- Email: [email protected]
Tick-borne encephalitis virus strains of Western Siberia
Virus Research 70 (2000) 1 – 12 www.elsevier.com/locate/virusres
Tick-borne encephalitis virus strains of Western Siberia V.N. Bakhvalova a, V.A. Rar b, S.E. Tkachev b, V.A. Matveev b, L.E. Matveev b, A.S. Karavanov b, A.K. Dobrotvorsky a, O.V. Morozova b,* a
Institute of Systematics and Ecology of Animals of Siberian Branch of Russian Academy of Sciences, Frunze Street 11, 630091 No6osibirsk 91, Russian Federation b No6osibirsk Institute of Bioorganic Chemistry of Siberian Branch of Russian Academy of Sciences, La6rentye6 Prospect 8, 630090 No6osibirsk 90, Russian Federation Received 10 February 2000; received in revised form 15 March 2000; accepted 19 May 2000
Abstract Tick-borne encephalitis virus (TBEV) strains were isolated from ticks in Western Siberia for 12 years. Molecular hybridization of the 46 viral RNA with the TBEV cDNA and oligonucleotide probes revealed differences between the Siberian and Far Eastern strains. A comparison of the viral E gene fragment nucleotide sequence showed 89 – 98% homology between Siberian TBEV strains, whereas their similarity with strains from other populations was less than 83%. However, the viral E and NS1 glycoprotein antigenic structures appeared to be conservative because of the degenerate genetic code. This was shown by enzyme-linked immunosorbent assay with the corresponding monoclonal antibodies (MAb). The single exception was the MAb 17C3 against nonstructural glycoprotein NS1, which could distinguish Siberian from Far Eastern strains. Moreover, the neurovirulence differed between strains from the two natural populations. Lower neuroinvasiveness of the Siberian strains in comparison with Far Eastern Sofyin strain might be caused by both E and NS1 glycoprotein mutations. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Tick-borne encephalitis virus strains; Genetic subtypes; Monoclonal antibodies; E and NS1 antigenic structures; Neurovirulence
1. Introduction TBEV causes dangerous central nervous system diseases in humans. General infection leads to development of meningitis or encephalitis with fatality ranging from 1 to 30%. Virus persistence in natural foci results in periodic outbreaks in the indigenous human population. * Corresponding author. Tel.: +7-3832-396226; fax: +73832-333677. E-mail address: [email protected] (O.V. Morozova).
The TBEV genome RNA is approximately 11 kb long (Pletnev et al., 1990). Complete nucleotide sequences of genome RNA from both the TBEV strains isolated in the Far East (Sofyin strain Pletnev et al., 1990) and strain 205 (Safronov et al., 1991)) as well as in West Europe (Neudorfl strain Mandl et al., 1989), strains Hypr and 263 (Wallner et al., 1996)) have been determined previously. For several other TBEV strains, nucleotide sequences of genome fragments have been described (Ecker et al., 1999; Zlobin et al.,
0168-1702/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 0 0 ) 0 0 1 7 4 - X
2
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
1996). Genome structures of Western Siberian viral strains have not been studied previously. The TBEV genome encodes three structural and seven nonstructural proteins. RNA is packaged into a nucleocapcid with a protein C having a molecular weight (Mr) of about 14 kDa, which is surrounded by a lipid envelope that contains a glycoprotein E of Mr 50 – 56 kDa and a small nonglycosylated protein M of Mr 7 – 9 kDa (Pletnev et al., 1990). The single NS1 nonstructural glycoprotein presents inside or on the surface of infected cells, or is secreted into cultural medium, thus inducing the production of the protective complement-fixing antibodies (Schlesinger et al., 1986). The TBEV strains are divided into European, Siberian and Far Eastern subtypes (Ecker et al., 1999). The degree of variation between strains within each subtype is low. However, a difference between the TBEV subtypes has been reported for different species of the Fla6i6iridae family (Ecker et al., 1999). In spite of numerous nucleotide changes of TBEV strain genomes, the corresponding proteins are highly conservative. This is due to the degenerate genetic code (Mandl et al., 1989; Pletnev et al., 1990). Comparative analysis of the TBEV E protein of different strains reveals clusters of amino acid variation distributed throughout. However, a major difference is observed within the B domain (Gritsun et al., 1995). Some E protein epitopes are different for European and Far Eastern strains (Pomelova et al., 1991; Tsekhanovskaya et al., 1993). Population features of the TBEV NS1 nonstructural glycoprotein have not been studied before. The present work is devoted to the comparative analysis of the genomes, the antigenic structures and the neurovirulence of the TBEV Western Siberian strains.
2. Materials and methods
2.1. TBEV strains From 1980 to 1992, TBEV strains were isolated from hungry ticks (Ixodes persulcatus) in the wood-steppe part of the Novosibirsk region in
West Siberia. These strains were supported by intracerebral infection of white mice weighing 6–7 g according to the procedure of Gromashevsky (1986). Identification of isolates was performed by means of hemagglutination test (Clarke and Casals, 1958) and biological neutralization using white mice (Habel, 1972; Deryabin et al., 1986). The TBEV Sofyin strain was obtained from the Ivanovsky Virology Institute (Moscow, Russia). These TBEV strains were stored at − 20°C and analyzed after 7–10 passages.
2.2. Neuro6irulence The neurovirulence of the TBEV strains was determined by titering using white mice weighing 6–7 g. The animals were infected both intracerebrally and subcutaneously (0.03 and 0.25 ml, respectively) with ten-times serial dilutions of 10% brain suspensions from infected white mice. Virus titers were calculated in lg LD50 using Kerber method (Semenov, 1965; Jawetz et al., 1982). The neuroinvasiveness index was estimated to be the difference of the intracerebral and subcutaneous titers (Levkovich et al., 1967).
2.3. RNA isolation Viral RNA were isolated from 10% brain suspensions of infected mice by phenol deproteinization, as previously described (Dobrikova et al., 1986).
2.4. Molecular hybridization of TBEV strains RNA with Sofyin strain cDNA probe The TBEV cDNA of the Sofyin strain isolated from a patient in the Far East has previously been cloned (Pletnev et al., 1990). Total RNA concentrations were estimated by measuring the optical densities at 260 nm. RNA from the intact mouse brain was used as a negative control of the hybridization. The positive control was homologous to the radioactive probe TBEV Sofyin strain RNA. Synthesis and characterization of radioactive labeled TBEV cDNA probe as well as hybridization conditions have been previously
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
3
described (Dobrikova et al., 1986; Shamanin et al., 1990).
scribed (Matveev et al., 1989; Matveeva et al., 1998).
2.5. Re6erse transcription– polymerase chain reaction
2.8. Enzyme-linked immunosorbent assay
5%-AGCCACAGGACACGTGTA-3% (E1)(1348 – 1365 nucleotides of Sofyin strain genome) and 5% - CATCTTGACCATGGGAGA - 3% (E2) (1486 – 1503 nucleotides of Sofyin strain genome) deoxyribo-oligonucleotides corresponding to the TBEV Sofyin strain E gene (Pletnev et al., 1990) were synthesized in the Novosibirsk Institute of Bioorganic Chemistry of Siberian Branch of Russian Academy of Sciences. Reverse transcription – polymerase chain reaction (RT-PCR) was carried out with E1 and E2 primers according to Godovikova et al. (1994).
