serum collected before the appearance of tbev - specific immunoglobulin m ( igm ) ( acute - phase serum ) was available from 2 of 11 tbe patients . the patients , both 38 years of age , included a man from vest - agder county , who had not been abroad during the last 4 weeks before disease , and a woman from hordaland county , who was bitten by a tick on bornholm island . high levels of tbev igm and moderate to high levels of tbev igg were detected in convalescent - phase sera from both patients by enzyme immunoassay ( enzygnost , dade behring , marburg , germany ) . ticks were collected by dragging a blanket in the field in areas where patients with tbe had been reported . all collected ticks were unfed . a total of 360 nymphs , adults , and larvae were collected in may and june 2003 , and 450 nymphs , adults , and larvae were collected from august 27 to october 8 , 2004 . acute - phase sera from the 2 patients and 810 ticks ( i. ricinus ) were examined for tbev . rna was extracted from serum samples and tick suspensions by using the qiaamp viral rna mini kit ( qiagen , germantown , md , usa ) and the tripure rna isolation system ( roche diagnostics , lewes , uk ) , respectively . for initial detection of tbev rna , the 5  noncoding region was amplified by nested rt - pcr ( 6 ) , and positive samples were amplified in the coding e protein region ( nucleotides [ nt ] 13231765 ) . rt was performed by using mmlv ( moloney murine leukemia virus ) rt kit ( invitrogen , carlsbad , ca , usa ) and reverse primer 827r ( nt 17771800 ) ( table ) according to manufacturer 's recommendations . pcr and nested pcr were performed by using primer pairs 283f1 ( nt 12331255 ) to 827r1 ( nt 17771800 ) and 349f2 ( nt 13011322 ) to 814r2 ( nt 17661787 ) , respectively ( table ) . pcr amplicons were purified and sequenced by using a dna sequencing kit ( abi prism , pe biosystems , foster city , ca , usa ) on a 3100 genetic analyzer ( pe biosystems ) . the phylip program package ( 7 ) was used to analyze the e protein gene sequence data : 500 bootstrap replicates ( phylip 's seqboot program ) were fed to the dnadist program ( with kimura 's 2-parameter option ) , distance matrixes were analyzed with the fitch program ( which used the fitch - margoliash algorithm ) , and the bootstrap support values for the tree were calculated with the consense program . consensus phylogenetic tree ( fitch - margoliash ) based on the e protein encoding sequence ( nucleotides 13231765 ) . newly identified tickborne encephalitis virus ( tbev ) strains from norway and denmark are underlined . abbreviations and accession numbers for viruses and tbev strains : powassan virus , powv , strain lb ( l06436 ) ; langat virus , lgtv , strain tp21 ( af253419 ) ; kyasanur forest disease virus , kfdv , ( x74111.1 ) ; omsk hemorrhagic fever virus , ohfv , ( x66694.1 ) ; tbev , strain ir991m4 ( ab049349 ) ; tbev , strain ir992m3 ( ab049350 ) ; tbev , strain ir992m7 ( ab049351 ) ; tbev , strain ir992f7 ( ab049352 ) ; tbev , strain ir992f13 ( ab049353 ) ; tbev , strain ir991m1 ( ab049348 ) ; tbev , strain aina ( af091006 ) ; tbev , strain vasilchenko ( af069066 ) ; tbev , strain latvia 196 ( aj415565 ) ; tbev , strain oshima ( ab022292 ) ; tbev , strain sofjin ( x03870.1 ) ; tbev , strain kh9810 ( ab022297 ) ; tbev , strain tblood ( af091019 ) ; tbev , strain rk1424 ( af091016 ) ; tbev , strain n132 ( af091013 ) ; tbev , strain 263 ( u27491 ) ; tbev , strain latvia-1271800 ( aj319586 ) ; tbev , strain hypr ( u39292 ) ; tbev , strain neudoerfl ( u27495 ) ; tbev , strain latvia-979300 ( aj319585 ) ; tbev , strain latvia-811000 ( aj319583 ) ; tbev , strain kumlinge - a52 ( x60286 ) ; tbev , strain latvia-836900 ( aj19584 ) ; tbev , strain latvia-1168600 ( aj319582 ) ; tbev , strain lithuania-100 ( aj414703 ) ; louping ill virus ( liv ) , strain uk369/t2 ( y07863.1 ) ; liv , strain turkey ( lo1265 ) ; liv , strain greece ( x77732 ) ; liv , strain spain ( x77470.1 ) ; liv , strain japan 3248/49/p10 ( m94956.1 ) . from 360 ticks collected in 2003 , only 1 pool ( 10 ticks ) was positive by amplification of the 5  noncoding region ; similarly , only 1 pool of 450 ticks collected in 2004 was rt - pcr - positive . from the positive tick pool in 2004 , we sequenced 179 nt of the highly conserved region in 5  region of tbev , enough to prove that the virus was tbev , not liv . no material from the tick pools thus , the overall virus prevalence in ticks was 0.3% in 2003 and 0.2% in 2004 , if we assume that only 1 tick in the positive pool was infected . a partial e gene sequence ( 443 bp ) was recovered from the 2 human samples . the identity on the nucleotide level between these sequences was 98.6% , and they showed 97.2%99.0% identity to other tbev strains within the tbev - eu subtype . the levels of sequence identity to strains belonging to tbev - fe and tbev - sib subtypes were 81.4%83.0% and 83.5%86.2% , respectively . phylogenetic analysis of these sequences showed that they belong to the tbev - eu subtype ( figure ) , which does not show clear , separated lineages correlating to geographic regions . the norwegian tbev strain clustered together with strain neudoerfl isolated in austria , and the denmark-1 strain clustered together with the group of strains from latvia , finland ( kumlinge ) , and estonia , albeit bootstrap supports of these clusterings were below the widely accepted confidence limit , 70% . the sequence identity between strains denmark-1 and neudoerfl was 99.0% ; between strains norway-1 and latvia9783 , the sequence identity was 98.7% . one of the 2 patients was infected in vest - agder county in norway , and the other on bornholm island , denmark . genetic analysis showed that the norwegian and danish strains belong to the tbev - eu subtype . although the sequences of norway-1 and denmark-1 strains showed the highest level of identity to the corresponding sequences of tbev - eu subtype , they were distinguishable from each other and also from the sequences of tbev - eu strains characterized previously . in tbe - endemic areas in europe and on bornholm island , 0.5%5% of ticks are infected with tbev ( 5 ) . in norway , where tbe is a rare disease , the prevalence is lower ( 0.2%0.3% ) . in denmark tbev has been detected in ticks by rt - pcr ( 5 ) , but to our knowledge , no reports of tbev findings in danish patients exist . the emergence of tbe in norway in the 1990s poses the question of whether these new endemic foci have become truly established recently or have remained unnoticed because of underdiagnosis . although the northern spread of tbev due to climate changes has been predicted ( 8) , other factors such as rates of contact between ticks and humans , abundance of ticks , and their amplifying hosts may play a role in tbe epidemiology . further monitoring of the tbe situation in norway both in patients and nature is needed to establish guidelines for preventive measures and vaccination programs in tbe - endemic areas . we report the first genome detection and characterization of tbev from persons with tbe in norway and denmark . our results showed that the norwegian and danish strains clustered with earlier reported strains of the tbev - eu subtype .
serum from 2 norwegians with tickborne encephalitis ( tbe ) ( 1 of whom was infected in denmark ) and 810 norwegian ticks were tested for tbe virus ( tbev ) rna by reverse transcription  polymerase chain reaction . 
 sequencing and phylogenetic analysis were performed . 
 this is the first genome detection of tbev in serum from norwegian patients .