Siberian subtype tickborne encephalitis virus, Finland.We isolated 11 Siberian subtype (programming) subtype - If S is a subtype of T then an expression of type S may be used anywhere that one of type T can and an implicit type conversion will be applied to convert it to type T. tickborne encephalitis encephalitis (ĕnsĕf'əlī`təs), general term used to describe a diffuse inflammation of the brain and spinal cord, usually of viral origin, often transmitted by mosquitoes, in contrast to a bacterial infection of the meninges virus (TBEV TBEV Tick-Borne Encephalitis Virus ) strains from Ixodes persulcatus Noun 1. Ixodes persulcatus - bites humans; a vector for Lyme disease spirochete hard tick, ixodid - ticks having a hard shield on the back and mouth parts that project from the head genus Ixodes, Ixodes - type genus of the family Ixodidae 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 Eastern Europe The countries of eastern Europe, especially those that were allied with the USSR in the Warsaw Pact, which was established in 1955 and dissolved in 1991. and Siberia. ********** Tickborne encephalitis (TBE) is a disease endemic in a zone extending from central and eastern Europe The term "Central and Eastern Europe" came into wide spread use, replacing "Eastern bloc", to describe former Communist countries in Europe, after the collapse of the Iron Curtain in 1989/90. to Siberia and Japan. Three subtypes of the causative agent tickborne encephalitis virus (TBEV) are known: the European, Siberian, and Far Eastern (1,2). The main vector for the European subtype is &odes ricinus, and for the other 2 subtypes, I. persulcatus (1,3-5). I. ricinus is found in Europe and Middle East (6), and I. persulcatus ranges from eastern Europe to China and Japan. The boundary between their distribution lies at the Russian side of the Finnish-Russian border (1,7). The distribution areas of both tick species overlap in eastern Europe (4,5) (Figure 1). I. persulcatus has not been reported from northern or western Europe Western Europe The countries of western Europe, especially those that are allied with the United States and Canada in the North Atlantic Treaty Organization (established 1949 and usually known as NATO). except for an engorged en·gorge v. en·gorged, en·gorg·ing, en·gorg·es v.tr. 1. To devour greedily. 2. To gorge; glut. 3. To fill to excess, as with blood or other fluid. v.intr. nymph nymph, in Greek mythology nymph (nĭmf), in Greek mythology, female divinity associated with various natural objects. It is uncertain whether they were immortal or merely long-lived. There was an infinite variety of nymphs. on a willow warbler (Zool.) a very small European warbler (Phylloscopus trochilus); - called also bee bird, haybird, golden wren, pettychaps, sweet William, Tom Thumb, and willow wren. See also: Willow (Phylloscopus troehilus) in northeastern Sweden in May 1992 (6). [FIGURE 1 OMITTED] In Finland, the TBE-endemic areas are mostly in the coastal regions: two thirds of cases come from the Aland Islands. Other TBE-endemic regions include the Archipelago of Turku, a focus in Isosaari (an island outside Helsinki), the Lappeenranta region in southeastern Finland, and the Archipelago of Kokkola in western Finland (9). The virus was found in the same areas (except for Isosaari) as early as the 1960s by screening antibodies to TBEV from cattle sera (10). The TBE focus in the Archipelago of Kokkola (63[degrees]50'N, 23[degrees]10'E), [approximately equal to] 300 km south of the Arctic Circle Arctic Circle, imaginary circle on the surface of the earth at 66 1-2°N latitude, i.e., 23 1-2° south of the North Pole. It marks the northernmost point at which the sun can be seen at the winter solstice (about Dec. , has a peculiar location; it is an