Innate and adaptive immunity in wild rodents spontaneously and experimentally infected with the tick-borne encephalitis virus

Journal Pre-proof Innate and adaptive immunity in wild rodents spontaneously and experimentally infected with the tick-borne encephalitis virus

Olga V. Morozova, Victor V. Panov, Valentina N. Bakhvalova PII:

S1567-1348(20)30019-8

DOI:

https://doi.org/10.1016/j.meegid.2020.104187

Reference:

MEEGID 104187

To appear in:

Infection, Genetics and Evolution

Received date:

18 September 2019

Revised date:

29 November 2019

Accepted date:

8 January 2020

Please cite this article as: O.V. Morozova, V.V. Panov and V.N. Bakhvalova, Innate and adaptive immunity in wild rodents spontaneously and experimentally infected with the tick-borne encephalitis virus, Infection, Genetics and Evolution(2019), https://doi.org/ 10.1016/j.meegid.2020.104187

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Journal Pre-proof Innate and adaptive immunity in wild rodents spontaneously and experimentally infected with the tick-borne encephalitis virus Olga V. Morozovaa,b,*, Victor V. Panovc, Valentina .N. Bakhvalovac

Ivanovsky Institute of Virology of the National Research Center of Epidemiology and Microbiology of N.F. Gamaleya of the Russian Ministry of Health, 16 Gamaleya Street, 123098, Moscow, Russian Federation, [email protected].

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Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency of Russia, 1a Malaya Pirogovskaya Street, 119435, Moscow, Russian Federation Institute of Systematics and Ecology of Animals of the Siberian Branch of the Russian Academy of Sciences, 11 Frunze Street, 630091, Novosibirsk, Russia, [email protected]

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* Corresponding author

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Abstract

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Two dominant species of wild small rodents trapped in Novosibirsk region, South-Western Siberia, Russia differed in their susceptibility to the tick-borne encephalitis virus (TBEV)

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infection. TBEV RNA average detection rate for Northern red-backed vole Myodes rutilus (Pallas, 1779) (82.2±5.8% blood samples and 63.1±2.7% organ samples) significantly exceeded the

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corresponding values for the striped field mouse Apodemus agrarius (Pallas, 1771) (47.0±8.7% blood and 24.5±2.8% organ samples) (p<0.001). Innate immunity may be one of possible reasons

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of the differences. Th1 cytokine gene expression distinguished between M. rutilus (12.5±8.5%) and A. agrarius (66.6±11.4%), whereas Th2 cytokine frequencies were statistically similar (81.8±12.2% and 100.0%, respectively). Polarization indexes (PI) of the innate immunity calculated as ratio of Th2 to Th1 cytokine RNA detection rates for both M. rutilus (6.5) and A. agrarius (1.5) suggested Th2 mainly humoral immune response against persistent TBEV in natural mammalian hosts. Therefore, the TBEV-induced antibodies were analyzed by ELISA and hemagglutination inhibition (HI) tests. The TBEV-specific antibodies were detected in 74.8±4.3% sera of M. rutilus and 67.3±6.8% of A. agrarius. Among them HI antibodies were found in 4.8±2.1% of the same analyzed sera of M. rutilus and in 6.0±3.4% blood samples of A. agrarius only. To model the TBEV persistence both M. rutilus and A. agrarius were infected with the suspensions of the TBEV-infected ticks with further observations during 4 subsequent months. Detection rate of the TBEV RNA and antigen E remained high during the whole period, however, pathogenic for laboratory suckling mice virus was isolated up to 8 days postinfection. At late stages of the persistent infection (1-4 months) the TBEV RNA detection rate in northern

Journal Pre-proof red-backed voles remained high 70.6±7.9% whereas in striped field mice significantly declined to 26.7±9.2% (p<0.001). Comparative analysis of the innate immunity of the wild rodents in 2 months postinfection showed similar frequencies of Th2 cytokine gene expression for M. rutilus (77.8±10.1%) and A. agrarius (71.4±12.5%) (p>0.05) but Th1 cytokine mRNA detection rates were different (44.4±12.5% and 85.7±9.7%, respectively) (p<0.05). In 2 months PI decreased from 6.5 until 1.75 for M. rutilus and from 1.5 until 0.83 for A. agrarius. Nevertheless, Th2 mainly humoral immune response was confirmed by direct detection of the TBEV-specific antibodies. HI and neutralizing antibodies were revealed in blood sera of the small rodents of both studied species in 30 days postinfection and remained at detectable levels during 4 months of observations. Accordingly, Th2 polarized innate immunity of small rodents might facilitate

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the TBEV intracellular persistence in the presence of HI and neutralization antibodies.

