the European sub-
type (Figure 3). The sequences obtained from each
given district of the Helsinki area showed local geo-
graphic clustering, but the strains from the different
districts did not share a common ancestor. The strains
from Espoo formed a monophyletic group that clus-
tered together with the strains from Kuutsalo, Kotka,
which is located approximately 140 km from Espoo
[38] and Bornholm Denmark [39]. Furthermore, this
group formed a sister clade to strains detected in Swit-
zerland [40]. The strains from Sipoo formed another
monophyletic group. Strains from Latvia [41] and
Estonia [42] formed an outgroup for this cluster. The
strain sequenced from bank vole brain (Jollas) clus-
tered together with strains from Latvia [41]. The
TBEV strain from Isosaari clustered together with
Figure 3. Maximum clade credibility tree constructed from complete coding sequences (A) and E genes (B).
EMERGING MICROBES AND INFECTIONS 679
strains from Slovenia [43], Czech Republic [44] and
Germany [45], as previously reported [37]. None of
the newly detected strains from Espoo, Sipoo or Jollas
grouped together with the previously detected TBEV
strain from Isosaari, Helsinki, which comprised the
fourth TBEV lineage within the Helsinki area.
The pairwise genetic differences of complete coding
sequences within the monophyletic clusters detected in
the Helsinki area ranged from 15 to 33 nucleotides
(translated to 2–8 amino acids) in Espoo and 8–12
nucleotides (2–4 amino acids) in Sipoo. The molecular
clock analyses based on complete coding sequences
suggested estimated time to the most recent common
ancestor (tMRCA) between 40.6 and 48.2 years [95%
HPD range 20.5–76.8] for Espoo cluster and 36.9–
42.2 years [95% HPD range 20.6–65.6] for Sipoo clus-
ter, depending on the molecular clock model used
(Table 2). The estimated tMRCAs for the Espoo/
Kotka/Bornholm cluster were between 254 and 280
years [95% HPD 163-418].
Discussion
The epidemiology of TBE has changed during the last
decade in Finland [10]. In the Helsinki area, the capital
area of Finland by the Gulf of Finland in the Baltic Sea,
TBEV had been known to be endemic on only one
outer island, Isosaari, since the 1990s [9], but no
other observations of local TBEV transmission were
made prior to the year 2011. In this study, we detected
an increasing trend of TBE incidence and found evi-
dence for local transmission and previously undetected
endemic foci of TBEV in the Helsinki area.
Finland is at the northernmost limit of the TBE
endemic area in Europe and here TBEV occurs in
endemic foci. Since 2008, new foci have appeared
annually in Finland [4,10–12,37,38]. Apart from the
focus of Isosaari Island, no TBEV was recognized in
the Helsinki area prior to 2011 when the first locally
acquired human cases acquired in the Helsinki area
were reported. In this study, we report an increased
incidence of TBE in the residents of the Helsinki
area. This increase was observed for both locally
acquired and total reported cases. The increase in
TBE incidence in the Helsinki area coincides with
that of the whole Finnish population, in which the inci-
dence has increased from 0.72to 1.11 between the years
2012–2016 (APC 14.4% [CI 0.7–28.1]) [46].
TBEV circulation in nature is very sensitive to var-
ious environmental factors such as microclimate and
host animal population density [21,47–49]. Intrigu-
ingly, the sequence variation within the two foci of
which more than one strain was sequenced was rather
high, suggesting that the introduction of TBEV into
these two foci has occurred most likely decades ago.Figure 3 Continued
680 T. SMURA ET AL.
According to climate models, the microclimate of the
Helsinki area has been suitable for TBEV circulation
already at the turn of the millennium [50]. It appears
to have taken decades before introductions of TBEV
into the area, ecological factors, and human contacts
with the infected ticks all coincided in space and time
for TBE to manifest as locally transmitted and notified
cases from foci within the Helsinki area.
Further, the phylogenetic analysis indicated that the
TBEV strains sequenced from the Sipoo archipelago,
Espoo and Helsinki (Jollas peninsula and Isosaari
Island) do not cluster together, although they are located
only 20 km apart. This suggests independent introduc-
tions of four TBEV strains to the region. The strains
from Espoo, on the other hand, are distantly related to
the TBEV strain found in Kuutsalo, Kotka (140 km
apart) [38]. However, the estimated tMRCAs for the
Espoo/Kotka cluster were 250–280 years. With the cur-
rent dataset, it cannot be concluded if these represent
two independent introductions into Finland, or one
introduction followed by a spreading of the virus
along the coastline. Generally, the independent intro-
duction of distinct TBEV lineages to a restricted geo-
graphical region is consistent with the studies
conducted in Central and Southern Europe, where sev-
eral TBEV lineages can be found in a given geographical
region [40,44,51,52]. Thus, TBEV typically displays a
high level of focus-specific clustering, but a low level
of clustering within larger geographical areas. Most
likely, however, the circulation of TBEV has been pre-
viously more limited in Finland and Scandinavia com-
pared to Central Europe and the Baltic states.