isolated focus, far from other TBE-endemic areas, and is the northernmost TBE-endemic area known. Furthermore, the recent TBE cases have been severe with sequelae sequelae Clinical medicine The consequences of a particular condition or therapeutic intervention (11,12). A cluster of cases in 2002 led us to study ticks in the Kokkola Archipelago for TBEV. The Study A total of 1,181 ticks were collected by flagging in the Archipelago of Kokkola in June 2004 (Table 1). In the tick-collecting areas 1-7, TBE patients had reported tick bites, and in areas 8-10, no TBE cases have been found. All the locations were islands or peninsulas within 20 km of each other. The ticks were homogenized ho·mog·e·nize v. ho·mog·e·nized, ho·mog·e·niz·ing, ho·mog·e·niz·es v.tr. 1. To make homogeneous. 2. a. To reduce to particles and disperse throughout a fluid. b. in pools of [approximately equal to] 10 with Dulbecco phosphate-buffered saline plus 0.2% bovine serum albumin serum albumin n. See seralbumin. (D-PBS-BSA) and sand to 122 pools. RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic was isolated from 100 [micro]L of the pools by TriPure Isolation Reagent (Roche Diagnostics Roche Diagnostics Division is a subsidiary of Hoffmann-La Roche which manufactures equipment and reagents for research and medical diagnostic applications. Internally, it is organized into six major business areas: Roche Applied Science, Roche Centralized Diagnostics, Roche , Espoo, Finland). The RNA was dissolved in 20 [micro]L diethyl pyrocarbonate-treated water, and 10 [micro]L was used for nested reverse transcription reverse transcription n. The process by which DNA is synthesized from an RNA template. (RT)-PCR, amplifying a 252-nt sequence from the TBEV-NS5 gene to detect TBEV-RNA according to Puchhammer-Stockl et al. (13), except that the outer forward primer used was 5'-GGAGGCTGAACAACTGCAC-3". TBEV-RNA was detected in 13 pools (each consisted of 10 adult ticks) (Table 1). Assuming that only 1 tick in a positive pool was positive for TBEV RNA, the overall TBEV prevalence was 1.1%. To isolate TBEV strains from the RT-PCR-positive tick pools, 20 [micro]L of the supernatant supernatant /su·per·na·tant/ (-na´tant) the liquid lying above a layer of precipitated insoluble material. supernatant the liquid lying above a layer of precipitated insoluble material. of the pools diluted 1:1 in D-PBS-BSA was injected intracerebrally into suckling suckling In mammals, the drawing of milk into the mouth from the nipple of a mammary gland. In human beings, it is referred to as nursing or breast-feeding. The word also denotes an animal that has not yet been weaned—that is, whose access to milk has not yet been NMRI NMRI Nuclear Magnetic Resonance Imaging NMRI Naval Medical Research Institute NMRI National Marshmallow Roasters Institute (Citrus Heights, CA) mice. One litter of suckling mice was used for each pool. The mice were followed for 14 days or until symptoms of illness appeared, and then they were killed. From 200 [micro]L of the homogenized mouse brains, diluted 1:5 with D-PBS-BSA, RNA was extracted by TriPure, and RT-PCR RT-PCR reverse transcriptase-polymerase chain reaction. See PCR1. for the partial TBEV-E gene was performed. The cDNA was produced with the reverse primer 5'-CCYCCAGCCARGAGRAAGC-3' by M-MuLV-RT enzyme (Fermentas, Vilnius, Lithuania), and subsequent PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) was performed with this and a forward primer 5"AACAGGGAYTTTGTCACTGGYACTC-3' by Taq DNA polymerase DNA polymerase /DNA po·lym·er·ase/ (pah-lim´er-as) any of various enzymes catalyzing the template-directed incorporation of deoxyribonucleotides into a DNA chain, particularly one using a DNA template. (Fermentas) (detailed RT-PCR protocol available from the