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Keywords: tick-borne encephalitis virus; Northern red-backed vole Myodes rutilus (Pallas, 1779); striped field mouse Apodemus agrarius (Pallas, 1771); cytokines; virus-neutralizing and

Abbreviations:

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hemagglutination inhibition antibodies.

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ELISA – enzyme-linked immunosorbent assay

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HI – hemagglutination inhibition IFN – interferon IL – interleukin

PI – polarization index

RT2-PCR – reverse transcription with real time PCR TBEV – tick-borne encephalitis virus

Introduction Epidemiology of vector-borne zoonotic diseases depends on the interactions of the pathogens with their hosts (Rizzoli et al., 2014). Natural reservoir hosts may respond to infections by a combination of resistance (expulsion of pathogens), tolerance (attenuation of pathology) (Jackson et al., 2014; Stone et al., 2017) and susceptibility. Little is currently known about tick-borne virus persistence in wild vertebrates in natural populations. Experimental

Journal Pre-proof laboratory conditions differ from a natural environment and linear laboratory mice lack the genetic diversity and environmental pressures characteristic of natural populations (Turner and Paterson, 2013). Therefore, examination of wild rodents may help to explain variations in infectious disease susceptibility and to estimate real risks of zoonosises. Increasing evidence points to the innate immunity involvement in explaining interspecies and inter-individual variations in susceptibility to infectious diseases. Innate immunity refers to nonspecific defense mechanisms induced within a few hours of an antigen's appearance, processing and presentation. Invading infectious agents are initially recognized by the innate system through pattern-recognition receptors (PRRs). After stimulation by the ligand, PRRs activate an intracellular signaling cascade, which initiates both innate and acquired specific

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immune responses. Antigenic stimulation of naive T cells in the presence of specific cytokines

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produced by innate cells induces activation, expansion and differentiation of T cells into distinct effector T cells (Abbas et al., 1996) that were previously classified into T helper (Th)1 or Th2

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subtypes depending on the cytokines they produced. Interferon (IFN) γ, interleukin (IL) 1β and IL12 induce Th1 response for clearing intracellular pathogens by activating effector functions of

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macrophages and inducing antibody class switching to IgG2a. In contrast, IL4 and IL10 are

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known to regulate pro-inflammatory cytokines and to induce Th2 cytokine gene expression to eliminate extracellular pathogens and promote antibody class switching to IgG1 and IgE. One of

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the striking features of Th1 and Th2 cells is that they cross-regulate functions of each other by producing antagonizing cytokines. After discovery of Th17 cells taking part in clearing extracellular pathogens and tissue inflammation, this paradigm had been revised (Jiang, 2011).

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Tick-borne encephalitis (TBE) is the most important flavivirus infection of the central nervous system in Eurasia. Its etiological agent, the tick-borne encephalitis virus (TBEV), circulates among vertebrate hosts and arthropod vectors. Ixodid ticks feed on more than 100 species of vertebrate hosts including mammals, reptiles, amphibians and birds (Filippova, 1985). Coevolution of viruses with their hosts toward less deleterious infections ensures the survival of both host and virus. Therefore, most wild vertebrates are susceptible and tolerant for both mosquito- and tick-borne flaviviruses. Resistance to flavivirus-induced diseases in wild mammals is conferred by the autosomal Flv-resistance gene identified as 2'-5' oligoadenylate synthetase 1b (oas1b) gene that expresses constitutively, does not require IFN for induction and inhibits flavivirus replication (Brinton and Perelygin, 2003). Small mammals mainly rodents (Rodentia) and insectivorous (Insectivora) with high reproduction rates and their life span even shorter than that of ixodid ticks are natural hosts for both immature ticks (mainly larvaes) and TBEV. Several small rodent species are highly competent in supporting the TBEV transmission. Regardless the sensitivity of vertebrate hosts to

Journal Pre-proof the TBEV their susceptibility estimated by viraemia after infection essentially differs (Levkovich et al., 1967; Chunikhin et al., 1990). Regardless of species variations and regional features lifelong persistence of apathogenic TBEV in the presence of the virus-specific antibodies is typical for small rodents and insectivorous (Bakhvalova et al., 2006; 2011; 2016 and references therein). Our research was aimed at cytokine gene expression analysis with calculation of polarization indexes of immune response and antibodies detection in wild small rodents Myodes rutilus and Apodemus agrarius spontaneously and experimentally infected with the TBEV.

