Anu Jääskeläinen

Siberian Subtype Tickborne Encephalitis Virus, Finland

We isolated 11 Siberian subtype tickborne encephalitis virus (TBEV) strains from Ixodes persulcatus ticks from a TBEV-endemic focus in the Kokkola Archipelago, western Finland. Thus I. persulcatus and the Siberian TBEV are reported in a focus considerably northwest of their previously known range in eastern Europe and Siberia.

Tick-borne Encephalitis Virus in Wild Rodents in Winter, Finland, 2008–2009

Rodents might maintain tick-borne encephalitis virus (TBEV) in nature through latent persistent infections. During 2 subsequent winters, 2008 and 2009, in Finland, we detected RNA of European and Siberian subtypes of TBEV in Microtus agrestis and Myodes glareolus voles, respectively. Persistence in rodent reservoirs may contribute to virus overwintering.

Tick-borne encephalitis virus in ticks in Finland, Russian Karelia and Buryatia.

Tick-borne encephalitis (TBE) is a central nervous system infection caused by a flavivirus [tick-borne encephalitis virus (TBEV)], transmitted by Ixodes ticks and endemic in a large region in Eurasia. We collected 2411 ticks from Finland and Russia in 2003-2008, screened them for TBEV by RT-PCR and isolated and analysed eight strains belonging to all three TBEV subtypes; in addition, we obtained two European-subtype strains from human serum samples. TBEV RNA prevalence in unengorged ticks was approximately 1 % both in the northernmost TBE-endemic areas of Europe in Finland and Russian Karelia, and in Siberia in Buryatia. In Finland, both Ixodes ricinus and Ixodes persulcatus ticks were found from distinct areas and, in Russian Karelia, were overlapping in the same study site. TBEV E and NS3 gene sequences obtained showed a variability of 0-4 % within European-subtype strains, 2-9 % for Siberian-subtype strains and 3-13 % for Far Eastern-subtype strains.

European subtype tick-borne encephalitis virus in Ixodes persulcatus ticks.

To the Editor: The northernmost tick-borne encephalitis (TBE) focus is in Simo, Finnish Lapland. Four TBE cases were confirmed during 2008–2009. Tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and is endemic to Eurasia from central Europe to the Far East. The virus has 3 subtypes: European (TBEV-Eur), Siberian (TBEV-Sib), and Far Eastern (TBEV-FE). TBEV-Eur is mainly transmitted by I. ricinus ticks (sheep ticks) and the 2 other subtypes by I. persulcatus ticks (taiga ticks). The range of I. ricinus ticks covers most of continental Europe and the British Isles; I. persulcatus ticks are distributed throughout eastern Europe and Asia to the People’s Republic of China and Japan. The transmission cycle of at least TBEV-Eur in nature is fragile and depends on microclimatic conditions. Thus, within the I. ricinus distribution area, TBE is endemic merely focally (1,2). In Finland, TBE foci are located by the sea or large lakes (Figure A1). Both vector tick species are found: I. ricinus ticks in the southern and central parts of the country, but I. persulcatus ticks are in scattered foci along the western coast, including the Kokkola archipelago and Narpio municipality, where they carry TBEV-Sib (3,4) (Figure A1). The first human TBE cases from Simo in Lapland (65°40′N, 24°54′E; Figure A1) were reported during 2008 (n = 2) and 2009 (n = 2). On the basis of interviews with the 2 patients from 2008, we collected 97 ticks and 17 bank voles from the 2 probable sites of infection during June 2009. From the rodents, we extracted blood from the heart and performed TBEV-antibody tests by immunofluorescence assay. The ticks were placed in 51 pools (1–3 ticks/pool). We isolated RNA from tick pools and rodent lungs and brains by TriPure Isolation Reagent (Roche Diagnostics, Indianapolis, IN, USA) and performed real-time reverse transcription–PCR (5) to detect TBEV RNA. For the positive tick pools, we confirmed the identification species by Ixodes mtDNA sequencing (6). Six of 51 tick pools (with a total of 97 I. persulcatus ticks) were positive for TBEV in real-time reverse transcription–PCR, resulting in 6% TBEV RNA prevalence. At least 1 organ was positive for TBEV RNA in as many as 15/17 bank voles, in line with our finding that TBEV RNA persists in rodents for months (7); 4 rodents had antibodies to TBEV. The TBEV RNA prevalence among ticks and rodents was relatively high, as is the incidence among humans (0.57 cases/year/1,000 inhabitants) in Simo, indicating a focus with high activity. We isolated 6 TBEV strains from suckling mice (experimental animal permit ESLH-2008–06558/Ym-23): 2 from I. persulcatus tick pools (Simo-38 and Simo-48; pools of 2 and 3 ticks, respectively), and 4 from TBEV antibody– and RNA-positive rodent lung–brain suspensions (Simo-2, -5, -7 and -9). Partial envelope (E) and nonstructural protein 3 genes (4) of the isolated TBEV strains were sequenced (accession nos. HQ228014–HQ228024, GenBank) and subjected to phylogenetic analysis (Figure A1). Within the 1208 nt from the E gene, Simo-38 and Simo-48 from ticks and Simo-9 from a bank vole were identical. Other sequences differed for 1 nt and Simo-2 for 1 aa compared with the others. All strains were monophyletic and belonged to the TBEV-Eur subtype. The partial nonstructural protein 3 gene sequences were identical, and the phylogenetic tree showed similar topography as for the E gene (not shown). The only tick species found in Simo was I. persulcatus, further widening its known distribution along the western coast of Finland (Figure A1). However, the virus subtype found in Simo was TBEV-Eur strain, the main vector of which is the I. ricinus tick. TBEV-Eur strains are commonly very closely related to each other and do not form clear geographic clusters (4). Thus, it is difficult to deduce the origin of the virus. The nearest TBE-endemic focus is the Kokkola archipelago, ≈200 km south (Figure A1), but there I. persulcatus ticks carry the TBEV-Sib strain (3). The nearest areas to which the TBEV-Eur strain is endemic are in southern Finland where only I. ricinus ticks have been found. Cattle serum samples were negative for antibodies to TBEV in the Simo area in the 1960s (8). The first human TBE cases from Simo were identified during 2008 and 2009. We isolated TBEV strains from ticks and rodents in 2009. Simo appears to be a recently established, and the northernmost, TBE focus known. TBEV may have been introduced to Simo from a geographically distinct location recently, likely within the past 50 years. TBE seems to be moving northward in Europe (9) and shifting upward to higher elevations in the mountains (10), apparently influenced by climate change. An altered microclimate favoring TBE circulation (1), in addition to introduction of the virus, could also explain the recent emergence of TBE in Simo. In conclusion, Simo in Finnish Lapland is a new TBE-endemic focus demonstrating northward movement of foci and an unusual combination of the TBEV-Eur strain and I. persulcatus ticks in an area with no evidence of cocirculation of tick species or TBEV subtypes.