Interaction of E and NS1 glycoproteins of TBEV strains with MAbs was studied by enzymelinked immunosorbent assay (ELISA) as previously described (Matveeva et al., 1998). To increase the sensitivity, 0.13 mg/ml 3,3%,5,5%-tetramethylbenzidin dihydrochloride (TMB) as a chromogen was added. The measuring of optical density at 450 nm was performed no later than 30 min after termination of the reaction. The last dilution of ascitic fluid producing two-times higher optical density of the studied sample in comparison with background was considered as MAb titer.
2.6. Sequencing 3. Results Nucleotide sequences of TBEV strains E gene fragment RT-PCR products were determined according to (Sanger et al., 1977) with radioactive labeled E1 and E2 primers, and T7-modified DNA polymerase (‘Promix’, Novosibirsk, Russia). The following modifications were made: reaction mixture (17.5 ml) containing 2 ml buffer, 100 mM Tris–HCl (pH 8.9), 500 mM KCl, 25 mM MgCl2, 1 U activity of enzyme, 1 pmol 5%-end labeled primer and 20 ng electrophoretically pure amplicon was divided into 4 ml samples in tubes with a 4 ml mixture of 200 mM 4dNTP and 3 mM corresponding ddNTP. The following cycling parameters were used: 94°C for 3 min, 54°C for 1.5 min, 72°C for 2 min, one cycle; 94°C for 1 min, 54°C for 1.5 min, 72°C for 2 min, 30 cycles. Reaction products were fractionated in 7% PAAG with 6 M urea. Nucleotide sequences were compared using the interactive program CLUSTALW (Higgins et al., 1994).
3.1. Molecular hybridization of TBEV strain RNA Genome RNAs of 46 TBEV strains of Western Siberia were investigated via molecular hybridization with [32P]-cDNA of TBEV of the Sofyin strain, which predominates in the Far East (Zlobin et al., 1996). The hybridization occurred both in water and 50% formamid solutions at 45, 55 and 65°C. Hybridization levels of RNA isolated from the Siberian strains with cDNA of the Sofyin strain probe in 50% formamid at 65°C were very low (Fig. 1). Hybridization with the virus-specific cDNA and oligonucleotide probes revealed essential genomic differences between the TBEV strains isolated from the Far Eastern and Siberian populations.
3.2. Nucleotide sequences of TBEV strain E gene fragments
2.7. Monoclonal antibodies Construction and characterization of monoclonal antibodies (MAbs) against the TBEV Sofyin strain E virion glycoprotein and NS1 nonstructural protein have been previously de-
To study genetic homology, six TBEV strains were chosen. Strains 228, 246 and 774 in periods of time when exceptionally high disease rate in humans was observed. Strains 400, 1441 and 1508 have been isolated during the minimal encephali-
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
4
The Lesopark-11 strain isolated by V.I. Zlobin in the Novosibirsk region in 1986 (Shamanin et al., 1990; Zlobin et al., 1996) was found to be a member of the same TBEV Siberian subtype. It is notable that there were no significant differences between E gene fragment structures of the TBEV strands isolated from ticks and from human patients. Genome fragments of the virus strains isolated during periods with different tick-borne encephalitis rates were also quite similar. Inside each TBEV subtype, the homology level was high: 98–100% between the Western European strains 263, Hypr and Neudoerfl; 89–98%
tis rate in Western Siberia. Nucleotide sequences of E gene (1376–1474 nucleotides), which corresponded to the part of E glycoprotein domain that induces neutralization and anti-hemagglutination antibody production (Tsekhanovskaya et al., 1993), were determined (Fig. 2). Comparison of the E gene fragment nucleotide sequences showed 89 – 98% homology between the TBEV Siberian strains (Table 1). All of the strains collected in the Novosibirsk region were similar to Aina/1448 strain (93 – 96% homology), which was isolated from human cerebrospinal fluid in Irkutsk region in 1963 (Pogodina et al., 1981).
Fig. 1. Results of the molecular hybridization of RNA from TBEV strains with radioactive labeled cDNA probe corresponding to Sofyin strain RNA in 50% formamid at the different temperatures. Table 1 Comparative analysis of the TBEV E gene fragment homology (%)
Sofyin 205 Aina/1448 228 246 400 774 1441 1508 Hypr Neudorfl
Sofyin
205
Aina/1448
228
246
400
774
1441
1508
Hypr
Neudorfl
100 94 79 81 78 78 81 80 80 78 77
94 100 79 79 80 76 80 80 82 76 75
79 79 100 95 94 94 93 94 96 83 82
81 79 95 100 90 96 97 98 92 82 81
78 80 94 90 100 89 90 91 97 81 80
78 76 94 96 89 100 94 95 91 81 80
81 80 93 97 90 94 100 98 92 80 79
80 80 94 98 91 95 98 100 93 81 80
80 82 96 92 97 91 92 93 100 83 82
78 76 83 82 81 81 80 81 83 100 98
77 75 82 81 80 80 79 80 82 98 100
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
5
Fig. 2. Nucleotide sequences of E gene fragments of TBEV strains. Conservative nucleotides are marked with * in the last lane. Sequences of the West Siberian strains are underlined and marked bold. (M) after the Siberian strain number means the TBEV strain isolated during the periods of the maximal disease rate in humans in Western Siberia. Far Eastern strains Sofyin and 205 are shown on the top. The Eastern Siberian strain Aina/1448 is before the Western Siberian strains 228, 246, 400, 774, 1441 and 1508. The strains 263, Hypr and neudorfl isolated in Western Europe are demonsrated on the bottom. Other TBEV strains have been found in Europe.
6
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
between the Siberian strains; and 94% for the Far Eastern strains 205 and Sofyin. Similarity of the E gene fragment between the Siberian and European strains was less than 83%, and that between the Siberian and Far Eastern strains was less than 81%. Moreover, the viral populations of West Europe and the Far East differed more essentially from each other (75 – 78% homology). Deduced amino acid sequences revealed 100% homology of the E protein fragment of the TBEV strains.
3.3. ELISA with MAbs against E protein Antigenic structures of E structural and NS1 nonstructural glycoproteins of TBEV strains were investigated by ELISA with the corresponding MAbs panel. To analyze the TBEV E protein antigenic structure, several MAbs against epitopes in different domains of the protein were used: 5F6, 4A6, 3B7, 7C2, 15B1 and 1H11 MAbs (E1 domain); 14D5, 13D6, 1B1, 11D1 and 10C2 MAbs (E2 domain); 2D1 and 5G10 MAbs (E3 domain) (Tsekhanovskaya et al., 1993). 10C2 MAb binds with E protein of all of the flaviviruses of the TBEV complex. The majority of MAbs recognize only particular flavivirus species. 5F6 and 4A6 MAbs are subtype specific. ELISA with MAbs against E protein demonstrated the similarity of this protein among the studied viral strains (Fig. 3). Data for other MAbs were similar. Viral E protein homology is known to be 93 – 96% among the TBEV strains and 36 – 42% for different flaviviruses (Mandl et al., 1989; Pletnev et al., 1990). This coincides with both the deduced amino acid sequences of the protein fragment (Fig. 2) and ELISA data for the TBEV Siberian strains (Fig. 3).