Materials and Methods

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Wild mammals and ticks. Monitoring of population dynamics of small mammals along with their TBEV infection rate was performed in aspen-birch and pine forests near Novosibirsk,

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Russia (5449' N, 8305' E, total area approximately 90 km2) on stationary site each year during

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the period of 1998-2011 using zoological and virological methods (Bakhvalova et al., 2006; 2016).

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Small mammals were trapped using live-traps (more than 20 traps with 10 m intervals between them) (Bakhvalova et al., 2006; 2016) from May to August each year. Samples of brain,

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spleen and blood clot from the animals were separately washed with 0.15 M NaCl and 10% suspensions in physiological solution were prepared for individual analysis.

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The research complied with the guidelines for work with experimental animals and protection of animals against cruelty (Animal Welfare Act no. 246/1992 Coll.) and was in

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accordance with the law N755 of the Russian Ministry of Health of 1977-12-08. Blood samples were taken from retroorbital sinus of the trapped wild rodents before and in 2, 4, 8, 16, 30, 60, 90 and 120 days after their experimental infection with the TBEV and stored at – 700 C.

TBEV detection. TBEV was detected using ELISA, reverse transcription with real time PCR (RT2-PCR) with primers and subtype-specific fluorescent hydrolysis probes corresponding to TBEV NS1 gene and bioassay as previously described (Bakhvalova et al., 2006; 2011; 2016). Cytokine gene expression was assayed in blood cells of the wild rodents by RT2-PCR with primer pairs specific to mRNA of rodent IFN γ, interleukin (IL) 1β, 4, 10 and 12, as previously described (Morozova et al., 2012). Polarization indexes (PI) were calculated as ratio of Th2 and Th1 cytokine RNA frequencies. TBEV-specific antibodies were determined in individual sera of the wild rodents stored for less one week at 4°С by means of HI with goose erythrocytes (Clarke and Casals, 1958) and

Journal Pre-proof neutralization test (Derjabin et al., 1986) using ICR mice (8-10 g), serum dilution 1:10 and 100 LD50 TBEV strain 2689 (GenBank (https://www.ncbi.nlm.nih.gov) accession number JQ693478). ELISA to detect antibodies against the TBEV was performed with the TBEV of strain Sofjin of the Far Eastern subtype and the strain Aina 1447 of the Siberian subtype from the infected porcine embryo kidney cells immobilized on the polystyrene plates. Immune complexes were revealed with secondary antibodies against mouse IgM and IgG conjugated with horseradish peroxidase (“BioSan”, Novosibirsk, Russia). Statistical comparisons were carried out using the Student’s t-test. In the text and tables mean values are represented with the Standard Error of the Mean (SEM) and percentages with the Standard Error of the Percentage (SEP) (Lakin, 1980; Sheskin, 2011). Correlation analysis

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was performed using Statistica 7.0 (StatSoft Inc.). In all cases, p-values <0.05 were considered to

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be significant.

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Results and discussion

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1. Natural TBEV infection of wild small rodents Dominant species of wild small rodents - Northern red-backed voles M. rutilus and field

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mice A. agrarius are the reservoir hosts of both TBEV and immature ixodid ticks with similar population dynamics and their involvement in feeding of ixodid ticks in the South-Western Siberia, Russia

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(Bakhvalova et al., 2006; 2011; 2016). TBEV RNA was detected by RT2-PCR in 207 brain or spleen samples from 328 analyzed organs of M. rutilus but only in 59 from 241 brain or spleen samples of A.

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agrarius trapped in the Novosibirsk region, South-Western Siberia, Russia during 1998-2011 years (Fig. 1). Noteworthy, that the average TBEV RNA detection rate in M. rutilus organs 63.1±2.7% (total range from 37.5±7.8% tо 100%) significantly (p<0.001) exceeded the corresponding value for A. agrarius ((24.5±2.8%) with fluctuations from 3.3±3.3% to 75.0±13.1%) (Fig.1). In blood cells the TBEV RNA detection in 82.2±5.8% of 45 samples from M. rutilus also surpassed one of A. agrarius (47.0±8.7% of 34 blood samples) (p<0.001). The TBEV infection rate of M. rutilus и and A. agrarius changed synchronically and unidirectionally (r=0.4) and did not depend on their population numbers (correlation coefficients r = - 0.05 and r = 0.13, respectively). Viral loads with TBEV of the Far Eastern, the Siberian and the European subtypes varied in a broad ranges and average values corresponded to several thousands of TBEV RNA copies in brain and spleen as previously described in detail (Bakhvalova et al., 2006; 2016). Quantities of the TBEV Far Eastern RNA and Siberian RNA in 1 ml of blood were as high as 2.4*10 5 and 2.4*102, respectively (Bakhvalova et al., 2016).

