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Complete Genome Sequences of Four European Subtype Strains of Tick-Borne Encephalitis Virus from Eastern Siberia, Russia Renat V. Adelshin,a,b Olga V. Melnikova,a Ludmila S. Karan,c Evgeny I. Andaev,a Sergey V. Balakhonova Irkutsk Anti-Plague Research Institute of Siberia and Far East, Rospotrebnadzor, Irkutsk, Russiaa; Irkutsk State University, Irkutsk, Russiab; Central Institute of Epidemiology, Rospotrebnadzor, Moscow, Russiac
Received 7 May 2015 Accepted 13 May 2015 Published 18 June 2015 Citation Adelshin RV, Melnikova OV, Karan LS, Andaev EI, Balakhonov SV. 2015. Complete genome sequences of four European subtype strains of tick-borne encephalitis virus from eastern Siberia, Russia. Genome Announc 3(3):e00609-15. doi:10.1128/genomeA.00609-15. Copyright © 2015 Adelshin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Renat V. Adelshin, [email protected].
T
here are three main subtypes of tick-borne encephalitis virus (TBEV): 1, Far-Eastern; 2, European (Western); and 3, Siberian (1). Each subtype dominates its respective area. However, there are many cases when the TBEV subtypes have been detected in unusual areas, such as the Siberian subtype in Europe (2, 3), the Far-Eastern subtype in Crimea (4, 5) and the European part of Russia (6), and the European subtype in South Korea (7). The Siberian TBEV subtype absolutely dominates in the eastern Siberia nowadays, the Far-Eastern subtype is now found not so often as 30 to 40 years ago, and the European subtype occurs in isolated cases (8). The TBEV strains of the European subtype reported to date in eastern Siberia have been isolated from small mammals and from a blood sample of the only recorded infected patient (8). We present here the complete genome sequences of four European subtype strains of TBEV isolated from Ixodes persulcatus ticks, collected by flagging in TBE natural foci throughout Irkutsk city suburbs, and from the brain of a shrew (Sorex sp.) captured in one of these foci. The strains were named IrkutskBR_99-08, IrkutskBR_1434-09, IrkutskBR_1456-09, and Sorex 18-10. The strain IrkutskBR_99-08 was isolated by infecting porcine embryo kidney cells with an enzyme-linked immunosorbent assay (ELISA)-negative male tick suspension. The strains IrkutskBR_1434-09 and IrkutskBR_1456-09 were isolated by intracerebral inoculation of newborn nonlinear laboratory mice with a poor ELISA-positive female tick suspension. The strain Sorex 18-10 was isolated by inoculating newborn nonlinear laboratory mice with 10% saline brain suspension. Each strain is highly neurotropic for laboratory mice whether inoculated through the intracerebral or peripheral route. Total RNA was extracted from brain tissue (10% suspension in saline) using the RiboPrep kit (ILS Ltd., Russia). Reverse transcription was performed by means of the Reverta-L 100 kit containing random hexanucleotides (Central Research Institute of Epidemiology, Moscow, Russia). Amplification of cDNA fragments was performed with a PCR kit (Sintol, Moscow, Russia) and a set of primers (see the GenBank submissions). The PCR products were sequenced with the ABI Prism BigDye Terminator version 1.1 cycle
May/June 2015 Volume 3 Issue 3 e00609-15
sequencing kit on the Genetic Analyzer 3500xl (Applied Biosystems). The sequences (10,471 bp) were aligned in the BioEdit version 7.0.5.3 (9). Phylogenetic analysis was performed using the MEGA5 program (10) using the maximum-likelihood method. Each nucleotide sequence was compared to that of the European prototype strain Neudoerfl (U27495), and the following identities were shown: 97.2% for IrkutskBR_99-08 and 97.3% for IrkutskBR_1434-09, IrkutskBR_1456-09, and Sorex 18-10. To summarize, we have provided the first complete genome sequences of three European subtype strains of TBEV isolated from taiga ticks (I. persulcatus) and from a shrew (Sorex sp.) in eastern Siberia. Nucleotide sequence accession numbers. The complete sequences have been deposited in GenBank under the accession numbers KP331441 (IrkutskBR_99-08), KP331442 (IrkutskBR_1434-09), KP331443 (IrkutskBR_1456-09), and KP938507 (Sorex 18-10). ACKNOWLEDGMENT This research was supported by Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor).