3.4. ELISA with MAbs against NS1 protein The following MAbs were selected for epitope analysis of TBEV NS1 protein: 4C4, 22H8 MAbs were specific for all the TBEV complex flaviviruses; 16B1, 20B4, 17C3 MAbs interacted with several species of the TBEV complex. 29G9 MAb were species specific (Matveeva et al.,
1998). Among the studied strains, 271, 396, 758, 766, 771 and 776 were isolated from ticks in Western Siberia during periods of maximal encephalitis rate in humans, while others in periods of low disease level. ELISA with MAbs against NS1 protein did not reveal the differences between TBEV strains from different populations with one single exception. MAb 17C3, with the epitope near C-terminal part of TBEV NS1 glycoprotein (data not shown), appeared to be able to distinguish the Siberian strains from Sofyin strain (Fig. 4). The data obtained were in agreement with the known high homology of NS1 nonstructural protein (98% homology between TBEV Far Eastern strains and 96–98% homology among West European strains; 90% similarity between glycoproteins of the viral strains from different populations and 37–44% homology for different flavivirus species (Pletnev et al., 1990)). According to our ELISA data (Figs. 3 and 4), NS1 nonstructural glycoprotein was shown to be more variable than virion glycoprotein E.
3.5. Neuro6irulence of TBEV strains In spite of the conservative amino acid sequences and the stable antigenic structures of the TBEV strains, biological properties can vary. Lower lethality level and less severe forms of tick-borne encephalitis for patients in Siberia in comparison with the Far East might indicate decreased neurovirulence of the TBEV Siberian strains. Intracerebral titers of the Western Siberian TBEV strains varied from 5.8 to 8.5 lg LD50. Subcutaneous titers ranged from 2.7 to 7.0 lg LD50. The neuroinvasiveness indexes were 0.3–4.3. Comparative analysis of neurovirulence of TBEV strains isolated from ticks in natural foci of Siberia (Pustovalova et al., 1984) and the Far East (Leonova et al., 1990b; Vereta et al., 1983) showed considerable similarity within the Siberian natural viral population and significant distinction from the Far Eastern strains (Tables 2 and 3). High virulent strains (perifiric titers, \ 6.0 lg LD50) prevailed in the Far East population. The Siberian strains had a moderate neurovirulence (titers, 4.1–5.9 lg LD50) (Table 3).
a
–, The absence of data in publications.
–
51.5 9 8.8
–
15.2 9 6.4
–
87.8 95.8
63.0 95.3
50.0 97.4
43.7 9 7.3
6.3
60.8 9 7.2
56.5 97.8
39.197.9
4.4
6.0. 97.8
45.0 97.9
55.0 98.0
0.0
–
12.2 95.8
26.2 96.4
28.4 9 6.7
37.5 9 7.8
4.9–3.5 lg LD50
]5.0 lg LD50
57.0 lg LD50
]9.0 lg LD50 8.9–7.1 lg LD50
Subcutaneous infection
Intracerebral infection
% strains in groups with different titers
Far East 1. Chabarovsk 33.39 8.3 region (near Amur river). 2. South –
Western Siberia 1. South-Eastern part of Western Siberia (Novosibirsk region) 2. Western part of Western Siberia (Tumen region) 3. East Ural (Ekaterinburg region)
Geographic location of natural focus
Table 2 Structure of TBEV natural populations neurovirulencea
–
0.0
10.8
10.8
2.5 9 2.5
B3.5 lg LD50
59.59 7.5
63.6 98.5
–
–
2.509 6.9
]6.0 lg LD50
0.0
7.193.9
33.3 9 7.2
–
–
12.595.3
54.0 lg LD50
36.4 9 6.4
–
–
62.5 9 7.8
5.9–4.1 lg LD50
Subcutaneous infection V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12 7
8
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
Fig. 3. ELISA results of the TBEV Western Siberian strains with mono- and polyclonal antibodies against E structural glycoprotein.
4. Discussion Our results suggest the existence of the TBEV Siberian subtype (Ecker et al., 1999). The viral strains from Western and Eastern Siberia appeared to have the similar genome and antigenic structures as well as the same biological properties. Further study will enable one to estimate the presence of this genetic subtype in different ecosystems. The stability of the TBEV E gene
during the 12-year period in isolated natural foci is exceptionally notable. This fact attests to the stability of the viral genome RNA in the absence of an RNA reparation system in host cells. The prolonged stability of viral genes could be the result of the elimination of defective virions in the process of natural selection. Revealed significant genetic differences of TBEV strains from different natural populations of Western Siberia and the Far East could be the
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
consequence of TBEV adaptation to certain ecological conditions. The Siberian TBEV population is known to be younger than the Far Eastern population (Vershinsky, 1984). Despite the heterogeneity of the E gene of the TBEV strains, the corresponding E protein is highly conservative (Pletnev et al., 1990; Leonova et al., 1990a). The flaviviral NS1 nonstructural glycoprotein is more variable than the E protein. Therefore, amino acid sequences of epitopes for
9
MAb 17C3 near the NS1 glycoprotein C-end were shown to be different between the Siberian and Far Eastern TBEV strains. Neurovirulence is known to be determined with envelope glycoprotein E, nonstructural proteins and/or the 3%-untranslated region of the genome. The envelope glycoprotein E plays the main role in flavivirus virulence. This is because the single amino acid changes result in the loss of virulence (McMinn, 1997). Mutations in NS1 glycoprotein
Fig. 3. (Continued)
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
10
Fig. 4. ELISA results of the TBEV Western Siberian strains with 17C3 MAb against NS1 nonstructural glycoprotein. Table 3 Average neurovirulence of TBEV strains of different natural populationa Geographic location of natural focus
TBEV titers (lg LD50)
Neuroinvasiveness index
Intracerebral infection
Subcutaneous infection
Western Siberia 1. South-Eastern part of Western Siberia (Novosibirsk region) 2. Western part of Western Siberia (Tumen region) 3. East Ural (Ekaterinburg region)
7.1 9 0.2 6.9 90.2 7.1 90.2
5.2 90.2 4.9 90.2 5.1 90.2
1.9 90.1 2.0 9 0.1 1.8 90.1
Far East 1. Amur region (Chabarovsk region) 2. South
8.6 90.2 –
6.3 90.1 –
2.3 90.2 –
a
–, The absence of data in publications.
