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a 15-year - old boy received 2 tick bites near the village of glubinnoe , in the northern part of primorsky district , on may 30 and june 1 , 2004 , respectively .
the first prodromal symptoms developed on june 8 . a high fever with strong headache and paresis of cervical muscles developed in the next 2 days . on june 11 , the patient was transported by emergency airplane from a local hospital to the regional clinical hospital no . 1 in vladivostok , where high fever with pronounced meningeal symptoms and complete disorientation in place and time were observed . coma and acute respiratory dysfunction due to paralysis of respiratory muscles developed the next day , and the patient was put on an automatic respirator .
virus strain glubinnoe/2004 was isolated from a brain sample from the patient by using pig kidney embryo ( pke ) cells .
one hundred microliters homogenized brain diluted 1:100 was applied onto pke cells in minimal essential medium supplemented with 2% fetal calf serum .
the cells were incubated at 37c for 4 days , and cell culture supernatants were used for second passage on pke cells .
the virus for sequencing and immunologic experiments was purified from infected pke cells on third passage by centrifugation on a sucrose gradient ( 11 ) .
the protocol of study was approved by the institutional review board of the institute of epidemiology and microbiology , vladivostok .
a panel of monoclonal antibodies ( mabs ) to tbev was used for elisa as described earlier ( 12 ) .
viral rna was extracted by using a ribo - sorb kit ( interlabservice inc . , moscow , russia ) ; then rna was transcribed to cdna and amplified by pcr .
the purified cdna fragment was used for sequencing in a beckman ceq2000xl sequencer ( beckman coulter , inc . ,
fifty primers were designed on the basis of the tbev sequence ( genbank dq989336 ) , allowing 150-bp overlap between adjacent pcr fragments .
each pcr fragment was independently amplified and sequenced 3. sequences were aligned with clustalx ( 13 ) .
molecular data were statistically processed by using the program mega ( 14 ) . the program phylip version 3.57 ( university of washington , seattle , wa , usa ) or puzzle version 4.0.2 ( university of munich , munich , germany ) was used for constructing a phylogenetic tree .
the nucleotide sequences of the viral isolates were compared with published complete polyprotein sequences of tbev ( table 1 ) .
we found profound differences in the nucleotide sequences between listed tbev strains and the glubinnoe/2004 strain .
the identity with 2 typical far eastern strains , 205 and sofjin - ho , was 95.2% and 94.9% , respectively ( table 1 ) .
the conservation of the strain s nucleotide sequences ranged from 82.7% to 95.2% , whereas amino acid sequences conservation ranged from 91.0% to 98.4% , depending on the type of tbev ( table 1 ) . to further delineate the genetic variation , we analyzed polyprotein sequences of 37 other flaviviruses in comparison with glubinnoe/2004 ( table 2 ) .
fifty - three and 57 amino acid ( aa ) substitutions were found when glubinnoe/2004 was compared to strains 205 and sofjin - ho , respectively , and 14 of these were unique substitutions for all studied flaviviruses .
most substitutions were located in the c - terminal hydrophobic domain ( cthd ) of proteins c , ns3 , and ns5 .
the cthd had 5 substitutions in 20 aa fragment ; ns3 , 10 substitutions , and ns5,16 substitutions .
we also found that 5 putative cleavage sites of polyprotein were modified ; the changes were located in viral c / cthd and anchored c / prm sites .
no substitutions were found in well - known features of protein e , such as the 12 cysteine residues , potential n - glycosylation sites , fusion peptide , and dexh core motif of the ns3 helicase .
a cysteine residue in position 4 of ns1 protein was replaced with a glycine ; this mutation was described previously only for sofjin - ho ( bab72162 ) .
nt , nucleotide ; aa , amino acid .  time of submission of the sequence to genbank . 
virus was isolated from a patient with chronic form of tbev infection ; a patient was infected in 1973 in tomsk ; the virus was isolated in moscow in 1985 from brain .
* position number corresponds to the polyprotein sequence glubinnoe/2004 of tickborne encephalitis virus ( tbev ) .
the following polyprotein sequences were used for polyprotein alignment : dq862460 ( tbev , glubinnoe/2004 ) ; abj74160 ( tbev , 205 ) ; aan73266 ( tbev , senzhang ) ; bab71943 ( tbev , oshima 510 ) ; bab72162 ( tbev , sofjin - ho ) ; af 069066 ( tbev , vasilchenko);aad34205 ( tbev , vasilchenko ) ; abf46836 ( tbev , ek-238 ) ; aao43537 ( tbev , zausaev ) ; np_043135   ( tbev , neudoerfl ) ; aaz80455 ( tbev , ts - variant of strain 263 ) ; aaa86739 ( tbev , 263 ) ; q01299 ( tbev , hypr ) ; abb90676 ( spanish sheep encephalitis virus ) ; bb90675 ( turkish sheep encephalitis virus ) ; aaq91606 ( ohfv , bogolubovka ) ; np 878909 ( ohfv , bogoluvovska ) ; aar98531 ( ohfv , kubrin ) ; abb90677 ( greek goat encephalitis virus ) ; np_044677 ( louping ill virus);np_620108 ( langat virus , tp21 ) ; aaf75260 ( langat virus , e5 ) ; p29837 ( langat virus , tp21);aaq91607 ( kyasanur forest disease virus ) ; np_722551 ( alkhurma virus ) ; np_620099 ( powassan virus);aal32169 ( deer tick virus ) ; abb90669 ( gadgets gully virus ) ; abb90671 ( karshi virus ) ;
yp_224133 ( karshi virus ) ; abe73208 ( karshi virus ) ; abb90673 ( royal farm virus ) ; abb90670 ( kadam virus ) ; abb90674 ( saumarez reef virus ) ; abb90668 ( meaban virus ) ; abb90672 ( tyuleniy virus ) ; np_689391 ) .