authors upon request). A region of 205 nt from the NS5 gene from the RT-PCR-positive tick pools and 1,225-nt stretch from the E gene from the brains of the infected suckling mice were sequenced (GenBank accession nos. in Table 2). Unexpectedly, based on the partial NS5 sequences from the RTPCR-positive tick pools (data not shown), the TBEV strains in Kokkola belonged to the Siberian subtype of TBEV. A phylogenetic tree based on the partial E gene sequences (1,076 nt) obtained from the TBEV isolates was prepared by the maximum likelihood method (Figure 2, scripts and datasets available from the authors upon request). Within the 1,076-nt stretch of the E gene, the Kokkola strains were [greater than or equal to] 99.6% identical to each other. The closest relatives were Latvia-1-96 (97% identical) and the Estonian strains Est54, Est3535, and EK328 (95%-96%). Consequently, the Siberian subtype strains isolated from Finland and nearby Baltic states form a lineage together within the Siberian subtype. Other Siberian subtype strains Vasilchenko, Aina, Zausaev, and TBEV228 showed 92%-94% identity, and the European and Far Eastern subtypes showed 84%-86% identity. However, the vector tick species for Siberian-type TBEV, I. persulcatus, was not known to exist in Finland. This knowledge led us to study the tick species more carefully. DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. was isolated from 20 tick pools by TriPure and resuspended in 100 [micro]L of TE (Tris-HCl 10 mmol/L, EDTA EDTA: see chelating agents. 1 mmol/L, pH [approximately equal to] 8). The tick species was determined as I. persulcatus by amplifying an average of 339 bp from mitochondrial mitochondrial pertaining to mitochondria. mitochondrial RNAs a unique set of tRNAs, mRNAs, rRNAs, transcribed from mitochondrial DNA by a mitochondrial-specific RNA polymerase, that account for about 4% of the total cell RNA that 16S RNA gene by PCR and subsequent sequencing according to Caporale et al. (14). Because the ticks were pooled and homogenized before species identification, 30 adult ticks from the same region collected later in the summer were examined microscopically. All these specimens were I. persulcatus by morphologic criteria. [FIGURE 2 OMITTED] Conclusions A TBE focus has existed in the Kokkola Archipelago at least since the 1960s when TBEV antibodies were detected in cattle (10), but the local TBE viruses have not been characterized. A cluster of severe human cases in the beginning of the 2000s prompted us to carry out the present study. In June 2004, we collected 1,181 ticks from the Archipelago of Kokkola and detected TBEV-RNA in 13 pools. Eleven were also positive in virus isolation. The sequences showed that the strains belonged to the Siberian subtype, whereas in the other Finnish TBE-endemic foci, only European subtype TBEV closely related to the central European strains has previously been detected (15). Furthermore, the tick species was I. persulcatus. Our results show that both I. persulcatus and the Siberian type TBEV are occurring several hundreds of kilometers further to northwest than what has been known previously. Because the 2 tick species are similar to the naked eye and in their behavior, and published data on distribution of Ixodes ticks in Finland are sparse and outdated, we cannot exclude the possibility that I. persulcatus also exists unnoticed elsewhere in Finland. More tick surveys and epidemiologic studies are needed to map the distribution areas of the 2 vector species and of the different TBEV subtypes in Finland. However, in our recent tick collections from Aland and southern (Isosaari, 60[degrees]N, 25[degrees]E) and eastern (Lappeenranta, 61[degrees]N, 28[degrees]E, and Joensuu, 62[degrees]N, 29[degrees]E) Finland, all ticks