Innate immunity

Journal Pre-proof The observed long-term differences of TBEV infection rates might be caused by innate immunity of the wild rodents. Gene expression of both Th1 cytokines (IL1β, IL12 and IFN γ) and Th2 cytokines (IL4 and IL10) was assayed by RT2-PCR in blood cells of wild rodents. Indeed, Th1 cytokine gene expression rate differed (p<0.01) for M. rutilus (12.5±8.5% (in 2 from 16 blood samples)) and A. agrarius (66.6±11.4% (12 from 18 analyzed samples)) whereas Th2 cytokine frequencies were statistically similar (81.8±12.2% (9 from 11 blood samples) and 100% (16 analyzed samples), respectively) (Fig. 2). PI=6.5 for M. rutilus and 1.5 for A. agrarius suggested Th2 mainly humoral immune response. Humoral immune response

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Comparative analysis of anti-TBEV IgM and IgG antibodies in spontaneously infected

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wild rodents was carried out by ELISA and HI test. High statistically similar detection rates of the TBEV-specific antibodies for M. rutilus (74.8±4.3%) and A. agrarius (67.3±6.8%) (Table 1)

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confirmed the involvements of the small rodents in circulation of the TBEV in natural populations (Fig.1) and proved Th2 polarized immune response. Seasonal dynamics showed that

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IgM frequencies declined from 81.5±7.6% in summer to 44.4±12.1% in fall (October-

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November) when inactive larvae and nymphs of ixodid ticks did not feed on the small mammals in the Western Siberia (Bakhvalova et al., 2006). Broad ranges of ELISA titers for M. rutilus (from 1:100 tо 1:800) and A. agrarius (from 1:100 to 1:400) were similar and corresponded well

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to similar high frequencies of Th2 cytokines (Fig. 2). However, HI antibodies with titers from 1:40 to 1:80 were found in 4.8±2.1% sera of M. rutilus and in 6.0±3.4% blood samples of A.

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agrarius only (Table 1). Difference between ELISA and HI test data can be resulted from various sensitivity limits of the methods and HI properties for part of total antibodies detected by ELISA.

During the long-term monitoring (1980-2013) of 1386 M. rutilus and 1115 A. agrarius blood sera HI antibodies were revealed in 15.7±1.0% smples of M. rutilus and 6.5±0.7% sera of A. agrarius (p<0.001); proportions of seropositive rodents of two species changed synchronically and unidirectionally (r=0.63; p<0.01) from 0 to 63.1±5.3% and from 0 to 33.3±8.0%, respectively (Fig. 3). Titers of HI antibodies varied in broad ranges from 1:10 to 1:320 for both rodent species with similar average values up to 1:40. However, correlation between relative population numbers and proportions of small rodents with HI antibodies was found for M. rutilus only (r = 0.36) rather not for A. agrarius (r = - 0.09). Humoral immune response was previously described to be induced by numerous endo- and ectoparasites in wild mammals in natural populations (Dobrotvorsky et al., 1998) as well as persistent TBEV (Bakhvalova et al., 2006). Earlier both ecological and epidemiological

Journal Pre-proof significance of mainly humoral immunoreactivity was evidently underestimated due to low sensitivity limits of used methods (Lokhmiller R.L., Moshkin M.P., 1999; Mak et al., 2002) Currently available data on cytokine gene expression (Fig. 2) and antibody detection using ELISA and HI test (Table 1, Fig. 3) revealed that Th2 polarized innate immunity of wild rodents might facilitate the TBEV persistence inside host cells in the presence of specific antibodies controlling free extracellular virions only (Bakhvalova et al., 2006; 2011). Taken together, the TBEV persistence in vertebrate and invertebrate reservoir hosts provides resistance of the parasitary system.

