Prevalence of Tick-Borne Encephalitis in Ixodes Ricinus and Dermacentor

VYTAUTAS MAGNUS UNIVERSITY

FACULTY OF NATURAL SCIENCES

DEPARTMENT OF BIOLOGY

BORIS MOSSE

Prevalence of Tick-Borne encephalitis in Ixodes ricinus

and Dermacentor reticulatus ticks in Belarus

Bachelor Thesis

“Biology and Genetics” study program, state code 6121DX011

Life Sciences

SUPERVISOR: ALGIMANTAS PAULAUSKAS

DEAN OF THE FACULTY: PROF. DR. SAULIUS MICKEVIČIUS

KAUNAS 2020

Experimental work was done during autumn semester 2019.

Reviewer of the bachelor’s thesis is:

The work is defended: at a remote public meeting of the Bachelor's Thesis Defense Commission on 18s of june, 2020, 9:00 remotely.

Vytautas Magnus University, Department of Biology.

Address of the department: Department of Biology, Faculty of Natural Sciences, Vytautas

Magnus University, Vileikos street 8, lt-44404, Kaunas, republic of Lithuania.

CONTENTS

SUMMARY ...... 4 INTRODUCTION ...... 5 Main goal of the work: ...... 5 Tasks of the work: ...... 5 LITERATURE REVIEW ...... 6 1. General biology of Ixodes ticks ...... 6 1.2. Classification ...... 6 1.2. Life cycles of ticks ...... 6 1.3 Geographical distribution and habitats of ticks ...... 7 1.4 Pathogens transmitted by Ixodes ticks ...... 8 2. Epidemiology of tick-borne pathogens...... 9 2.1 Lyme borreliosis and tick-borne encephalitis in Europe ...... 9 2.2 Low prevalent pathogens distribution ...... 10 2.3. Ticks distribution and epidemiological situation of Tick-Borne Pathogens in Belarus . 11 3 Tick-borne encephalitis virus ...... 14 3.1 Classification of tick-borne encephalitis virus ...... 14 3.2 Genome and structural characteristics of tick-borne encephalitis virus ...... 15 3.3 Tick-borne encephalitis pathogenesis and virus replication ...... 16 3.4 Tick-borne encephalitis virus natural cycles ...... 17 3.5 Prevalence rate of tick-borne encephalitis virus in ticks ...... 17 MATERIALS AND METHODS ...... 18 1. Collection of questing ticks ...... 18 2. Identification of ticks ...... 18 3. RNA extraction from tick samples ...... 20 4. Real-time reverse transcription-PCR ...... 22 5. Pooled prevalence calculations ...... 23 RESULTS AND DISCUSSION ...... 23 CONCLUSIONS ...... 26 REFERENCES ...... 28

SUMMARY

With an annual average number of more than 2,900 cases, tick-borne encephalitis is the vector-borne diseases with largest impact on human health in Europe. Over the last decades, Belarus contributes a highly endemic area for major tick- borne diseases comparing to other European countries. To evaluate the existence of encephalitis virus endemic foci we determined the prevalence of TBEV in I. ricinus and D. reticulatus ticks collected from different locations in Belarus.

A total of 52 ticks were collected at two locations of Belarus in south and north of Minsk region during April-June 2019. The number of places planned for this research was significantly reduced due to the coronavirus pandemia and border closure. Samples were screened for the presence of specific TBEV RNA by quantitative real-time PCR. Ticks positive for encephalitis virus were not detected.

Parallel study held at the same period by Belarus Centre for Epidemiology and Microbiology detected seven positive samples of I. ricinus and D. reticulatus from four collection sites. The TBEV prevalence was estimated as 2.23% in I. ricinus and 1,75% in D. reticulatus. Thus, encephalitis continues to be a priority tick-borne infection in Belarus.

