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Experimental infection caused by two variants of one tick-borne encephalitis virus strain: similar virulence, but different influence on lymphocyte subpopulation structure

Oxana V. Motuzova1, Elina A. Akhmatova2, Vasily G. Khomenkov2, Nelli K. Akhmatova2, Olga V. Lebedinskaya3* and Galina G. Karganova1
  • 1 SBSI «Chumakov Institute of Poliomyelitis and Viral Encephalitides», Russia
  • 2 I.I. Mechnikov Research Institute for Vaccines and Sera, Russia
  • 3 Acad. E.A. Wagner Perm State Medical University, Russia

Tick-borne encephalitis virus (TBEV) is mammalian tick-borne flavivirus. Virus replication in the central nervous system (CNS) leads to serious neurological disorders, such as meningitis, encephalitis or meningoencephalitis. At present, the pathogenetic mechanisms of tick-borne encephalitis, including the virus interaction with the immune system actively studied, but are still not completely clear. Previously, in experiments on laboratory animals it has been shown that TBEV strains might vary differ in their pathogenic characteristics. For example, during the infection caused by peripheral infection by strain Sofjin observed shorter life expectancy and higher mortality, compared to the other strains, such as Oshima [2]. How virus virulence is associated with the immune response features during the flavivirus infection remains poorly studied. Like other viruses, TBEV has the ability to modify the influence of the immune system and hide from its recognizing. For example, the virus leads to formation of intracellular vesicles which may protect the replicative form of the viral RNA from cell recognition through RIG-I and MDA-5 ways, and thus delay the start of IFN I induction. TBEV have the same ability to replicate in the different immune system cell types. It has been shown that T cells can support the TBEV strains replication: Absettarov, Neudorfl, Hypr 71. There is information about the interaction of dendritic cells and TBEV [1]. However, these data are fragmented, molecular-cellular mechanisms of interaction of the virus with the microorganism are poorly studied, in particular, little is known about the influence of effectors both innate and adaptive immunity for TBE. Previously, we showed that TBEV could exist as a heterogeneous population comprising variants with a selective advantage for reproduction in ticks or in mammals [4]. Such variants of the same strain may vary differ in their pathogenic characteristics and, accordingly, the ability to induce the antiviral immune response. Nature of innate and adaptive immune response activation may depend on the ratio of such variants in the population. Purpose: To study the immunophenotypical features of mice lymphocytes in experimental infection caused by one strain of TBEV and variant received from this strain population. Matherials and methods. Experimental animals. Mice Balb/c (SPF, female, 15-16 g) obtained from the breeding of laboratory animals "Pushchino" contained in the vivarium conditions FSUE on manufacture of bacterial and viral preparations of Chumakov Institute of poliomyelitis and viral encephalitides Viruses. TBEV: strain EK-328 of Siberian subtype and variant 58, received from this strain population by cloning one plaque. The viruses differ from each other by three amino acids in the non-structural region (proteins NS2A and NS4A) [4]. Earlier experiments in mice demonstrated a high virulence of viruses, but showed differences in their sensitivity to IFN-α and IFN-α induction time. Animals were infected intraperitoneally with 0.3 ml of 1.000.000 PFU of virus as porcine embryo kidney (PEK) cells cultural fluid. In each group 6 mice was studied. Study of mice spleen lymphocytes subpopulation structure was performed by flow cytometry using monoclonal antibodies against the different lymphocyte subpopulations antigens. Single-cell suspensions of splenocytes were prepared by grinding the spleen with the plunger of a disposable syringe, passing the ground spleen through nylon mesh, and suspending the cells in PBS. Mouse spleen single-cell suspensions were stained with fluorochrom- conjugated anti-mouse: CD3, CD4, CD8, CD19, I-EK (MHC class II) – FITC; CD25, NK1.1 – PE; Foxp3 – APC, SD45 – PerC7 monoclonal antibodies (e-Bioscience, USA) at 4°C for 30 min. Then, erythrocytes were lysed with a red blood cell lysis buffer (BioLegend, USA). After washing with PBS, the samples were fixed with a fixation solution (BioLegend, USA) and analyzed by flow cytometry (Cytomix FC-500, Beckman Coulter, USA with the CXP software). The cell population gate was determined by the front and side light-scattering and cell size; 10 000 cells per gate were found. The group tests were compared using the Mann–Whitney rank sum test for independent samples. The P value of ≤0.05 was considered statistically significant (STATISTICA 8 software). Results