Diagnosis of Tick-Borne Encephalitis by a μ-Capture Immunoglobulin M-Enzyme Immunoassay Based on Secreted Recombinant Antigen Produced in Insect Cells

ABSTRACT Acute tick-borne encephalitis is diagnosed by detection of IgM antibodies to tick-borne encephalitis virus (TBEV) (genus Flavivirus) in patient serum. TBEV membrane (M) and envelope (E) proteins have previously been shown to form virus-like particles when expressed in mammalian cells. We expressed the prM/M and E proteins in insect cells with a recombinant baculovirus system and obtained antigenic protein secreted into the cell culture medium, as evidenced by detection by a panel of five monoclonal antibodies to TBEV E protein. According to the sedimentation pattern in sucrose gradient centrifugation, the proteins were most likely secreted as virus-like particles. A μ-capture immunoglobulin M-enzyme immunoassay (IgM-EIA) test was developed and compared to a commercially available TBEV-IgM test (Progen) based on inactivated purified TBEVs. With a panel of 100 TBEV-IgM-negative, 50 TBEV-IgM-positive, and seven dengue virus-IgM-positive serum samples from our diagnostic laboratory, a sensitivity of 100% and specificity of 99% were obtained, and the correlation coefficient of EIA absorbances with the reference test was 0.93. The antigen was also suitable for IgG antibody detection in an immunofluorescent assay format. This is the first time that secreted, fully antigenic E protein has been produced in insect cells for this arthropod-borne flavivirus.

First report on tick-borne pathogens and exoskeletal anomalies in Ixodes persulcatus schulze (Acari: Ixodidae) collected in Kokkola coastal region, Finland

Abstract Thirty ticks collected in Kokkola coastal region (64°N, 23°E) in 2004 were identified as Ixodes persulcatus Schulze and studied for the presence of tick-borne pathogens and exoskeletal anomalies. One of the ticks was positive for tick-borne encephalitis virus. Ticks positive for several species of Borrelia were also detected, including B. afzelii (n = 12), B. garinii (n = 7), B. burgdorferi sensu stricto (n = 2), some other species of B. burgdorferi sensu lato group (n = 3), Ehrlichia muris (n =5) and Babesia microti (n = 1). The total proportion of infected ticks was 63% (19 of 30), of which 12 were multiply infected. Forty percent of the ticks had exoskeletal anomalies, and these ticks had more multiple infections (6/12) than morphologically normal ones (6/18). In conclusion, this small sample size suggests that several important tick-borne pathogens apparently circulate in the newly detected focus of I. persulcatus in the Kokkola region. The results warrant further epidemiological studies on the incidence and prevalence of tick-borne diseases and on mapping the distribution of different Ixodes species in Finland.