can also alter the virulence. Thus, deletion of the first NS1 glycosylation site decreases the virulence, whereas the absence of the second glycosylation site results in a small increase of the virulence (McMinn, 1997). Amino acid substitution near the C-end of the NS1 glycoprotein (Arg299Ala299) causes low yield of virus, while mutations in the N-terminal part are lethal (Muylaert et al., 1997). Taking into account the similarity of the E gene fragment of all the studied
Western Siberian TBEV strains with Aina/1448 strain, amino acid sequences of Aina/1448 and Sofyin strains were compared. The following results were obtained: 15 from 496 amino acid residues (3%) appeared to differ. According to our data, lower virulence of TBEV Siberian strains in comparison with the Far Eastern strains might be the result of changes not only in E protein, but also in the C-terminal part of NS1 glycoprotein. Neurovirulence of the viral strains from different
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
populations coincided with frequencies of different forms of tick-borne encephalitis in Siberia and the Far East (Pustovalova et al., 1984). To summarize the available data, one could conclude the geographic variability of TBEV strains between different natural foci and the relative stability inside the natural population. Acknowledgements The present work was supported with grants of the Russian Fund of Fundamental Research (N 98-04-49499) and the Integration Program of Fundamental Research of Siberian Branch of Russian Academy of Science. References Clarke, D.H., Casals, J., 1958. Techniques for hemagglutination and hemagglutination-inhibition with arthropodborne viruses. Am. J. Trop. Med. Hygiene 7, 561–573. Deryabin, P.G., Lebedeva, G.A., Loginova, N.V., 1986. Neutralization reaction of togaviruses using mice and tissue cultures. In: S.Ya. Gaidamovich (Ed.), Arbovirus (Methods of Laboratory and Natural Research). Moscow, pp. 120 – 126 (in Russian). Dobrikova, E.Yu., Pletnev, A.G., Shamanin, V.A., 1986. Detection of tick-borne encephalitis virus in human blood and individual ticks by the method of molecular hybridization of nucleic acids. Voprosi virusologii 6, 739–742 (in Russian). Ecker, M., Allison, S.L., Meixner, T., Heinz, F.X., 1999. Sequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and Asia. J. Gen. Virol. 80, 179 – 185. Godovikova, T.S., Orlova, T.N., Dobrikova, E.Yu., Shamanin, V.A., Zarytova, V.F., Vorobyeva, N.V., Serdukova, N.A., Shamanina, M.Yu., Petruseva, I.O., Pitsenko, N.D., 1994. High sensitive nonradioactive detection of tick-borne encephalitis virus. Bioorganicheskaya Chim. 20, 1196 – 1205 (in Russian). Gritsun, T.S., Holmes, E.C., Gould, E.A., 1995. Analysis of flavivirus envelope proteins reveals variable domains that reflect their antigenicity and may determine their pathogenesis. Virus Res. 35, 307–321. Gromashevsky, V.L., 1986. Arbovirus isolation methods. In: S.Ya. Gaidamovich (Ed.), Arbovirus (Methods of Laboratory and Natural Research). Moscow, pp. 90–93 (in Russian). Habel, K., 1972. Neutralization reaction of viruses. In: K. Habel, N.P. Salzman (Eds.), Virology and Molecular Biology Methods. Mir, Moscow, 237 pp. (in Russian).
11
Higgins, D., Thompson, J., Gibson, T., Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673 – 4680. Jawetz, E., Melnik, J., Adelberg, E., 1982. Medicinal Microbiology Manual. Medicine, Moscow, 198 pp. (in Russian). Leonova, G.N., Matveev, L.E., Pletnev, A.G., 1990a. Study of tick-borne encephalitis virus strains isolated on the south of Far East using monoclonal antibodies. Vopr. Virusol. 5, 421 – 423 (in Russian). Leonova, G.N., Muratkina, G.N., Kruglyak, S.P., 1990b. Study of neurovirulence of tick-borne encephalitis virus strains isolated on the South of Far East. Vopr. Virusol. 5, 399 – 401 (in Russian). Levkovich, E.N., Pogodina, V.V., Zasuchina, G.D., Karpovich, L.G., 1967. Viruses of Tick-Borne Encephalitis Virus Complex. Medicine, Moscow, pp. 240 – 245 (in Russian). Mandl, C.W., Heinz, F.X., Stockl, E., Kunz, C., 1989. Genome sequence of tick-borne encephalitis virus (western subtype) and comparative analysis of nonstructural proteins with other flaviviruses. Virology 173, 291 – 301. Matveev, L.E., Godovikov, A.A., Karavanov, A.S., Pletnev, A.G., Rubin, S.G., Semashko, I.G., Tsekhanovskaya, N.A., Chumakov, M.P., Pressman, E.K., 1989. Monoclonal antibodies against tick-borne encephalitis virus glycoprotein: preliminary characterization. Vopr. Virusol. 34, 694 – 698 (in Russian). Matveeva, V.A., Dobrikova, E.Yu., Tsekhanovskaya, N.A., Popova, R.V., Pressman, E.K., Karavanov, A.S., Matveev, L.E., 1998. Construction and characterization of the monoclonal antibodies against tick-borne encephalitis virus nonstructural proteins. Vopr. Virusol. 3, 134 – 137 (in Russian). McMinn, 1997. The molecular basis of virulence of the encephalitogenic flaviviruses, J. Gen. Virol. 78, 2711 – 2722. Muylaert, I.R., Galler, R., Rice, C.M., 1997. Genetic analysis of the yellow fever virus NS1 protein: identification of a temperature-sensitive mutation which blocks RNA accumulation. J. Virol. 71, 291 – 298. Pletnev, A.G., Yamshchikov, V.F., Blinov, V.M., 1990. Nucleotide sequence of the genome and complete amino acid sequence of the polyprotein of tick-borne encephalitis virus. Virology 174, 250 – 263. Pogodina, V.V., Bochkova, N.G., Koreshkova, G.V., 1981. Properties of tick-borne encephalitis virus strains of Aina/ 1448 serotype. Vopr. Virusol. 6, 741 – 746 (in Russian). Pomelova, V.G., Gaidamovich, S.Y., Stephenson, J.R., Lavrova, N.A., Sveshnikova, N.A., 1991. Antigenic analysis of tick-borne encephalitis complex viruses by time-resolved fluoro immunoassay with monoclonal antibodies. J. Virol. Methods 31, 293 – 300. Pustovalova, V.Ya., Katin, A.A., 1984. Characteristics of the reasons of the different epidemiologic appearances of tickborne encephalitis in West Siberia and Far East. J. Microbiol. Epidemiol. Immunol. 3, 46 – 48 (in Russian).
12
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12 Tsekhanovskaya, N.A., Matveev, L.E., Rubin, S.G., Karavanov, A.S., Pressman, E.K., 1993. Epitope analysis of tick-borne encephalitis (TBE) complex viruses using monoclonal antibodies to envelope glycoprotein of TBE virus (persulcatus subtype). Virus Res. 30, 1 – 16. Vereta, L.A., Vorobyeva, R.N., Kovaleva, E.N., Nikolaeva, S.P., 1983. Influence of tick-borne encephalitis virus strains persisting in nature on immunity of people near Amur river. Vopr. Virusol. 5, 601 – 604 (in Russian). Vershinsky, B.V., 1984. Paleogeographic factors of tick-borne encephalitis natural foci appearance and the modern structure of nosoareal. In: Parasitology. Nauka, Leningrad, 138 pp. (in Russian). Wallner, G., Mandl, C.W., Ecker, M., Holzmann, H., Stiasny, K., Kunz, C., Heinz, F.X., 1996. Characterization and complete genome sequences of high- and low-virulence variants of tick-borne encephalitis virus. J. Gen. Virol. 77, 1035 – 1042. Zlobin, V.I., Mamaev, L.V., Djioev, Yu.P., Kozlova, I.V., 1996. Genetic types of tick-borne encephalitis virus. J. Infect. Pathol. 3, 13 – 17 (in Russian).
Safronov, P.F., Netesov, S.V., Mikryukova, T.P., Blinov, E.G., Osipova, N., Kiseleva, N.N., Sandakhchieev, L.S., 1991. Tick-borne encephalitis virus strain 205: nucleotide sequence of genes and complete amino acid sequence of viral proteins. Mol. Genet. 4, 23–29. Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 – 5467. Schlesinger, J.J., Brandriss, M.W., Cropp, C.B., Monath, T.P., 1986. Protection against yellow fever in monkeys by immunization with yellow fever virus nonstructural protein NS1. J. Virol. 60, 1153 – 1155. Semenov, B.F. 1965. Mathematical methods in virological research. In: P.F. Zdrodovsky, I.M. Sokolov (Eds), Manual for Laboratory Diagnostics of Viral and Rickettsial Diseases. Moscow, pp. 208–218 (in Russian). Shamanin, V.A., Pletnev, A.G., Rubin, S.G., Zlobin, V.I., 1990. Differentiation of strains of tick-borne encephalitis virus by means of RNA–DNA hybridization. J. Gen. Virol. 71, 1505 – 1515.