INTRODUCTION

Ticks are obligate haematophagous (blood-feeding) ectoparasites that feed on a variety of vertebrate host animals, including mammals, birds, reptiles, and sometimes amphibians. They transmit a wide range of disease-causing organisms and are of great medical and veterinary importance. In Europe the diseases transmitted by ticks are considered the most important vector-borne diseases. Concerning impact on human health the two most widespread are Lyme borreliosis and tick-borne encephalitis. They also play a major role in the transmission of other tick-born ediseases, in particular anaplasmosis, babesiosis, tularemia, and rickettsiosis. The wide distribution of bacterial, virus and protozoal agents of human pathogenicity and the extreme range of their prevalence in ticks indicate the need for comprehensive studies on ticks and tick- borne pathogens in particular in Eastern Europe countries.

Main goal of the work: To evaluate the existence of encephalitis virus endemic foci and determine the prevalence of TBEV in ticks collected from different locations in Belarus.

Tasks of the work: (i) Collect data about distribution of I. ricinus and D. reticulatus in Belarus.

(ii) Check the presence of I. persulcatus in Belarus

(iii) Make statistical analysis of the prevalence of TBE in this species.

LITERATURE REVIEW

1. General biology of Ixodes ticks

1.2. Classification Ticks are arthropods closely related to mites, with which they form the subclass Acari within the class Arachnida. Members of the Acari are the only known parasitic arachnids. The 896 known species of ticks can be further divided into three families, the Argasidae (soft ticks, 193 species), the Ixodidae (hard ticks, 702 species), and the Nutalliellidae (which consist of only one species from Africa, Nuttalliella namaqua) (Guglielmone et al., 2010). Hard ticks can be easily identified by their visible mouthparts, and by the presence of the scutum, a plate-like shield on the dorsal surface. They normally feed only once during each of the three parasitic life stages (larva, nymph, adult female) and remain attached to the host for a long time (Gray, 2002). The name of the type species Ixodes ricinus is derived from the greek word "ixos" for the European mistletoe plant (Viscum album). Soft ticks lack a hardened upper surface and have less conspicuous mouthparts than ixodes ticks. They feed repeatedly, each time taking only small amounts of blood, and remain normally in the close vicinity of their hosts, such as in their nests and burrows (endophilic behaviour) (Gray, 2002). While some argasid species do transmit diseases, ixodes ticks are by far more important vectors for several zoonoses (infectious diseases that can be transmitted between animals and humans) and are considered the most important ectoparasites of livestock (Gray, 2002). Following morphological comparisons and molecular studies a group of 21 Ixodes species was suggested named as Ixodes ricinus-persulcatus group (Filippova, 2002). This complex contains all four Ixodes species (I. ricinus, I. persulcatus, I. scapularis, and I. pacificus) that are the main vectors for Lyme borreliosis (LB)-causing spirochaetes. Based on phylogeographic data, it was assumed that the European population of this tick species is genetically mostly homogeneous (Noureddine et al., 2011). Later, a phylogeographic genetic structure was revealed by use of mitochondrial genes multi-sequence analysis at least in the case of geographically distant populations (Dinnis et al., 2014).

1.2. Life cycles of ticks The life cycles of ixodes ticks, during which there are four stages (egg, larva, nymph, and adult), take between one and six years. Each of the three parasitic stages seeks the hosts, attaches, feeds and drops off. Most ixodes species require the new hosts for each feeding stage 6

(i.e. they are three-host ticks). Hosts may be confined to one single species or can come from a wide range of vertebrate animals, as in the case of I. ricinus. Exophilic ticks such as I. ricinus actively seek their hosts by questing in the vegetation. The duration of the feeding ranges from 2- 6 days for larvae, 3-8 days for nymphs, and 6-12 days for adult females, while adult males rarely feed. The feeding is followed by a digestion period and the moulting of larvae and nymphs to the next stage, while adult females begin to produce a single batch of 1,000-10,000 eggs, after which they die. In total, ixodes ticks spend only a small amount of time on their hosts, but over 90% of their life in long periods of resting and development, mostly hidden in the vegetation layer or in the host's nest. The developmental rate, host seeking period and diapause are strongly influenced by climatic factors. Therefore, seasonal climatic changes have major effects on the composition and activity of tick populations (Gray et al., 2009). Fig. 1 shows the life cycle of I. ricinus, including the duration of each moulting process, and groups of host animals that each stage most commonly feeds on. As indicated by the human symbol in the center, each postembryonic stage is capable of infesting humans when encountered.