and discussion Study of spleen lymphocyte subpopulation structure of mice infected with strain EK-328 and variant 58 revealed the differences. The strain EK-328 induced increase of the number of T cells (CD45/CD3) in 1.8 times as compared with intact mice (from 32.5 to 59.4%), probably due to increasing the number of T helper CD3/CD4 (from 2.5 to 43.8%) and less CTLs (cytotoxic lymphocytes CD3/CD8) – from 8 to 12.3%. Antigen CD3 is a marker of mature T-lymphocytes and presented on T medullary tymocytes and peripheral blood lymphocytes. This antigen is highly specific and CD3 detection is clearly pointing to the belonging of T-lymphocytes to the mature stages of these cells differentiation. The main factor preventing the active virus proliferation is mature cytotoxic lymphocytes (CD8 T cells, CTL). These cells have an important property - specificity of action on target cells, i.e., the ability to destroy cells infected with viral particles [5]. It is typical of EK-328 is the induction of B lymphocyte population (CD45/CD19, p<0.05) from 14.2 to 23.2% (in 1.6 times). This accompanied by decrease in the number of NK cells (CD16/32) (in 3.5 times, from 7.3 to 2.1%) due to increased values of NKT cells compared to the control group. It is known that there are natural killer T-cells (NKT) in the activated lymphocytes population, along with CTL and NK cells. These cells are expressed both NK cells markers (CD16, CD56) and T-cell differentiation antigens (CD3, CD4, CD8). This lymphocytes subpopulation is found mainly during the infectious process in the liver and lungs, but virtually absent in peripheral blood [3]. In our studies EK-328 induced lymphocyte activation in 1.5 times (p<0.05), as evidenced by the clones appearance with early activation marker CD45/CD25 (14%). This was accompanied by a decrease (p <0.05) of T-regulatory cells (T-regs – CD4/CD25/Foxp3,) in 2.2 times (from 0.9 to 0.4%), which play an important role in induction preventing and control of immune responses, as well promoting to the formation of immune tolerance [4]. Variant 58 58 in contrast to EK-328, did not affect the T lymphocytes amount, but also provided suppressive effect on NK cells (from 7.3 to 3%). It is now known that NK-cells play an important biological role in immune surveillance mechanisms against tumor cells, parasites and virus-infected cells, by regulating the proliferation and differentiation of bone marrow cells, the elimination of the aging somatic cells, modulation of innate immune cells, activation or suppression of the T- and B-lymphocytes proliferation as well as maturation and generation of virus-specific cytotoxic T-lymphocytes, which can be considered as an essential component nonspecific defense and cell-mediated immune response [3] Variant 58 caused decline of NKT cells from 0.4% to 0.1% (p<0,05) and T-regulatory cells from 0.9 to 0.5%. (p <0.05). Variant 58 similar EK-328 strain increased (p<0.05) cell early activation marker's (CD45/CD25) level in 1.5-1.7 times (15.3%). Thus, the studied strains of TBEV differ in their effect on lymphocyte subpopulation structure of infected mice, providing different effect. EK-328, in contrast to the variant 58, activated CD45/CD3 T-lymphocytes, CD3/CD4 T-helper cells, CD3/CD8 cytotoxic T cells and CD45/CD19 B-lymphocytes. Both viruses in one way or another caused decline of NK cells and T-reg cells amount. Interest from the point of view of the relationship of macro- and micro-organism is to determine the characteristics of interaction between different population's components of TBEV with the host immune system, in particular the comprehensive estimation of the virus influence on the activation and differentiation markers expression. Thus, we have shown that virus strain and variant from its population which have the similar virulence can alter the differentiation and activation molecules expression on the innate and adaptive immunity effectors in different ways and thus modulate effector functions of innate and adaptive immunity.

References

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Keywords: Tick-borne encephalitis, lymphocyte subpopulation structure, immunophenotype, innate immunity, Adaptive Immunity

Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.

Presentation Type: Poster Presentation

Topic: Infectious and parasitic diseases

Citation: Motuzova OV, Akhmatova EA, Khomenkov VG, Akhmatova NK, Lebedinskaya OV and Karganova GG (2015). Experimental infection caused by two variants of one tick-borne encephalitis virus strain: similar virulence, but different influence on lymphocyte subpopulation structure. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00184

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Received: 06 May 2015; Published Online: 14 Sep 2015.

* Correspondence: Ms. Olga V Lebedinskaya, Acad. E.A. Wagner Perm State Medical University, Perm, Russia, lebedinska@mail.ru