.
Tick-borne encephalitis virus strains of Western Siberia V.N. Bakhvalova a, V.A. Rar b, S.E. Tkachev b, V.A. Matveev b, L.E. Matveev b, A.S. Karavanov b, A.K. Dobrotvorsky a, O.V. Morozova b,* a
Institute of Systematics and Ecology of Animals of Siberian Branch of Russian Academy of Sciences, Frunze Street 11, 630091 No6osibirsk 91, Russian Federation b No6osibirsk Institute of Bioorganic Chemistry of Siberian Branch of Russian Academy of Sciences, La6rentye6 Prospect 8, 630090 No6osibirsk 90, Russian Federation Received 10 February 2000; received in revised form 15 March 2000; accepted 19 May 2000
Abstract Tick-borne encephalitis virus (TBEV) strains were isolated from ticks in Western Siberia for 12 years. Molecular hybridization of the 46 viral RNA with the TBEV cDNA and oligonucleotide probes revealed differences between the Siberian and Far Eastern strains. A comparison of the viral E gene fragment nucleotide sequence showed 89 – 98% homology between Siberian TBEV strains, whereas their similarity with strains from other populations was less than 83%. However, the viral E and NS1 glycoprotein antigenic structures appeared to be conservative because of the degenerate genetic code. This was shown by enzyme-linked immunosorbent assay with the corresponding monoclonal antibodies (MAb). The single exception was the MAb 17C3 against nonstructural glycoprotein NS1, which could distinguish Siberian from Far Eastern strains. Moreover, the neurovirulence differed between strains from the two natural populations. Lower neuroinvasiveness of the Siberian strains in comparison with Far Eastern Sofyin strain might be caused by both E and NS1 glycoprotein mutations. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Tick-borne encephalitis virus strains; Genetic subtypes; Monoclonal antibodies; E and NS1 antigenic structures; Neurovirulence
1. Introduction TBEV causes dangerous central nervous system diseases in humans. General infection leads to development of meningitis or encephalitis with fatality ranging from 1 to 30%. Virus persistence in natural foci results in periodic outbreaks in the indigenous human population. * Corresponding author. Tel.: +7-3832-396226; fax: +73832-333677. E-mail address: [email protected] (O.V. Morozova).
The TBEV genome RNA is approximately 11 kb long (Pletnev et al., 1990). Complete nucleotide sequences of genome RNA from both the TBEV strains isolated in the Far East (Sofyin strain Pletnev et al., 1990) and strain 205 (Safronov et al., 1991)) as well as in West Europe (Neudorfl strain Mandl et al., 1989), strains Hypr and 263 (Wallner et al., 1996)) have been determined previously. For several other TBEV strains, nucleotide sequences of genome fragments have been described (Ecker et al., 1999; Zlobin et al.,
0168-1702/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 0 0 ) 0 0 1 7 4 - X
2
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
1996). Genome structures of Western Siberian viral strains have not been studied previously. The TBEV genome encodes three structural and seven nonstructural proteins. RNA is packaged into a nucleocapcid with a protein C having a molecular weight (Mr) of about 14 kDa, which is surrounded by a lipid envelope that contains a glycoprotein E of Mr 50 – 56 kDa and a small nonglycosylated protein M of Mr 7 – 9 kDa (Pletnev et al., 1990). The single NS1 nonstructural glycoprotein presents inside or on the surface of infected cells, or is secreted into cultural medium, thus inducing the production of the protective complement-fixing antibodies (Schlesinger et al., 1986). The TBEV strains are divided into European, Siberian and Far Eastern subtypes (Ecker et al., 1999). The degree of variation between strains within each subtype is low. However, a difference between the TBEV subtypes has been reported for different species of the Fla6i6iridae family (Ecker et al., 1999). In spite of numerous nucleotide changes of TBEV strain genomes, the corresponding proteins are highly conservative. This is due to the degenerate genetic code (Mandl et al., 1989; Pletnev et al., 1990). Comparative analysis of the TBEV E protein of different strains reveals clusters of amino acid variation distributed throughout. However, a major difference is observed within the B domain (Gritsun et al., 1995). Some E protein epitopes are different for European and Far Eastern strains (Pomelova et al., 1991; Tsekhanovskaya et al., 1993). Population features of the TBEV NS1 nonstructural glycoprotein have not been studied before. The present work is devoted to the comparative analysis of the genomes, the antigenic structures and the neurovirulence of the TBEV Western Siberian strains.
2. Materials and methods
2.1. TBEV strains From 1980 to 1992, TBEV strains were isolated from hungry ticks (Ixodes persulcatus) in the wood-steppe part of the Novosibirsk region in
West Siberia. These strains were supported by intracerebral infection of white mice weighing 6–7 g according to the procedure of Gromashevsky (1986). Identification of isolates was performed by means of hemagglutination test (Clarke and Casals, 1958) and biological neutralization using white mice (Habel, 1972; Deryabin et al., 1986). The TBEV Sofyin strain was obtained from the Ivanovsky Virology Institute (Moscow, Russia). These TBEV strains were stored at − 20°C and analyzed after 7–10 passages.
2.2. Neuro6irulence The neurovirulence of the TBEV strains was determined by titering using white mice weighing 6–7 g. The animals were infected both intracerebrally and subcutaneously (0.03 and 0.25 ml, respectively) with ten-times serial dilutions of 10% brain suspensions from infected white mice. Virus titers were calculated in lg LD50 using Kerber method (Semenov, 1965; Jawetz et al., 1982). The neuroinvasiveness index was estimated to be the difference of the intracerebral and subcutaneous titers (Levkovich et al., 1967).
2.3. RNA isolation Viral RNA were isolated from 10% brain suspensions of infected mice by phenol deproteinization, as previously described (Dobrikova et al., 1986).
2.4. Molecular hybridization of TBEV strains RNA with Sofyin strain cDNA probe The TBEV cDNA of the Sofyin strain isolated from a patient in the Far East has previously been cloned (Pletnev et al., 1990). Total RNA concentrations were estimated by measuring the optical densities at 260 nm. RNA from the intact mouse brain was used as a negative control of the hybridization. The positive control was homologous to the radioactive probe TBEV Sofyin strain RNA. Synthesis and characterization of radioactive labeled TBEV cDNA probe as well as hybridization conditions have been previously
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
3
described (Dobrikova et al., 1986; Shamanin et al., 1990).
scribed (Matveev et al., 1989; Matveeva et al., 1998).
2.5. Re6erse transcription– polymerase chain reaction
2.8. Enzyme-linked immunosorbent assay
5%-AGCCACAGGACACGTGTA-3% (E1)(1348 – 1365 nucleotides of Sofyin strain genome) and 5% - CATCTTGACCATGGGAGA - 3% (E2) (1486 – 1503 nucleotides of Sofyin strain genome) deoxyribo-oligonucleotides corresponding to the TBEV Sofyin strain E gene (Pletnev et al., 1990) were synthesized in the Novosibirsk Institute of Bioorganic Chemistry of Siberian Branch of Russian Academy of Sciences. Reverse transcription – polymerase chain reaction (RT-PCR) was carried out with E1 and E2 primers according to Godovikova et al. (1994).
Interaction of E and NS1 glycoproteins of TBEV strains with MAbs was studied by enzymelinked immunosorbent assay (ELISA) as previously described (Matveeva et al., 1998). To increase the sensitivity, 0.13 mg/ml 3,3%,5,5%-tetramethylbenzidin dihydrochloride (TMB) as a chromogen was added. The measuring of optical density at 450 nm was performed no later than 30 min after termination of the reaction. The last dilution of ascitic fluid producing two-times higher optical density of the studied sample in comparison with background was considered as MAb titer.