Fig. 1. Life cycle of Ixodes ricinus. Taken from Parola & Raoult (2001).

1.3 Geographical distribution and habitats of ticks Ticks occur on every continent and are even found in marine colonies of seabirds across the Northern and Southern hemispheres (I. uriae) (Olsen et al., 1993). The four Ixodes species that are mainly responsible for the transmission of LB and other zoonoses are member of the Ixodes ricinus-persulcatus species complex and occur in temperate regions of the Northern hemisphere (Fig. 2) (Gray, 2002). The Taiga tick, I. persulcatus, has the widest distribution of these four species, ranging from Poland in Central Europe across the entire region of boreal 7 forests in Eastern Europe and Asia to Japan and Korea (Keirans et al., 1999). The sheep tick, I. ricinus occurs in temperate regions of Europe between the latitudes 65° N and 39° N, and extends eastwards from the British Isles (10° W) across Europe into Asia at a longitude of 60° E, partially overlapping with the distribution of I. persulcatus in Central and Eastern Europe (Gray, 1991). I. ricinus has also been found in North Africa, although the taxonomic status of this population may require further evaluation (Noureddine et al., 2011). I. ricinus as a species with limited mobility is transported mainly by its hosts. Obviously, the flying bird species may transport the ticks (containing tick-borne pathogens) for large distances (Waldenstrom et al., 2007). According to long-term climatic data, the expansion of ticks can be explained by increasing average annual temperature associated with global climatic changes (Daniel et al., 2009).

Fig. 2. Geographical distribution of four Ixodes species, that are the main vectors for LB in humans. Taken from Swanson et al., 2006.

1.4 Pathogens transmitted by Ixodes ticks Apart from mosquitoes, ticks are the second most important vectors of human diseases worldwide, and the main disease vectors in the Northern hemisphere (Parola, Raoult, 2001). Globally they are also the most significant vector of diseases affecting livestock (Jongejan, Uilenberg, 2004). Ticks transmit a wider variety of pathogens than any other group of arthropod vectors, including viruses, bacteria (including spirochaetes, i.e. spiralshaped bacteria with a flexible cell wall), and protozoans. I. ricinus together with three other members of the I. ricinus- persulcatus group (I. persulcatus, I. scapularis, I. pacificus), is responsible for the transmission of several viral, bacterial, and protozoan diseases in humans. These include tick-borne 8 encephalitis (TBE) in mainland Europe and Asia, human granulocytic anaplasmosis, Lyme borreliosis (LB), and babesiosis. TBE can be potentially fatal and causes over 10,000 cases of human infections each year in mainland Europe. LB was first identified in and named after Old Lyme, Connecticut, USA (Kurtenbach et al., 2006), and was later shown to be caused by a spirochaete transmitted by ticks (Burgdorfer et al., 1982) that was named Borrelia burgdorferi. Apart from the agents of LB and TBE, this species may transmit Anaplasma phagocytophilum, an intracellular bacterium causing human granulocytic anaplasmosis, pathogenic for immunocompromised patients, Francisella tularensis that can also be transmitted by aerosol, causing tularaemia in humans, spotted fever rickettsiae (Rickettsia helvetica, R. monacensis, R. aeschlimanii), protozoan pathogens of the genus Babesia (B. divergens) mainly of veterinary significance but able to cause infection at least in immunocompromised humans, Babesia venatorum, a newly defined species with two human disease cases reported in asplenic patients, Babesia microti pathogen of human previously assumed rare in Europe, but recently receiving more attention (Anna L. Reye et al. 2013). In Europe, I. ricinus is far the most important vector tick species transmitting diseases of humans. Nevertheless, recently there have been changes recorded in the distribution and abundance of other tick species with a potential to attack and transmit diseases to humans. Changes in the occurrence of Dermacentor sp. (D. reticulatus) ticks were reported from various European countries. The shift in the distribution of the vectors is associated with increased exposition to the pathogens they transmit. This tick species may also attack human and be involved in transmission of R. slovaca and R. raoultii (Parola et al., 2013), B. burgdorferi and TBE virus (Biernat et al., 2014).