Siberian subtype tick-borne encephalitis virus in Ixodes ricinus in a newly emerged focus, Finland

The first tick-borne encephalitis (TBE) cases in Kotka, Finland appeared in 2010. Altogether ten human cases have been diagnosed by 2014. Four had long-lasting sequelae. We collected 195 Ixodes ricinus ticks, nine rodents, and eleven shrews from the archipelago of Kotka in 2011. Three Siberian subtype TBE virus (TBEV) strains were isolated from the ticks and three mammals were positive for TBEV antibodies. The archipelago of Kotka is a newly emerged TBE focus of Siberian subtype TBEV circulating notably in I. ricinus. The patients had on average longer hospitalization than reported for the European subtype infection.

First evidence of established populations of the taiga tick Ixodes persulcatus (Acari: Ixodidae) in Sweden

BackgroundThe tick species Ixodes ricinus and I. persulcatus are of exceptional medical importance in the western and eastern parts, respectively, of the Palaearctic region. In Russia and Finland the range of I. persulcatus has recently increased. In Finland the first records of I. persulcatus are from 2004. The apparent expansion of its range in Finland prompted us to investigate if I. persulcatus also occurs in Sweden.MethodsDog owners and hunters in the coastal areas of northern Sweden provided information about localities where ticks could be present. In May-August 2015 we used the cloth-dragging method in 36 localities potentially harbouring ticks in the Bothnian Bay area, province Norrbotten (NB) of northern Sweden. Further to the south in the provinces Västerbotten (VB) and Uppland (UP) eight localities were similarly investigated.ResultsIxodes persulcatus was detected in 9 of 36 field localities in the Bothnian Bay area. Nymphs, adult males and adult females (n = 46 ticks) of I. persulcatus were present mainly in Alnus incana - Sorbus aucuparia - Picea abies - Pinus sylvestris vegetation communities on islands in the Bothnian Bay. Some of these I. persulcatus populations seem to be the most northerly populations so far recorded of this species. Dog owners asserted that their dogs became tick-infested on these islands for the first time 7–8 years ago. Moose (Alces alces), hares (Lepus timidus), domestic dogs (Canis lupus familiaris) and ground-feeding birds are the most likely carriers dispersing I. persulcatus in this area. All ticks (n = 124) from the more southern provinces of VB and UP were identified as I. ricinus.ConclusionsThe geographical range of the taiga tick has recently expanded into northern Sweden. Increased information about prophylactic, anti-tick measures should be directed to people living in or visiting the coastal areas and islands of the Baltic Bay.

Fatal Tick-Borne Encephalitis Virus Infections Caused by Siberian and European Subtypes, Finland, 2015

In most locations except for Russia, tick-borne encephalitis is mainly caused by the European virus subtype. In 2015, fatal infections caused by European and Siberian tick-borne encephalitis virus subtypes in the same Ixodes ricinus tick focus in Finland raised concern over further spread of the Siberian subtype among widespread tick species.

Test based on subtype-specific μ-capture IgM immunoassay can distinguish between infections of European and Siberian subtypes of tick-borne encephalitis virus.

BACKGROUND In many European countries (including Finland, Estonia, Latvia and Russia) two subtypes of tick-borne encephalitis virus (TBEV) occur with overlapping geographic distribution yet with apparently different severity and persistence of symptoms. However, it has not usually been possible to distinguish these infections in the laboratory, as TBEV RNA or sequences have rarely been retrieved from patients seeking medical care in the second phase of infection when the neurological symptoms occur, and serological tests have so far not been able to discriminate between the subtype-specific responses. OBJECTIVES The aim of this study was to assess the applicability of a μ-capture enzyme immunoassay (EIA) based on TBEV prME subviral particles produced in mammalian cells from Semliki-Forest virus replicons (SFV-prME EIA) to distinguish reactivity to European and Siberian strains of TBEV. STUDY DESIGN Altogether 54 TBEV IgM positive acute human serum samples and 6 positive cerebrospinal fluid (CSF) samples from different regions of Finland were tested in EIA with subtype-specific antigens and TBEV-IgM subtype-specific index ratios were determined. RESULTS All 30 samples from patients whose transmission had occurred in foci where only Siberian subtype of TBEV is occurring had an index ratio of more than 1.8, whereas all 30 acute TBE samples from an area where only European subtype circulates had an index ratio below 1.5. CONCLUSIONS We conclude that the assay is a useful tool to distinguish between acute infections of European and Siberian strains of TBEV, and should help in further studies of the clinical outcome of these two subtypes.