REFERENCES 1. Ecker M, Allison SL, Meixner T, Heinz FX. 1999. Sequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and Asia. J Gen Virol 80:179 –185. 2. Tonteri E, Jääskeläinen AE, Tikkakoski T, Voutilainen L, Niemimaa J, Henttonen H, Vaheri A, Vapalahti O. 2011. Tick-borne encephalitis virus in wild rodents in winter, Finland, 2008 –2009. Emerg Infect Dis 17:72–75. http://dx.doi.org/10.3201/eid1701.100051. 3. Jääskeläinen AE, Tikkakoski T, Uzcátegui NY, Alekseev AN, Vaheri A, Vapalahti O. 2006. Siberian subtype tickborne encephalitis virus, Finland. Emerg Infect Dis 12:1568 –1571. http://dx.doi.org/10.3201/ eid1210.060320. 4. Evstaf’ev IL. 2001. Results of the 20-year study of tick-borne encephalitis in Crimea. Zh Mikrobiol Epidemiol Immunobiol 2:53–57. (In Russian.) 5. Iurchenko OA, Vinograd NA, Dubina DA. 2012. Molecular genetic characterization of tick-borne encephalitis in Crimea. Vopr Virusol 3:40 – 43. (In Russian.)
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Three tick-borne encephalitis virus (TBEV) strains were isolated from Ixodes persulcatus ticks, and one was isolated from a shrew in the territory of eastern Siberia (Russia). The level of sequence identity compared to Neudoerfl (the European prototype strain) is 97.2 to 97.3%.
Adelshin et al.
6. Druzhinina TA, Pogodina VV, Bochkova NG, Yushchenko GV, Skorodumova LV, Sokolova IA, Barmotina TP. 2002. Tick-borne encephalitis in the Yaroslavl region: epidemiological aspects and prophylaxis. Epidemiol Infect Dis 5:13–16. (In Russian.) 7. Ko S, Kang JG, Kim SY, Kim HC, Klein TA, Chong ST, Sames WJ, Yun SM, Ju YR, Chae JS. 2010. Prevalence of tick-borne encephalitis virus in ticks from southern Korea. J Vet Sci 11:197–203. http://dx.doi.org/ 10.4142/jvs.2010.11.3.197. 8. Demina TV, Dzhioev YP, Verkhozina MM, Kozlova IV, Tkachev SE, Plyusnin A, Doroshchenko EK, Lisak OV, Zlobin VI. 2010. Genotyping
and characterization of the geographical distribution of tick-borne encephalitis virus variants with a set of molecular probes. J Med Virol 82: 965–978. http://dx.doi.org/10.1002/jmv.21765. 9. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 95–98. 10. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. http://dx.doi.org/10.1093/molbev/msr121.
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May/June 2015 Volume 3 Issue 3 e00609-15
Complete Genome Sequences of Four European Subtype Strains of Tick-Borne Encephalitis Virus from Eastern Siberia, Russia Renat V. Adelshin,a,b Olga V. Melnikova,a Ludmila S. Karan,c Evgeny I. Andaev,a Sergey V. Balakhonova Irkutsk Anti-Plague Research Institute of Siberia and Far East, Rospotrebnadzor, Irkutsk, Russiaa; Irkutsk State University, Irkutsk, Russiab; Central Institute of Epidemiology, Rospotrebnadzor, Moscow, Russiac
Received 7 May 2015 Accepted 13 May 2015 Published 18 June 2015 Citation Adelshin RV, Melnikova OV, Karan LS, Andaev EI, Balakhonov SV. 2015. Complete genome sequences of four European subtype strains of tick-borne encephalitis virus from eastern Siberia, Russia. Genome Announc 3(3):e00609-15. doi:10.1128/genomeA.00609-15. Copyright © 2015 Adelshin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Renat V. Adelshin, [email protected].