2. Epidemiology of tick-borne pathogens

2.1 Lyme borreliosis and tick-borne encephalitis in Europe With an annual average number of more than 85,000 cases of LB and almost 2,900 cases of TBE, these two diseases are the vector-borne diseases with largest impact on human health in Europe (ECDC meeting report, 2012). The data have a character of a rough estimate rather than a precise number. The situation is more complicated in the case of LB due to the multi- symptomatic character of the disease, confusing situation regarding chronic or post-lyme syndrome associated conditions, variable laboratory test performance and result interpretation (Rizzoli et al., 2011). In contrast, disease case based surveillance of TBE seems to be much more precise thanks to the relatively conservative clinical picture, reliable laboratory diagnostic tools and hence case definition. Based on data from the EU member states, the incidence of TBE in 9

2007 ranged from 0.03 (Italy) to 10.4 (Estonia) cases per 100,000 inhabitants (Stefanoff et al., 2011). From a long-term perspective, the highest incidence is recorded in the Baltic area (Estonia, Latvia, Lithuania). Over the last decades, Estonia, Latvia and Lithuania contribute a highly endemic area for major tick- borne diseases comparing to other countries of European Union. The annual incidence rates for TBE remain above 5 cases per 100,000 population, with decade average rates over 10 for each country. Lyme borreliosis (LB) incidence is significantly higher – decade average is 73,7 cases per 100 000 population, with maximum rates remaining over 100 reported from Estonia. Both TBE and LB are mandatory notifiable diseases in all three countries. From at least 2010 for Latvia and 2013 for Estonia, ehrlichiosis is also included in the annually notifiable communicable diseases lists, prepared by national health agencies (Geller et al., 2018). In the neighbouring countries of Belarus, the prevalence of Borrelia species in questing Ixodes ticks ranges from 6.2% in Poland, 11% in Lithuania to 40.7% in Russia and 18–51% in

Latvia (Reye AL et al. 2013).

2.2 Low prevalent pathogens distribution Anaplasma phagocytophilum is a rickettsial agent pathogenic for humans, causing human granulocytic anaplasmosis. Although the incidence of anaplasmosis in Europe is not well documented, clinical cases seem to be rare. In ticks, a prevalence of 2.9% in Lithuania, 2.9–8.7% in Poland, 3.6% in the Ukraine and 5.0% in Russia have been observed (Radzijevskaja et al.

2008, Grzeszczuk et al. 2004). Coxiella burnetii is the causative agent of Q fever in humans, but it also affects domestic and wild ruminants, leading to increased rates of abortion. Although ticks are competent vectors of this pathogen, so far Q fever outbreaks have been linked to exposure to infected animals and their products. Despite only limited studies the prevalence of C. burnetii in European ticks, does not seem to exceed 2.6% (Sprong H et al. 2011). Another tick-borne pathogen that can also be transmitted by aerosol is Francisella tularensis, causing tularaemia in humans. Prevalence rates in ticks are at least in France and Germany below 1.6% and like Q fever, tularaemia outbreaks are usually linked to the exposure to contaminated biological matter (faeces, blood, milk, etc.) rather than tick bites (Franke J et al.

2010). Bartonella species can be transmitted to humans by various arthropods including ticks and cause diseases like trench fever and cat scratch disease. The prevalence of these bacteria in ticks can vary as much as from 3.7% in Poland to 40% in Russia and France (Podsiadly E et al. 2009). Besides these bacterial agents, also human pathogenic protozoans can be transmitted by tick bites. At least two Babesia species, namely B. microti and B. divergens, are known to cause 10 babesiosis in humans, whereas for B. venatorum human pathogenicity is suspected. In Poland, 3.5% to 5.4% of ticks were found to be infected with B. microti, whereas 1.6% of ticks from

Russia were infected with B. venatorum (Wojcik-Fatla A et al. 2009).