were I. ricinus. Some researchers have found indications that the Siberian TBEV might cause more severe or more persistent forms of TBE than the European subtype (4), and 3 of 5 recent human TBE cases in Kokkola have been severe (11,12). However, the number of cases studied from Kokkola is too small for firm conclusions on the severity of the local disease. Acknowledgments We thank all who participated in the tick collecting in Kokkola Archipelago in June 2004: Anna Katz, Tytti Manni, Niina Putkuri, Auli Saarinen, Pirjo Sarjakivi, Elina Tikkakoski, and Mikko Tikkakoski. We also thank Simo Raisanen for delivering more tick material from the region; Pirjo Sarjakivi and Tytti Manni for excellent technical assistance; and Lorenza Beati, Thomas G. T. Jaenson, and Juhani Terhivuo for help in studying the morphology of ticks. The Helsinki Biomedical bi·o·med·i·cal adj. 1. Of or relating to biomedicine. 2. Of, relating to, or involving biological, medical, and physical sciences. Graduate School provided half of A. Jaaskelainen's salary since the beginning of January 2005, and the Finnish Funding Agency for Technology and Innovation provided other financial support from grant nos. 2343/31/05 and 1696/31/04. Baxter Oy provided financial support for the tick safari to Kokkola. References (1.) Ecker M, Allison SL, Meixner T, Heinz FX. Sequence analysis and genetic classification of tick-borne encephalitis viruses tick-borne encephalitis virus n. An arbovirus of the genus Flavivirus that occurs in two subtypes, Central European and Eastern, causing two forms of encephalitis; it is transmitted by ticks. from Europe and Asia. J Gen Virol. 1999;80:179-85. (2.) Hayasaka D, Ivanov L, Leonova GN, Goto A, Yoshii K, Mizutani T, et al. Distribution and characterization of tick-borne encephalitis viruses from Siberia and far-eastern Asia. J Gen Virol. 2001;82:1319-28. (3.) Lundkvist K, Vene S, Golovljova I, Mavtchoutko V, Forsgren M, Kalnina V, et al. Characterization of tick-borne encephalitis virus from Latvia: evidence for co-circulation of three distinct subtypes. J Med Virol. 2001;65:730-5. (4.) Chattel chattel (chăt`əl), in law, any property other than a freehold estate in land (see tenure). A chattel is treated as personal property rather than real property regardless of whether it is movable or immovable (see property). RN, Attoui H, Butenko AM, Clegg JC, Deubel V, Frolova TV, et al. Tick-borne virus diseases of human interest in Europe. Clin Microbiol Infect. 2004; 10:1040-55. (5.) Golovljova I, Vene S, Sjolander KB, Vasilenko V, Plyusnin A, Lundkvist A. Characterization of tick-borne encephalitis virus from Estonia. J Med Virol. 2004;74:580-8. (6.) Jaenson TG, Talleklint L, Lundqvist L, Olsen B, Chirico J, Mejlon H. Geographical distribution, host associations, and vector roles of ticks (Acari: Ixodidae, Argasidae) in Sweden. J Med Entomol. 1994;31:240-56. (7.) Ohman C. The geographical and topographical distribution of Ixodes ricinus in Finland. Acta Societatis Pro Fauna et Flora Fennica. 1961;76:1-25. (8.) International Scientific Working Group on Tick-Borne Encephalitis. 2006 Jan 11 [cited 2006 Mar 14]. Available from http://www.tbeinfo.com/tbe.aspx (9.) Wahlberg P, Saikku P, Brummer-Korvenkontio M. Tick-borne viral encephalitis viral encephalitis Viral meningoencephalitis Neurology, infectious disease A general term for nonpurulent–'aseptic' viral infection of the CNS Etiology Coxsackie A and B–eg, A7, enterovirus 71, herpes simplex, etc Clinical If the viral load is extreme, in Finland. The clinical features of Kumlinge disease during 1959-1987. J Intern Med. 1989;225:173-7. (10.) Tuomi J, Brummer-Korvenkontio M. Antibodies against viruses of the tick-borne encephalitis group in cattle sera in Finland. Ann Med Exp Biol Fenn. 1965;43:149-54. (11.) Marjelund S, Tikkakoski T, Tuisku S, Raisanen S. Magnetic resonance imaging magnetic resonance imaging (MRI), noninvasive diagnostic technique that uses nuclear magnetic resonance to produce cross-sectional images of organs and other internal body structures. findings and outcome in severe tick-borne encephalitis. Report of four cases and review of the literature. Acta Radiol. 