2.6. Sequencing 3. Results Nucleotide sequences of TBEV strains E gene fragment RT-PCR products were determined according to (Sanger et al., 1977) with radioactive labeled E1 and E2 primers, and T7-modified DNA polymerase (‘Promix’, Novosibirsk, Russia). The following modifications were made: reaction mixture (17.5 ml) containing 2 ml buffer, 100 mM Tris–HCl (pH 8.9), 500 mM KCl, 25 mM MgCl2, 1 U activity of enzyme, 1 pmol 5%-end labeled primer and 20 ng electrophoretically pure amplicon was divided into 4 ml samples in tubes with a 4 ml mixture of 200 mM 4dNTP and 3 mM corresponding ddNTP. The following cycling parameters were used: 94°C for 3 min, 54°C for 1.5 min, 72°C for 2 min, one cycle; 94°C for 1 min, 54°C for 1.5 min, 72°C for 2 min, 30 cycles. Reaction products were fractionated in 7% PAAG with 6 M urea. Nucleotide sequences were compared using the interactive program CLUSTALW (Higgins et al., 1994).
3.1. Molecular hybridization of TBEV strain RNA Genome RNAs of 46 TBEV strains of Western Siberia were investigated via molecular hybridization with [32P]-cDNA of TBEV of the Sofyin strain, which predominates in the Far East (Zlobin et al., 1996). The hybridization occurred both in water and 50% formamid solutions at 45, 55 and 65°C. Hybridization levels of RNA isolated from the Siberian strains with cDNA of the Sofyin strain probe in 50% formamid at 65°C were very low (Fig. 1). Hybridization with the virus-specific cDNA and oligonucleotide probes revealed essential genomic differences between the TBEV strains isolated from the Far Eastern and Siberian populations.
3.2. Nucleotide sequences of TBEV strain E gene fragments
2.7. Monoclonal antibodies Construction and characterization of monoclonal antibodies (MAbs) against the TBEV Sofyin strain E virion glycoprotein and NS1 nonstructural protein have been previously de-
To study genetic homology, six TBEV strains were chosen. Strains 228, 246 and 774 in periods of time when exceptionally high disease rate in humans was observed. Strains 400, 1441 and 1508 have been isolated during the minimal encephali-
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
4
The Lesopark-11 strain isolated by V.I. Zlobin in the Novosibirsk region in 1986 (Shamanin et al., 1990; Zlobin et al., 1996) was found to be a member of the same TBEV Siberian subtype. It is notable that there were no significant differences between E gene fragment structures of the TBEV strands isolated from ticks and from human patients. Genome fragments of the virus strains isolated during periods with different tick-borne encephalitis rates were also quite similar. Inside each TBEV subtype, the homology level was high: 98–100% between the Western European strains 263, Hypr and Neudoerfl; 89–98%
tis rate in Western Siberia. Nucleotide sequences of E gene (1376–1474 nucleotides), which corresponded to the part of E glycoprotein domain that induces neutralization and anti-hemagglutination antibody production (Tsekhanovskaya et al., 1993), were determined (Fig. 2). Comparison of the E gene fragment nucleotide sequences showed 89 – 98% homology between the TBEV Siberian strains (Table 1). All of the strains collected in the Novosibirsk region were similar to Aina/1448 strain (93 – 96% homology), which was isolated from human cerebrospinal fluid in Irkutsk region in 1963 (Pogodina et al., 1981).
Fig. 1. Results of the molecular hybridization of RNA from TBEV strains with radioactive labeled cDNA probe corresponding to Sofyin strain RNA in 50% formamid at the different temperatures. Table 1 Comparative analysis of the TBEV E gene fragment homology (%)
Sofyin 205 Aina/1448 228 246 400 774 1441 1508 Hypr Neudorfl
Sofyin
205
Aina/1448
228
246
400
774
1441
1508
Hypr
Neudorfl
100 94 79 81 78 78 81 80 80 78 77
94 100 79 79 80 76 80 80 82 76 75
79 79 100 95 94 94 93 94 96 83 82
81 79 95 100 90 96 97 98 92 82 81
78 80 94 90 100 89 90 91 97 81 80
78 76 94 96 89 100 94 95 91 81 80
81 80 93 97 90 94 100 98 92 80 79
80 80 94 98 91 95 98 100 93 81 80
80 82 96 92 97 91 92 93 100 83 82
78 76 83 82 81 81 80 81 83 100 98
77 75 82 81 80 80 79 80 82 98 100
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
5
Fig. 2. Nucleotide sequences of E gene fragments of TBEV strains. Conservative nucleotides are marked with * in the last lane. Sequences of the West Siberian strains are underlined and marked bold. (M) after the Siberian strain number means the TBEV strain isolated during the periods of the maximal disease rate in humans in Western Siberia. Far Eastern strains Sofyin and 205 are shown on the top. The Eastern Siberian strain Aina/1448 is before the Western Siberian strains 228, 246, 400, 774, 1441 and 1508. The strains 263, Hypr and neudorfl isolated in Western Europe are demonsrated on the bottom. Other TBEV strains have been found in Europe.
6
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
between the Siberian strains; and 94% for the Far Eastern strains 205 and Sofyin. Similarity of the E gene fragment between the Siberian and European strains was less than 83%, and that between the Siberian and Far Eastern strains was less than 81%. Moreover, the viral populations of West Europe and the Far East differed more essentially from each other (75 – 78% homology). Deduced amino acid sequences revealed 100% homology of the E protein fragment of the TBEV strains.
3.3. ELISA with MAbs against E protein Antigenic structures of E structural and NS1 nonstructural glycoproteins of TBEV strains were investigated by ELISA with the corresponding MAbs panel. To analyze the TBEV E protein antigenic structure, several MAbs against epitopes in different domains of the protein were used: 5F6, 4A6, 3B7, 7C2, 15B1 and 1H11 MAbs (E1 domain); 14D5, 13D6, 1B1, 11D1 and 10C2 MAbs (E2 domain); 2D1 and 5G10 MAbs (E3 domain) (Tsekhanovskaya et al., 1993). 10C2 MAb binds with E protein of all of the flaviviruses of the TBEV complex. The majority of MAbs recognize only particular flavivirus species. 5F6 and 4A6 MAbs are subtype specific. ELISA with MAbs against E protein demonstrated the similarity of this protein among the studied viral strains (Fig. 3). Data for other MAbs were similar. Viral E protein homology is known to be 93 – 96% among the TBEV strains and 36 – 42% for different flaviviruses (Mandl et al., 1989; Pletnev et al., 1990). This coincides with both the deduced amino acid sequences of the protein fragment (Fig. 2) and ELISA data for the TBEV Siberian strains (Fig. 3).