2.3. Ticks distribution and epidemiological situation of Tick-Borne Pathogens in Belarus Belarus is a landlocked country in North-Eastern Europe with a population of 9.4 million, 70% of which reside in urban areas. Its terrain is generally flat, with an average elevation of 160m above sea level and about 40% of the country is covered by forest. The most common tick species in Belarus are Ixodes ricinusand and Dermacentor reticulatus. There is one more medically important tick species circulating in the neighbouring countries of Belarus – Ixodes persulcatus. The sympatric zone of I. ricinus and I. persulcatus covers the southern and eastern parts of Estonia, the eastern part of Latvia, and a small part of northern Lithuania (Paulauskas et al. 2016) (Figure 3). According to this data I. persulcatus ticks can also be present only in the very northern part of Belarus.

Figure 3. Green dots indicate tick sampling sites. Grey area indicates I. ricinus allopatry zone. Dashed area represents the sympatric zone of I. ricinus and I. persulcatus according to Paulauskas et al. 2016.

Prevalence of tick-borne bacterial pathogens was investigated in Ixodes ricinus and Dermacentor reticulatus ticks from different geographical locations in Belarus (Reye AL et al. 2013). In total, 553 ticks belonging either to the species Ixodes ricinus (59.1%; 327/553) or Dermacentor reticulatus (40.9%; 226/553), were collected. Most ticks were collected in Gomel region (53.7%; 297/553), followed by Brest (14.8%; 82/553) and Minsk region (14.3%; 79/ 553). Only few ticks were collected in Grodno (8.1%; 45/553), Mogilev (5.6%; 31/553) and Vitebsk region (3.4%; 19/553). The overall prevalence of infected questing ticks from Belarus (counting mixed infected ticks only once) was 37.7% (171/ 453). Questing I. ricinus displayed an infection prevalence of 32.9% (95/289), which was significantly lower than for D. reticulatus (46.3%; 76/164; p,0.01) (Table 1). The overall prevalence of infections in feeding ticks was 32% (32/100); significantly fewer feeding I. ricinus ticks were positive in the pathogen detection PCRs (13.2%; 5/38) than feeding D. reticulatus ticks (43.5%; 27/62; p,0.001). On a regional level, there were considerable differences in the total prevalence of infected ticks, ranging from 10.5% in Vitebsk to 46.3% in Brest.

Table 1. Pathogen prevalence in questing and feeding ticks.

Overall, the pathogen prevalence in ticks was 30.6% for I. ricinus and 45.6% for D. reticulatus. The majority of infections were caused by members of the spotted-fever group rickettsiae (24.4%), 9.4% of ticks were positive for Borrelia burgdorferi sensu lato, with Borrelia afzelii being the most frequently detected species (40.4%). This survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. Another study includes the identification and typing of pathogens in ixodes ticks collected in Belarus regions in 2017 (Красько и др, 2018). The results of a study of 504 samples are presented in Table 2. DNA of Lyme disease pathogens was detected in 156 individuals (30.1%), rickettsia in 170 (33.7%), tick-borne encephalitis virus in 83 samples (16.47%). 104 (20.6%) is the causative agent of human anaplasmosis, 107 (21.2%) is the causative agent of ehrlichiosis. At the same time, 94 samples, which is 18.65% of all the ticks studied, contained several pathogens (mixed infection) and only 97 ticks (19.2%) did not detect pathogenic microorganisms. These data are close to those of the 2015 and 2016 seasons. A special feature is the increase in the number of ticks containing the causative agents of anaplasmosis and, especially, ehrlichiosis, 21.2% compared with 5.98% in 2016 and 2.3% in 2015. Mixed infection of ticks remains at the same level - 18.65% in 2017 against 17.7% in 2015 and 21.74% in 2016. Infection with Lyme disease causative agents remains practically unchanged - about 30% of all ticks examined and tick-borne encephalitis - about 14-16%.