T
here are three main subtypes of tick-borne encephalitis virus (TBEV): 1, Far-Eastern; 2, European (Western); and 3, Siberian (1). Each subtype dominates its respective area. However, there are many cases when the TBEV subtypes have been detected in unusual areas, such as the Siberian subtype in Europe (2, 3), the Far-Eastern subtype in Crimea (4, 5) and the European part of Russia (6), and the European subtype in South Korea (7). The Siberian TBEV subtype absolutely dominates in the eastern Siberia nowadays, the Far-Eastern subtype is now found not so often as 30 to 40 years ago, and the European subtype occurs in isolated cases (8). The TBEV strains of the European subtype reported to date in eastern Siberia have been isolated from small mammals and from a blood sample of the only recorded infected patient (8). We present here the complete genome sequences of four European subtype strains of TBEV isolated from Ixodes persulcatus ticks, collected by flagging in TBE natural foci throughout Irkutsk city suburbs, and from the brain of a shrew (Sorex sp.) captured in one of these foci. The strains were named IrkutskBR_99-08, IrkutskBR_1434-09, IrkutskBR_1456-09, and Sorex 18-10. The strain IrkutskBR_99-08 was isolated by infecting porcine embryo kidney cells with an enzyme-linked immunosorbent assay (ELISA)-negative male tick suspension. The strains IrkutskBR_1434-09 and IrkutskBR_1456-09 were isolated by intracerebral inoculation of newborn nonlinear laboratory mice with a poor ELISA-positive female tick suspension. The strain Sorex 18-10 was isolated by inoculating newborn nonlinear laboratory mice with 10% saline brain suspension. Each strain is highly neurotropic for laboratory mice whether inoculated through the intracerebral or peripheral route. Total RNA was extracted from brain tissue (10% suspension in saline) using the RiboPrep kit (ILS Ltd., Russia). Reverse transcription was performed by means of the Reverta-L 100 kit containing random hexanucleotides (Central Research Institute of Epidemiology, Moscow, Russia). Amplification of cDNA fragments was performed with a PCR kit (Sintol, Moscow, Russia) and a set of primers (see the GenBank submissions). The PCR products were sequenced with the ABI Prism BigDye Terminator version 1.1 cycle
May/June 2015 Volume 3 Issue 3 e00609-15
sequencing kit on the Genetic Analyzer 3500xl (Applied Biosystems). The sequences (10,471 bp) were aligned in the BioEdit version 7.0.5.3 (9). Phylogenetic analysis was performed using the MEGA5 program (10) using the maximum-likelihood method. Each nucleotide sequence was compared to that of the European prototype strain Neudoerfl (U27495), and the following identities were shown: 97.2% for IrkutskBR_99-08 and 97.3% for IrkutskBR_1434-09, IrkutskBR_1456-09, and Sorex 18-10. To summarize, we have provided the first complete genome sequences of three European subtype strains of TBEV isolated from taiga ticks (I. persulcatus) and from a shrew (Sorex sp.) in eastern Siberia. Nucleotide sequence accession numbers. The complete sequences have been deposited in GenBank under the accession numbers KP331441 (IrkutskBR_99-08), KP331442 (IrkutskBR_1434-09), KP331443 (IrkutskBR_1456-09), and KP938507 (Sorex 18-10). ACKNOWLEDGMENT This research was supported by Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor).
REFERENCES 1. Ecker M, Allison SL, Meixner T, Heinz FX. 1999. Sequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and Asia. J Gen Virol 80:179 –185. 2. Tonteri E, Jääskeläinen AE, Tikkakoski T, Voutilainen L, Niemimaa J, Henttonen H, Vaheri A, Vapalahti O. 2011. Tick-borne encephalitis virus in wild rodents in winter, Finland, 2008 –2009. Emerg Infect Dis 17:72–75. http://dx.doi.org/10.3201/eid1701.100051. 3. Jääskeläinen AE, Tikkakoski T, Uzcátegui NY, Alekseev AN, Vaheri A, Vapalahti O. 2006. Siberian subtype tickborne encephalitis virus, Finland. Emerg Infect Dis 12:1568 –1571. http://dx.doi.org/10.3201/ eid1210.060320. 4. Evstaf’ev IL. 2001. Results of the 20-year study of tick-borne encephalitis in Crimea. Zh Mikrobiol Epidemiol Immunobiol 2:53–57. (In Russian.) 5. Iurchenko OA, Vinograd NA, Dubina DA. 2012. Molecular genetic characterization of tick-borne encephalitis in Crimea. Vopr Virusol 3:40 – 43. (In Russian.)
Genome Announcements
genomea.asm.org 1
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Three tick-borne encephalitis virus (TBEV) strains were isolated from Ixodes persulcatus ticks, and one was isolated from a shrew in the territory of eastern Siberia (Russia). The level of sequence identity compared to Neudoerfl (the European prototype strain) is 97.2 to 97.3%.
Adelshin et al.
6. Druzhinina TA, Pogodina VV, Bochkova NG, Yushchenko GV, Skorodumova LV, Sokolova IA, Barmotina TP. 2002. Tick-borne encephalitis in the Yaroslavl region: epidemiological aspects and prophylaxis. Epidemiol Infect Dis 5:13–16. (In Russian.) 7. Ko S, Kang JG, Kim SY, Kim HC, Klein TA, Chong ST, Sames WJ, Yun SM, Ju YR, Chae JS. 2010. Prevalence of tick-borne encephalitis virus in ticks from southern Korea. J Vet Sci 11:197–203. http://dx.doi.org/ 10.4142/jvs.2010.11.3.197. 8. Demina TV, Dzhioev YP, Verkhozina MM, Kozlova IV, Tkachev SE, Plyusnin A, Doroshchenko EK, Lisak OV, Zlobin VI. 2010. Genotyping
and characterization of the geographical distribution of tick-borne encephalitis virus variants with a set of molecular probes. J Med Virol 82: 965–978. http://dx.doi.org/10.1002/jmv.21765. 9. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 95–98. 10. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. http://dx.doi.org/10.1093/molbev/msr121.
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