3.4. ELISA with MAbs against NS1 protein The following MAbs were selected for epitope analysis of TBEV NS1 protein: 4C4, 22H8 MAbs were specific for all the TBEV complex flaviviruses; 16B1, 20B4, 17C3 MAbs interacted with several species of the TBEV complex. 29G9 MAb were species specific (Matveeva et al.,
1998). Among the studied strains, 271, 396, 758, 766, 771 and 776 were isolated from ticks in Western Siberia during periods of maximal encephalitis rate in humans, while others in periods of low disease level. ELISA with MAbs against NS1 protein did not reveal the differences between TBEV strains from different populations with one single exception. MAb 17C3, with the epitope near C-terminal part of TBEV NS1 glycoprotein (data not shown), appeared to be able to distinguish the Siberian strains from Sofyin strain (Fig. 4). The data obtained were in agreement with the known high homology of NS1 nonstructural protein (98% homology between TBEV Far Eastern strains and 96–98% homology among West European strains; 90% similarity between glycoproteins of the viral strains from different populations and 37–44% homology for different flavivirus species (Pletnev et al., 1990)). According to our ELISA data (Figs. 3 and 4), NS1 nonstructural glycoprotein was shown to be more variable than virion glycoprotein E.
3.5. Neuro6irulence of TBEV strains In spite of the conservative amino acid sequences and the stable antigenic structures of the TBEV strains, biological properties can vary. Lower lethality level and less severe forms of tick-borne encephalitis for patients in Siberia in comparison with the Far East might indicate decreased neurovirulence of the TBEV Siberian strains. Intracerebral titers of the Western Siberian TBEV strains varied from 5.8 to 8.5 lg LD50. Subcutaneous titers ranged from 2.7 to 7.0 lg LD50. The neuroinvasiveness indexes were 0.3–4.3. Comparative analysis of neurovirulence of TBEV strains isolated from ticks in natural foci of Siberia (Pustovalova et al., 1984) and the Far East (Leonova et al., 1990b; Vereta et al., 1983) showed considerable similarity within the Siberian natural viral population and significant distinction from the Far Eastern strains (Tables 2 and 3). High virulent strains (perifiric titers, \ 6.0 lg LD50) prevailed in the Far East population. The Siberian strains had a moderate neurovirulence (titers, 4.1–5.9 lg LD50) (Table 3).
a
–, The absence of data in publications.
–
51.5 9 8.8
–
15.2 9 6.4
–
87.8 95.8
63.0 95.3
50.0 97.4
43.7 9 7.3
6.3
60.8 9 7.2
56.5 97.8
39.197.9
4.4
6.0. 97.8
45.0 97.9
55.0 98.0
0.0
–
12.2 95.8
26.2 96.4
28.4 9 6.7
37.5 9 7.8
4.9–3.5 lg LD50
]5.0 lg LD50
57.0 lg LD50
]9.0 lg LD50 8.9–7.1 lg LD50
Subcutaneous infection
Intracerebral infection
% strains in groups with different titers
Far East 1. Chabarovsk 33.39 8.3 region (near Amur river). 2. South –
Western Siberia 1. South-Eastern part of Western Siberia (Novosibirsk region) 2. Western part of Western Siberia (Tumen region) 3. East Ural (Ekaterinburg region)
Geographic location of natural focus
Table 2 Structure of TBEV natural populations neurovirulencea
–
0.0
10.8
10.8
2.5 9 2.5
B3.5 lg LD50
59.59 7.5
63.6 98.5
–
–
2.509 6.9
]6.0 lg LD50
0.0
7.193.9
33.3 9 7.2
–
–
12.595.3
54.0 lg LD50
36.4 9 6.4
–
–
62.5 9 7.8
5.9–4.1 lg LD50
Subcutaneous infection V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12 7
8
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
Fig. 3. ELISA results of the TBEV Western Siberian strains with mono- and polyclonal antibodies against E structural glycoprotein.
4. Discussion Our results suggest the existence of the TBEV Siberian subtype (Ecker et al., 1999). The viral strains from Western and Eastern Siberia appeared to have the similar genome and antigenic structures as well as the same biological properties. Further study will enable one to estimate the presence of this genetic subtype in different ecosystems. The stability of the TBEV E gene
during the 12-year period in isolated natural foci is exceptionally notable. This fact attests to the stability of the viral genome RNA in the absence of an RNA reparation system in host cells. The prolonged stability of viral genes could be the result of the elimination of defective virions in the process of natural selection. Revealed significant genetic differences of TBEV strains from different natural populations of Western Siberia and the Far East could be the
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
consequence of TBEV adaptation to certain ecological conditions. The Siberian TBEV population is known to be younger than the Far Eastern population (Vershinsky, 1984). Despite the heterogeneity of the E gene of the TBEV strains, the corresponding E protein is highly conservative (Pletnev et al., 1990; Leonova et al., 1990a). The flaviviral NS1 nonstructural glycoprotein is more variable than the E protein. Therefore, amino acid sequences of epitopes for
9
MAb 17C3 near the NS1 glycoprotein C-end were shown to be different between the Siberian and Far Eastern TBEV strains. Neurovirulence is known to be determined with envelope glycoprotein E, nonstructural proteins and/or the 3%-untranslated region of the genome. The envelope glycoprotein E plays the main role in flavivirus virulence. This is because the single amino acid changes result in the loss of virulence (McMinn, 1997). Mutations in NS1 glycoprotein
Fig. 3. (Continued)
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
10
Fig. 4. ELISA results of the TBEV Western Siberian strains with 17C3 MAb against NS1 nonstructural glycoprotein. Table 3 Average neurovirulence of TBEV strains of different natural populationa Geographic location of natural focus
TBEV titers (lg LD50)
Neuroinvasiveness index
Intracerebral infection
Subcutaneous infection
Western Siberia 1. South-Eastern part of Western Siberia (Novosibirsk region) 2. Western part of Western Siberia (Tumen region) 3. East Ural (Ekaterinburg region)
7.1 9 0.2 6.9 90.2 7.1 90.2
5.2 90.2 4.9 90.2 5.1 90.2
1.9 90.1 2.0 9 0.1 1.8 90.1
Far East 1. Amur region (Chabarovsk region) 2. South
8.6 90.2 –
6.3 90.1 –
2.3 90.2 –
a
–, The absence of data in publications.
can also alter the virulence. Thus, deletion of the first NS1 glycosylation site decreases the virulence, whereas the absence of the second glycosylation site results in a small increase of the virulence (McMinn, 1997). Amino acid substitution near the C-end of the NS1 glycoprotein (Arg299Ala299) causes low yield of virus, while mutations in the N-terminal part are lethal (Muylaert et al., 1997). Taking into account the similarity of the E gene fragment of all the studied
Western Siberian TBEV strains with Aina/1448 strain, amino acid sequences of Aina/1448 and Sofyin strains were compared. The following results were obtained: 15 from 496 amino acid residues (3%) appeared to differ. According to our data, lower virulence of TBEV Siberian strains in comparison with the Far Eastern strains might be the result of changes not only in E protein, but also in the C-terminal part of NS1 glycoprotein. Neurovirulence of the viral strains from different
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12
populations coincided with frequencies of different forms of tick-borne encephalitis in Siberia and the Far East (Pustovalova et al., 1984). To summarize the available data, one could conclude the geographic variability of TBEV strains between different natural foci and the relative stability inside the natural population. Acknowledgements The present work was supported with grants of the Russian Fund of Fundamental Research (N 98-04-49499) and the Integration Program of Fundamental Research of Siberian Branch of Russian Academy of Science. References Clarke, D.H., Casals, J., 1958. Techniques for hemagglutination and hemagglutination-inhibition with arthropodborne viruses. Am. J. Trop. Med. Hygiene 7, 561–573. Deryabin, P.G., Lebedeva, G.A., Loginova, N.V., 1986. Neutralization reaction of togaviruses using mice and tissue cultures. In: S.Ya. Gaidamovich (Ed.), Arbovirus (Methods of Laboratory and Natural Research). Moscow, pp. 120 – 126 (in Russian). Dobrikova, E.Yu., Pletnev, A.G., Shamanin, V.A., 1986. Detection of tick-borne encephalitis virus in human blood and individual ticks by the method of molecular hybridization of nucleic acids. Voprosi virusologii 6, 739–742 (in Russian). Ecker, M., Allison, S.L., Meixner, T., Heinz, F.X., 1999. Sequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and Asia. J. Gen. Virol. 80, 179 – 185. Godovikova, T.S., Orlova, T.N., Dobrikova, E.Yu., Shamanin, V.A., Zarytova, V.F., Vorobyeva, N.V., Serdukova, N.A., Shamanina, M.Yu., Petruseva, I.O., Pitsenko, N.D., 1994. High sensitive nonradioactive detection of tick-borne encephalitis virus. Bioorganicheskaya Chim. 20, 1196 – 1205 (in Russian). Gritsun, T.S., Holmes, E.C., Gould, E.A., 1995. Analysis of flavivirus envelope proteins reveals variable domains that reflect their antigenicity and may determine their pathogenesis. Virus Res. 35, 307–321. Gromashevsky, V.L., 1986. Arbovirus isolation methods. In: S.Ya. Gaidamovich (Ed.), Arbovirus (Methods of Laboratory and Natural Research). Moscow, pp. 90–93 (in Russian). Habel, K., 1972. Neutralization reaction of viruses. In: K. Habel, N.P. Salzman (Eds.), Virology and Molecular Biology Methods. Mir, Moscow, 237 pp. (in Russian).