we also performed phylogenetic analysis of the fully sequenced tbev strains and glubinnoe/2004 ( figure 1 ) and analyzed 100 different protein e gene sequences available in genbank ( data not shown ) . both phylogenetic trees clearly demonstrated that glubinnoe/2004 belonged to the eastern type and formed a separate clade ( branch or group ) within the type
the time of divergence of glubinnoe/2004 from the oshima - sofjin and senzhang groups was calculated by using the average substitution rate analysis ( 15 ) .
we estimate that glubinnoe/2004 and senzhang group diverged 300470 years ago and glubinnoe/2004 and oshima - sofjin group , 320490 years ago .
our findings suggest that parallel evolution of different genetic groups of tbev occurred in the relatively small far eastern region of russia .
phylogenetic tree , based on complete polyprotein sequences of tickborne encephalitis virus ( tbev ) .
the multiple sequence alignments were obtained with clustalx , and the tree was constructed by the neighbor - joining method .
the following sequences were used for the phylogenetic tree : dq862460 ( tbev , glubinnoe/2004 ) abj74160 ( tbev , 205 ) ; aan73266 ( tbev , senzhang ) ; bab71943 ( tbev , oshima 510 ) ; bab72162 ( tbev , sofjin - ho ) ; af069066 ( tbev , vasilchenko ) ; aao43537 ( tbev , zausaev ) ; np_043135 ( tbev , neudoerfl ) ; aaa86739 ( tbev , 263 ) ; q01299 ( tbev , hypr ) ; abb90676 ( spanish sheep encephalitis virus ) ; bb90675 ( turkish sheep encephalitis virus ) ; np_044677 ( louping ill virus);nc_001672 ( tbev ) ; ay217093 ( tbev , mdj-01 ) ; ay968065 ( yellow fever virus , uganda ) ; dq675533 ( dengue 3 ) .
the growth curves for glubinnoe/2004 and 205 viruses in pke cells at 37c are shown in figure 2 .
the maximum difference in virus yields ( in 50% tissue culture infective doses [ tcid50]/ml ) between glubinnoe/2004 and 205 viruses was 2.1 tcid50/ml at 12 h postinfection and nearly 10 from 15 to 36 h. by 72 h postinfection , pke cells infected by glubinnoe/2004 and 205 were completely lysed .
we also evaluated the replication of viruses by directly measuring viral e protein levels by using 2 mabs .
this finding correlated with a production rate of infectious virions with delay close to 6 h , which demonstrates that the glubinnoe/2004 strain may produce more infectious virions in the early stage of infection of a pke cell .
the discovered mutations in 4 of 5 cleavage sites in viral polyprotein are required for cleavage of structural viral proteins .
this finding may explain the more robust formation of infectious virions in the early stages of infection . growth curves and e protein synthesis of glubinnoe/2004 and 205 in pig kidney embryo ( pke ) cells : monolayer of pke cells were infected with glubinnoe/2004 and 205 of tickborne encephalitis virus ( tbev ) .
the cells were frozen and thawed 3 to release the virus , and infectious titers were determined by the viral cytopathic effect ( in 50% tissue culture infective doses [ tcid50]/ml ) assay in pke cells .
elisa using 10h10 and biotin - labeled eb1 anti  protein e monoclonal antibodies was used to determine the e protein levels in virus - infected cells . 1 , glubinnoe/2004 , virus yield ; 2 , 205 , virus yield ; 3 , glubinnoe/2004 , e protein synthesis ; 4 , 205 , e protein synthesis .
serologic data demonstrate that strain glubinnoe/2004 has epitopes well recognized by 14 anti - tbev mabs ( data not shown ) . based on these data
, we concluded that all protein e epitopes of strain 205 virus are also present on glubinnoe/2004 virions and are not affected by 4 aa substitutions found in protein e. this would suggest that modern vaccines against tbe and diagnostic immunologic kits will be effective against this novel variant .
we have genetically characterized tbev isolate glubinnoe/2004 by determining its complete coding nucleotide sequence and comparing it with most of the available tbev sequences .
we found 53 and 57 aa substitutions in comparison with strains 205 and sofjin - ho , respectively , and 14 of these were unique for 37 flaviviruses .
most substitutions were located in the cthd of proteins c , ns3 , and ns5 .
phylogenetic analysis showed that glubinnoe/2004 appears to be a separate lineage within the eastern type of tbev .
we estimate that strain glubinnoe/2004 diverged from eastern tbev in senzhang group 300470 years ago and from viruses in the oshima - sofjin group 320490 years ago .
five putative cleavage sites of the viral polyprotein were changed , including 4 sites responsible for processing of structural proteins .
these data suggest that glubinnoe/2004 could be used for production of vaccines , instead of strains 205 and sofjin , and for development of diagnostic kits .
this conclusion is supported by immunologic data with anti - tbev mabs , which demonstrated that viral glycoprotein e has a conserved antigenic structure typical of the eastern type of tbev .
we isolated a novel strain of tickborne encephalitis virus ( tbev ) , glubinnoe/2004 , from a patient with a fatal case in russia . 
 we sequenced the strain , whose landmark features included 57 amino acid substitutions and 5 modified cleavage sites . 
 phylogenetically , glubinnoe/2004 is a novel variant that belongs to the eastern type of tbev .