2004;45:88-94. (12.) Marjelund S, Jaaskelainen A, Tikkakoski T, Tuiskru S, Vapalahti O. Gadolinium gadolinium (gădəlĭn`ēəm), metallic chemical element; symbol Gd; at. no. 64; at. wt. 157.25; m.p. 1,312°C;; b.p. 3,233°C;; sp. gr. 7.898 at 25°C;; valence +3. enhancement of the cauda equina--a new MR imaging finding in a myeloradiculitic form of tick-borne encephalitis. AJNR AJNR American Journal of Neuroradiology Am J Neuroradiol. 2006;27:995-7. (13.) Puchhammer-Stockl E, Kunz C, Mandl CW, Heinz FX. Identification of tick-borne encephalitis virus ribonucleic acid Ribonucleic acid (RNA) One of the two major classes of nucleic acid, mainly involved in translating into proteins the genetic information that is carried in deoxyribonucleic acid (DNA). in tick suspensions and in clinical specimens by a reverse transcription-nested polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is assay, clin Diagn Virol. 1995;4:321-6. (14.) Caporale DA, Rich SM, Spielman A, Telford SR III, Kocher TD. Discriminating between Ixodes ticks by means of mitochondrial DNA sequences. Mol Phylogenet Evol. 1995;4:361 5. (15.) Han X, Aho M, Vene S, Peltomaa M, Vaheri A, Vapalahti O. Prevalence of tick-borne encephalitis virus in Ixodes ricinus ticks in Finland. J Med Virol. 2001;64:21-8. Address for correspondence: Anu Jaaskelainen, Department of Virology virology, study of viruses and their role in disease. Many viruses, such as animal RNA viruses and viruses that infect bacteria, or bacteriophages, have become useful laboratory tools in genetic studies and in work on the cellular metabolic control of gene expression , Haartman Institute, PO Box 21 (Haartmaninkatu 3), FIN-00014, University of Helsinki The University of Helsinki is not to be confused with the Helsinki University of Technology. The University of Helsinki (Finnish: Helsingin yliopisto, Swedish: Helsingfors universitet , Helsinki, Finland; email: anu.jaaskelainen@ helsinki.fi Anu E. Jaaskelainen, * Tapani Tikkakoski, ([dagger]) Nathalie Y. Uzcategui, * Andrey N. Alekseev, ([double dagger]) Antti Vaheri, * ([section]) and Olli Vapalahti * ([section]) ([paragraph]) * Haartman Institute, University of Helsinki, Helsinki, Finland; ([dagger]) Keski-Pohjanmaa Central Hospital, Kokkola, Finland; ([double dagger]) Russian Academy of Sciences Russian Academy of Sciences (Russian: Росси́йская Акаде́мия Нау́к, , St. Petersburg, Russia; ([section]) HUSLAB Hospital District of Helsinki and Uusimaa, Helsinki, Finland; and ([paragraph]) Faculty of Veterinary Medicine veterinary medicine, diagnosis and treatment of diseases of animals. An early interest in animal diseases is found in ancient Greek writings on medicine. Veterinary medicine began to achieve the stature of a science with the organization of the first school in the , University of Helsinki, Helsinki, Finland Ms Jaaskelainen is a PhD candidate at Helsinki Biomedical Graduate School and conducts research in the Department of Virology, University of Helsinki. Besides the molecular epidemiology molecular epidemiology Molecular medicine An evolving field that combines the tools of standard epidemiology–case studies, questionnaires and monitoring of exposure to external factors with the tools of molecular biology–eg, restriction endonucleases, of tickborne encephalitis virus, her interests include developing recombinant antigens for flavivirus diagnostics.