2. Experimental TBEV infection of trapped wild rodents

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To model the TBEV persistence the trapped wild rodents (preliminary checked for the

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absence of the TBEV RNA in their blood using RT2-PCR) were subcutaneously injected with the suspensions of the TBEV-infected ticks with subsequent detection of the viral RNA and antigen

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E, infectious TBEV as well as analysis of cytokine genes expression, HI and neutralizing antibodies during 4 subsequent months postinfection. Detection rate of the TBEV RNA and

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antigen E remained high during whole period of observations, however pathogenic for laboratory

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suckling mice virus was isolated mainly at the early stage of the experimental infection of the small rodents up to 8 days postinfection (Fig. 4). The similar viraemic period (2-8 days) with

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high virus titers was earlier observed in different species of small mammals including field mice, red voles, common voles etc. (Tick-Borne Encephalitis (TBE) and its Immunoprophylaxis, 1996 and references therein). At late stages of TBEV persistent infection (1-4 months) the TBEV

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RNA detection rate in red-backed voles remained high 70.6±7.9% whereas in field mice significantly declined (p<0.001 in comparison with early period) to 26.7±9.2%. Comparative analysis of innate immunity of the wild rodents in 2 months postinfection showed similar frequencies of Th2 cytokine gene expression for M. rutilus (77.8±10.1%) and A. agrarius (71.4±12.5%) (p>0.05) but Th1 cytokine mRNA detection rates were different (p<0.05) (44.4±12.5% and 85.7±9.7%, respectively) (Fig. 5). Meanwhile, part of animals with IL 1β mRNA was significantly higher (p<0.05) (Table 2) for A. agrarius than for M. rutilus that might cause lower levels of both spontaneous (Fig. 1) (Bakhvalova et al., 2006; 2011; 2016) and experimental model TBEV infection (Fig. 4) of A. agrarius compared to M. rutilus. One should note that original PI of M. rutilus before the experimental infection of the wild rodents with the TBEV was 6.5 with decrease until 1.75 in 2 months postinfection whereas for A. agrarius the originally relatively low PI changed from 1.5 until 0.83. Nevertheless, the data suggested the Th2 mainly humoral immune response which was confirmed by direct detection of the TBEVspecific antibodies.

Journal Pre-proof Virus-neutralizing and HI antibodies were revealed in blood sera of both rodent species in 30 days postinfection and remained at detectable levels during 4 months of observations (Fig. 6).

Conclusion Statistically significant (p<0.001) differences of the TBEV spontaneous natural infection rates, susceptibility and the virus persistence between two small rodent species - M. rutilus and A. agrarius dominating in the Western Siberian endemic region correlated with Th1 cytokine gene expression in the wild animals after their natural and experimental infection. Consequently,

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Th2 polarized innate immunity of small rodents might facilitate the TBEV intracellular persistence in the presence of HI and neutralization antibodies. Taken together, the TBEV

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persistence in vertebrate and invertebrate reservoir hosts provides resistance of the parasitary

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system.

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3. Bakhvalova VN.Panov VV.Morozova OV. Tick-borne encephalitis virus quasispecies rearrangements in ticks and mammals. In: Flavivirus encephalitis. (Daniel Růžek. ed.) InTech. 2011: 213-234. Available at: http://www.intechopen.com/articles/show/title/tickborne-encephalitis-virus-quasispecies-rearrangements-in-ticks-and-mammals 4. Bakhvalova, V.N., Chicherina, G.S, Potapova, O.F., Panov, V.V., Glupov V.V., Potapov, M.A., Seligman, S.J., Morozova, O.V., 2016. Tick-Borne Encephalitis Virus Diversity in Ixodid Ticks and Small Mammals in South-Western Siberia, Russia. Vector Borne and Zoonotic Diseases 6: 541-549. DOI:10.1089/vbz.2015.1834. 5. Brinton, M.A. and Perelygin, A.A., 2003. Genetic resistance to flaviviruses. Advances in Virus Res. 60: 43-85. 6. Clarke D.H., Casals J. Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Amer. J. Trop. Med. Hyg. 1958; 7(5): 561-573. 7. Chunikhin, S.P., 1990. Experimental study of tick-borne encephalitis virus ecology. Vopr. Virusol. 3: 183-188 (In Russian).

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Journal Pre-proof 19. Sheskin, D.J., 2011. Handbook of Parametric and Nonparametric Statistical Procedures. 5th ed. Chapman & Hall/CRC, New York. 1926 p. 20. Stone, B.L., Tourand, Y., Brissette, C.A., 2017. Brave New Worlds: The Expanding Universe of Lyme Disease. Vector Borne Zoonotic Dis. 17(9): 619-629. doi: 10.1089/vbz.2017.2127. 21. Tick-Borne Encephalitis (TBE) and its Immunoprophylaxis. Immuno AG, Vienna, Austria, 1996. 22. Turner, A.K., Paterson, S., 2013. Wild rodents as a model to discover genes and pathways underlying natural variation in infectious disease susceptibility. Parasite

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Immunol. 35(11): 386-395. doi: 10.1111/pim.12036.