Table 2. Infectiousness of ixodes ticks collected on the territory of the Republic of Belarus in the 2017 season by pathogenic microorganisms Region DNA/RNA Borrelia Encephalitis Rickettsia Anaplasmosis Ehrlihia Mixed Not extracted Burgdorferi spp. muris infection infected Sensu lato Brest 72 37 11 14 14 15 18 18 region Gomel 72 21 19 23 17 17 11 8 region Minsk 72 23 4 22 6 12 8 15

Minsk 72 20 7 13 18 18 10 15 region Mogilev 72 14 17 28 8 19 12 16 region Grodno 72 15 9 36 14 11 12 15 region Vitebsk 72 26 16 34 27 15 23 10 region Total 504 156 83 170 104 107 94 97

3 Tick-borne encephalitis virus

3.1 Classification of tick-borne encephalitis virus The causative agent of tick-borne encephalitis is a virus belonging to the family Flaviviridae, genus Flavivirus. After complete genome sequencing, phylogenetic relationships of viruses in this genus were analyzed in detail. The sequence data are generally concordant with the previous classification based on antigenic properties. Presently, a generally accepted classification scheme of the genus correlates with ecological properties of the viruses such as main vector and main reservoir host species. The genus consists of a large group of mosquito- borne flaviviruses (including West Nile virus, Yellow fever, Dengue), a group of tick-borne viruses and a group of viruses with no known vector (Gaunt et al., 2001). Tick-borne flaviviruses are further sub-divided into two groups according to preferred host: avian tick-borne flaviviruses and mammalian tick-borne flaviviruses including: Tick-borne encephalitis virus (TBEV). Among TBEV strains three virus subtypes can be differentiated: European subtype (TBEV-Eu), Far Eastern subtype (TBEV-FE) and Siberian subtype (TBEV- Sib). Although high degree of similarity in antigenic properties as well as in nucleotide sequence (maximum 5.6 % difference in deduced amino acid sequence of the E-protein) was revealed, the subtypes differ in the severity of the disease they cause, their main vector species and in the geographic distribution (Ecker et al., 1999). TBEV-Eu is distributed widely throughout eastern, central and western Europe (excluding Iberian Peninsula, UK, Ireland and Benelux) and in parts of southern Europe and Scandinavia. TBEV-FE occurs from east Europe through Russia and China to Japan and TBEV-Sib is distributed mainly in western Siberia (Stefanoff et al., 2011). In the Baltic's and parts of Russia, where both vectors occur, TBEV-Eu and TBEV-FE co-circulate (Golovljova et al., 2004). Occasionally, strains of Siberian subtype were detected in Latvia (Mavtchoutko et al., 2000) and Estonia (Golovljova et al., 2004). The main vector of TBEV-Eu is I. ricinus, while TBEV-FE and TBEV-Sib are mostly transmitted by I. persulcatus (Charrel et al., 2004). Strains of the European subtype of TBEV cause usually a milder disease with lower mortality rate (1-5 %) compared to TBEV-FE (5-35 %). Infection with TBEV-Sib as well has milder course than TBEV-FE. Differences in the disease severity could be alternatively explained by factors other than biological properties of the virus – by different case definition or level of medical care (Mandl, 2005).

3.2 Genome and structural characteristics of tick-borne encephalitis virus TBEV has a spherical virion of approximately 50 nm in diameter containing an icosaedric capsid surrounded by a host cell-derived lipid bilayer (Chambers et al., 1990) (Figure 4). The virion RNA is infectious and serves as both the genome and the viral messenger RNA. The whole genome is translated into a polyprotein, which is processed co- and post-translationally by host and viral proteases.

Figure 4. TBEV spherical virion of approximately 50 nm in diameter containing an icosaedric capsid.