11
Higgins, D., Thompson, J., Gibson, T., Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673 – 4680. Jawetz, E., Melnik, J., Adelberg, E., 1982. Medicinal Microbiology Manual. Medicine, Moscow, 198 pp. (in Russian). Leonova, G.N., Matveev, L.E., Pletnev, A.G., 1990a. Study of tick-borne encephalitis virus strains isolated on the south of Far East using monoclonal antibodies. Vopr. Virusol. 5, 421 – 423 (in Russian). Leonova, G.N., Muratkina, G.N., Kruglyak, S.P., 1990b. Study of neurovirulence of tick-borne encephalitis virus strains isolated on the South of Far East. Vopr. Virusol. 5, 399 – 401 (in Russian). Levkovich, E.N., Pogodina, V.V., Zasuchina, G.D., Karpovich, L.G., 1967. Viruses of Tick-Borne Encephalitis Virus Complex. Medicine, Moscow, pp. 240 – 245 (in Russian). Mandl, C.W., Heinz, F.X., Stockl, E., Kunz, C., 1989. Genome sequence of tick-borne encephalitis virus (western subtype) and comparative analysis of nonstructural proteins with other flaviviruses. Virology 173, 291 – 301. Matveev, L.E., Godovikov, A.A., Karavanov, A.S., Pletnev, A.G., Rubin, S.G., Semashko, I.G., Tsekhanovskaya, N.A., Chumakov, M.P., Pressman, E.K., 1989. Monoclonal antibodies against tick-borne encephalitis virus glycoprotein: preliminary characterization. Vopr. Virusol. 34, 694 – 698 (in Russian). Matveeva, V.A., Dobrikova, E.Yu., Tsekhanovskaya, N.A., Popova, R.V., Pressman, E.K., Karavanov, A.S., Matveev, L.E., 1998. Construction and characterization of the monoclonal antibodies against tick-borne encephalitis virus nonstructural proteins. Vopr. Virusol. 3, 134 – 137 (in Russian). McMinn, 1997. The molecular basis of virulence of the encephalitogenic flaviviruses, J. Gen. Virol. 78, 2711 – 2722. Muylaert, I.R., Galler, R., Rice, C.M., 1997. Genetic analysis of the yellow fever virus NS1 protein: identification of a temperature-sensitive mutation which blocks RNA accumulation. J. Virol. 71, 291 – 298. Pletnev, A.G., Yamshchikov, V.F., Blinov, V.M., 1990. Nucleotide sequence of the genome and complete amino acid sequence of the polyprotein of tick-borne encephalitis virus. Virology 174, 250 – 263. Pogodina, V.V., Bochkova, N.G., Koreshkova, G.V., 1981. Properties of tick-borne encephalitis virus strains of Aina/ 1448 serotype. Vopr. Virusol. 6, 741 – 746 (in Russian). Pomelova, V.G., Gaidamovich, S.Y., Stephenson, J.R., Lavrova, N.A., Sveshnikova, N.A., 1991. Antigenic analysis of tick-borne encephalitis complex viruses by time-resolved fluoro immunoassay with monoclonal antibodies. J. Virol. Methods 31, 293 – 300. Pustovalova, V.Ya., Katin, A.A., 1984. Characteristics of the reasons of the different epidemiologic appearances of tickborne encephalitis in West Siberia and Far East. J. Microbiol. Epidemiol. Immunol. 3, 46 – 48 (in Russian).
12
V.N. Bakh6alo6a et al. / Virus Research 70 (2000) 1–12 Tsekhanovskaya, N.A., Matveev, L.E., Rubin, S.G., Karavanov, A.S., Pressman, E.K., 1993. Epitope analysis of tick-borne encephalitis (TBE) complex viruses using monoclonal antibodies to envelope glycoprotein of TBE virus (persulcatus subtype). Virus Res. 30, 1 – 16. Vereta, L.A., Vorobyeva, R.N., Kovaleva, E.N., Nikolaeva, S.P., 1983. Influence of tick-borne encephalitis virus strains persisting in nature on immunity of people near Amur river. Vopr. Virusol. 5, 601 – 604 (in Russian). Vershinsky, B.V., 1984. Paleogeographic factors of tick-borne encephalitis natural foci appearance and the modern structure of nosoareal. In: Parasitology. Nauka, Leningrad, 138 pp. (in Russian). Wallner, G., Mandl, C.W., Ecker, M., Holzmann, H., Stiasny, K., Kunz, C., Heinz, F.X., 1996. Characterization and complete genome sequences of high- and low-virulence variants of tick-borne encephalitis virus. J. Gen. Virol. 77, 1035 – 1042. Zlobin, V.I., Mamaev, L.V., Djioev, Yu.P., Kozlova, I.V., 1996. Genetic types of tick-borne encephalitis virus. J. Infect. Pathol. 3, 13 – 17 (in Russian).
Safronov, P.F., Netesov, S.V., Mikryukova, T.P., Blinov, E.G., Osipova, N., Kiseleva, N.N., Sandakhchieev, L.S., 1991. Tick-borne encephalitis virus strain 205: nucleotide sequence of genes and complete amino acid sequence of viral proteins. Mol. Genet. 4, 23–29. Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 – 5467. Schlesinger, J.J., Brandriss, M.W., Cropp, C.B., Monath, T.P., 1986. Protection against yellow fever in monkeys by immunization with yellow fever virus nonstructural protein NS1. J. Virol. 60, 1153 – 1155. Semenov, B.F. 1965. Mathematical methods in virological research. In: P.F. Zdrodovsky, I.M. Sokolov (Eds), Manual for Laboratory Diagnostics of Viral and Rickettsial Diseases. Moscow, pp. 208–218 (in Russian). Shamanin, V.A., Pletnev, A.G., Rubin, S.G., Zlobin, V.I., 1990. Differentiation of strains of tick-borne encephalitis virus by means of RNA–DNA hybridization. J. Gen. Virol. 71, 1505 – 1515.
.