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Legends Figure 1. The TBEV RNA detection rate in organs of small rodents trapped in the Novosibirsk region, South-Western Siberia, Russia. Figure 2. Comparison of Th1 and Th2 cytokine gene expression frequencies in blood cells of wild rodents. Figure 3. Monitoring of HI antibody rates in wild rodents. Figure 4. Detection rate of the TBEV RNA, antigen E and the infectious virus pathogenic for laboratory suckling mice.

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Figure 5. Relative amounts (%) of wild rodents containing Th1 and Th2 cytokines in blood cells

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in 2 months postinfection.

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Figure 6. Relative amounts (%) of seropositive blood samples with the TBEV-specific antibodies in the trapped wild mammals after their experimental infection with the TBEV in tick suspensions.

Journal Pre-proof Table 1. Comparison of the TBEV-specific antibodies in trapped wild rodents

Proportions (%) of wild rodents with anti-TBEV antibodies Species

ELISA (IgM and/or IgG)

HI

74.8 ± 4.3 (77/103)

4.8 ± 2.1 (5/105)

67.3 ± 6.8 (33/49)

6.0 ± 3.4 (3/50)

M. rutilus

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A. agrarius

Journal Pre-proof Table 2. Cytokine gene expression rate in blood cells of wild small mammals in 2 months postinfection. Frequencies of cytokine mRNA detection (% and ratio of RT2-PCR positive samples to analyzed samples) Тh1 cytokines Th2 cytokines IL12 IL4 IL10 IL1 IFN

A. agrarius (14 samples)

22.2 ± 10.1* (4/18)

22.2 ± 10.1 (4/18)

22.2 ± 10.1 (4/18)

57.1 ± 13.7* (8/14)

28.6 ± 12.5 (4/14)

28.6 ± 12.5 (4/14)

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M. rutilus (18 samples)

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Species

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Note: * statistical significance p <0.05.

22.2 ± 10.1 (4/18)

77.8 ± 10.1 (14/18)

28.6 ± 12.5 (4/14)

71.4 ± 12.5 (10/14)

Journal Pre-proof A. agrarius

M. rutilus

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Proportions (%) of the animals with positive results of RT-PCR

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Years of observations

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region, South-Western Siberia, Russia.

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Fig. 1. The TBEV RNA detection rate in organs of small rodents trapped in the Novosibirsk

100 80

p <0.01

M. rutilus 60 40

A. agrarius

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Proportions (%) of animals with RNA of cytokines

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Fig. 2. Comparison of Th1 and Th2 cytokine gene expression frequencies in blood cells of wild

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rodents.

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r = 0.63; p < 0.01

60 50 40 30 20 10

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Years of observations

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Fig. 3. Monitoring of HI antibody rates in wild rodents.

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Proportions (%) of the animals with TBEV specific HI antibodies

A.agrarius

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Proportion (%) of the animals with TBEV RNA, antigen E and the infectious virus pathogenic for laboratory suckling mice.

Myodes rutilus 100 80

Pathogenic TBEV

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TBEV RNA

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Fig. 4. Detection rate of the TBEV RNA, antigen E and the infectious virus pathogenic for laboratory suckling mice.

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Fig. 5. Relative amounts (%) of wild rodents containing Th1 and Th2 cytokines in blood cells in

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Virus-neutralizing antibodies

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Fig. 6. Relative amounts (%) of seropositive blood samples with the TBEV-specific antibodies in the trapped wild mammals after the experimental infection with the TBEV in tick suspensions.

Journal Pre-proof Author contributions Olga V. Morozova was responsible for RT2-PCR, ELISA and manuscript preparation. Victor V. Panov carried out trapping of wild rodents and their species identification.

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Valentina N. Bakhvalova performed hemagglutination inhibition and neutralization tests as well as experimental infection of wild rodents with the tick-borne encephalitis virus together with manuscript editing.

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There are no conflicts to declare.

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Th2:Th1 cytokine gene expression ratio suggested mainly humoral immune response. Spontaneous and experimental TBEV infection induced antibodies in wild rodents. TBEV persisted in wild mammals in the presence of specific antibodies.

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Infection rate of Myodes rutilus with the TBEV exceeded one of Apodemus agrarius. Th1 cytokine RNA frequencies in M. rutilus were lower than in A. agrarius.