Our results, together with suggestions of long dis-
tance dispersal events based on molecular epidemiology
studies of TBEV [37,53] and findings of TBEV in ticks
carried by migratory birds [54–58], are in line with the
hypothesis that the initial introduction of TBEV has
occurred via migratory birds, and followed with a sub-
sequent local expansion of the established foci.
While all strains characterized from the Helsinki
region were of the European subtype, both European
and Siberian subtypes are known to circulate in Fin-
land. Further, both virus subtypes have been found in
both Ixodes ricinus and I. persulcatus vector species
[4,11]. A previous study based on a large collection of
ticks in 2015 demonstrated that only Ixodes ricinus
was present in the southern coast of Finland, including
the Helsinki area [5]. Tick collections not targeted at
the suspected TBEV foci found TBEV prevalence in
I. ricinus to be 0.2% [5]. Compared to this, the preva-
lences in the suspected foci reported in this study
were high. TBEV prevalence was found to be 5.6% in
ticks collected in Sipoo in the garden of a TBEV-
infected patient. Also, one out of 35 mammals was
TBEV RNA positive and six out of 35 mammals
TBEV antibody positive in the Jollas peninsula of Hel-
sinki, where three human cases of TBE have been
recorded. Out of 655 ticks collected from various
locations in Espoo, thirteen TBEV RNA positive ticks
were found from sites where human cases have been
reported, and only one from elsewhere. These results
are consistent with the well-known very focal distri-
bution of TBEV in nature. Even within the Helsinki
area, TBE human cases, TBEV infection markers in
wildlife samples and the presence of TBEV RNA in
ticks seem to concentrate to only a few foci.
This study provides a description of the early events
of establishment and subsequent spread of TBE foci in
an urban area, as evidenced by diverse data including
patient interviews, environmental sampling, TBEV
screening, full-genome sequencing and sequence
analysis. The findings are in concordance with obser-
vations on the changing epidemiology of TBE in the
Nordic countries and suggest the further potential for
TBEV emergence. Our results therefore call for further
monitoring of the infection pressure, using One Health
approaches to inform public health measures such as
risk assessment and vaccine recommendations.
Acknowledgements
We are very grateful to Johanna Martikainen and Mira
Utriainen for excellent laboratory assistance. We thank
Petri Kangaspunta for his help with collecting samples in
the Santahamina military island. We thank TBE patients
for their kind co-operation and help in assessing sampling
sites. We acknowledge CSC−IT Center for Science Ltd.
(Espoo, Finland) for the allocation of computational
resources.
Disclosure statement
No potential conflict of interest was reported by Otso Huitu,
Lauri Kareinen, Joni Uusitalo, Ruut Uusitalo, Tuula Han-
nila-Handelberg, Liina Voutilainen, Simo Nikkari, Tarja
Table 2. The tMRCAs and mean rates of evolution (substitutions/site/year) estimated using two clock models and two demographic
models.
Strict clock Log normal relaxed clock
Constant population Bayesian skyline Constant population Bayesian skyline
tMRCA 95% HPD tMRCA 95% HPD tMRCA 95% HPD tMRCA 95% HPD
Espoo 48.2 23.9–76.8 43.6 22.4–65.2 45.3 22.4–73.3 40.6 20.5–62.5
Sipoo 42.2 22.0–65.6 38.8 21.6–58.6 39.7 21.2–62.4 36.9 20.6–56.7
Espoo/Bornholm/Kuutsalo 280.4 162.3–421.0 276.8 172.5–398 258.1 151.6–400.6 253.6 148.4–369
Mean rate 2.0476E-5 1.1958–2.9353E-5 2.0436E-5 1.3012–2.8399E-5 2.2377E-5 1.2916–3.3221E-5 2.2466E-5 1.3782–3.1689E-5
Co-efficient of variation 0.0927 7.7115–16.6E-3 0.0994 1.65–0.17.72E-3
EMERGING MICROBES AND INFECTIONS 681
Sironen and Jussi Sane. Elina Tonteri and Anu Jääskeläinen
have received lecturing fees from Pfizer Ltd. for giving a lec-
ture in the International Scientific Working Group on Tick-
Borne Encephalitis (ISW-TBE) meeting. For Teemu Smura
and Suvi Kuivanen, the travel costs for attending Inter-
national Scientific Working Group on Tick-Borne Encepha-
litis (ISW-TBE) meeting have been paid by Pfizer Ltd. Janne
Castren is employee and shareholder of Archipelago doctors
Ltd involved in providing TBEV vaccinations. Gabriel von
Troil is employee of Archipelago doctors Ltd. Olli Vapalahti
has received honoraria and travel costs from Pfizer Ltd. Olli
Vapalahti’s research group has received royalties from TBE
diagnostic tests from Reagena Ltd.
Funding
This work was supported by the Academy of Finland (Suo-
men Akatemia) [grant number 316264], the Jane and
Aatos Erkko Foundation (Jane ja Aatos Erkon Säätiö),
Orion Research Foundation (Orionin Tutkimussäätiö),
Sigrid Jusélius Foundation (Sigrid Juséliuksen Säätiö) and
Helsinki University Central Hospital (Helsingin ja Uuden-
maan Sairaanhoitopiiri).
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