Table 1. Tick collection sites in Kokkola Archipelago, June 2004 *
TBE case-patient,
Location sex/age/y No. ticks (n/f/m) No. tick pools
1 M/48/2004 (12) 184 (19/87/78) 19
2 M/21/2002 (11) 80 (24/25/31) 8
3 F/24/2002 (11) 158 (2/87/69) 16
4 F/12/2002 (11) 474 (16/227/231) 48
5 F/73/2002 (11) 41 (2/22/17) 5
6 M/6/2003 166 (6/83/77) 17
7 M/7/2003 6(0/3/3) 1
8 No known cases 55 (1/25/29) 6
9 No known cases 9(2/5/2) 1
10 No known cases 8(0/6/2) 1
Tick pools positive Tick pools positive
Location in RT-PCR ([dagger]) in virus isolation ([dagger])
1 4, 8, 9 8, 9
2 25, 26 25, 26
3 39 39
4 79, 81, 84, 85, 86, 102, 118 79, 81, 84, 86, 102, 118
5 -- ND
6 -- ND
7 -- ND
8 -- ND
9 -- ND
10 -- ND
* TBE, tickborne encephalitis; RT-PCR, reverse transcription-polymerase
chain reaction; n, nymphs; f, adult females; m, adult males, ND, not
determined.
([dagger]) Nos. indicate the tick pool numbers, the same as used in the
phylogenetic tree.
Table 2. TBE virus strains compared by sequence analysis *
GenBank
Strain Geographic origin accession no.
Kokkola 4 Location 1, Kokkola DQ451297 ([dagger])
Kokkola 8 Location 1, Kokkola DQ451298, ([dagger])
DQ451286 ([double dagger])
Kokkola 9 Location 1, Kokkola DQ451299, ([dagger])
DQ451287 ([double dagger])
Kokkola 25 Location 2, Kokkola DQ451300, ([dagger])
DQ451288 ([double dagger])
Kokkola 26 Location 2, Kokkola DQ451301, ([dagger])
DQ451289 ([dagger])
Kokkola 39 Location 3, Kokkola DQ451302, ([dagger])
DQ451290 ([double dagger])
Kokkola 79 Location 4, Kokkola DQ451303 ([dagger])
DQ451291 ([double dagger])
Kokkola 81 Location 4, Kokkola DQ451304, ([dagger])
DQ451292 ([double dagger])
Kokkola 84 Location 4, Kokkola DQ451305, ([dagger])
DQ451293 ([double dagger])
Kokkola 85 Location 4, Kokkola DQ451306 ([dagger])
Kokkola 86 Location 4, Kokkola DQ451307, ([dagger])
DQ451294 ([double dagger])
Kokkola 102 Location 4, Kokkola DQ451308, ([dagger])
DQ451295 ([double dagger])
Kokkola 118 Location 4, Kokkola DQ451309, ([dagger])
DQ451296 ([double dagger])
Iso40 Isosaari, Finland AJ298323
Kumlinge A 52 Aland, Finland X60286
Est54 Estonia DQ393773
Est3535 Estonia D0393774
Est2546 Estonia DQ393779
Est3476 Estonia DQ393776
Latvia 1-96 Latvia AJ415565
RK1424 Latvia AF091016
Neudoerfl Austria U27495
Hypr Czech Republic U39292
263 Czech Republic U27491
Zausaev Siberia, Russia AF527415
Vasilchenko Novosibirsk, Russia L40361
Aina Irkutsk, Russia AF091006
EK-328 Estonia DQ486861
TBEV228 Novosibirsk region, Russia DQ385498
TBEV1467 Novosibirsk region, Russia AY753582
Sofjin-HO Primorskii Kray, Russia AB062064
Oshima 5-10 Hokkaido, Japan ABO62063
LIV United Kingdom NC 001809
OHFV, strain Russia AY193805
Bogoluvovska
Langat Malaysia AF253419
Powassan, LB United States NC 003687
strain
* TBE, tickborne encephalitis; LIV, Louping ill virus; OHFV, Omsk
hemorrhagic fever virus.
([dagger]) Accession no. for the partial NS5 gene sequence.
([double dagger]) Accession no. for the partial E gene sequence.
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