The genome consists of approximately 10-11 kb of single stranded RNA of positive polarity carrying type 1 cap on the 5´end (to allow translation) or a genome-linked protein (Chambers et al., 1990). The genome 3' terminus is not polyadenylated but forms a loop structure. The genome contains single open reading frame (ORF) flanked on both sides by untranslated regions (UTR). UTRs are probably included in the regulation of replication, translation or packaging of the virus.

The ORF is translated into a single polyprotein (approximately 3400 amino acids) and subsequently cleaved by cellular and viral proteases into 3 structural proteins (capsid protein C, pre-membrane protein prM, envelope protein E) and 7 nonstructural proteins (glycoprotein NS1, subunits of viral protease NS2A, NS2B, viral protease/helicase NS3, NS4A, NS4B, RNA dependent RNA polymerase NS5) (Figure 5). Protein C builds up the capsid, proteins M (cleaved prM protein) and E are integrated in the lipid envelope (Gritsun et al., 2003b).

Figure 5. TBEV single polyprotein cleaved into 3 structural proteins and 7 nonstructural proteins.

3.3 Tick-borne encephalitis pathogenesis and virus replication After inoculation of the virus into the skin by the vector tick, the virus is transported via Langerhans cells to lymphatic system and lymphatic nodules. Subsequent virus replication is followed by viremic phase and the virus is spread hematogenously to different organs. The virus is highly neurotropic, however particular mechanism directing the virus into central nervous system (CNS) is not known yet. Neurons represent the main target cells for the virus. The damage to CNS is partly due to the virus action itself and partly due to the host immune response (Ruzek et al., 2009). The cell receptors of the virus as well as the mechanism of passing through the blood-brain barrier remain unknown. The virus is internalized by the means of receptor-mediated endocytosis. Protein E triggers the fusion with the membrane of endosome under low pH. After the viral coat is resolved the transcription begins. Whole genome copies of negative and positive polarity are produced. The genome RNA serves as well as mRNA and is translated into a single polyprotein, which is subsequently cleaved. Proteins are processed in association with the endoplasmic reticulum. Capsid and envelope are assembled and premature virions are produced. Maturation occurs in the Golgi apparatus by cleavage of the prM protein enabling the formation of E protein homodimers. A mature virion is able to leave the cell by the means of membrane fusion (Mandl, 2005).

3.4 Tick-borne encephalitis virus natural cycles TBEV is ecologically classified as an arbovirus – arthropod-borne virus. Arboviruses were defined as viruses transmitted by arthropod vector from a viremic vertebrate host to another. Viral replication occurs in both – vertebrate host and arthropod vector. Ixodes ricinus is the main vector of TBEV in Europe. The virus survives in the tick transstadially and is, although with relatively low efficiency, transmitted transovarially. In general, the virus circulates in natural foci between the developmental stages of I. ricinus and its hosts, however other vectors may contribute. Common way of arbovirus transmission involves ingestion of the blood of a viremic host by the tick and transfer of the virus during subsequent feeding to another host. TBEV-infected natural hosts usually undergo a very short viremic phase, which obviously affects the probability of the virus transmission. Apart from viremic transmission, virus may be transferred from one tick to another in the absence of viremia by the means of co-feeding. Co-feeding transmission occurs between ticks feeding in immediate vicinity to each other – localized transmission occurs without virus circulation in blood. This mechanism was first described for Thogoto virus in 1987, later it was confirmed also for TBEV in I. persulcatus and I. ricinus ticks (Labuda et al., 1997). Currently, this way of transmission is considered an important mechanism of TBEV maintenance in natural cycles. Interestingly, co-feeding transmission can occur even on immune hosts. This fact remarkably shifts the insight into TBEV circulation since the number of available hosts is much higher than previously estimated (excluding immune hosts). Moreover, this way of transmission may be enhanced by immunomodulative factors present in tick saliva – so called “saliva activated transmission”). Although the importance of co-feeding in TBEV transmission cycles is generally accepted, the precise mechanism is still under discussion. Some authors consider spring co-incidence of larvae and nymphs the fundamental prerequisite for the emergence of TBEV foci. Co-feeding transmission from infected nymphs to uninfected larvae (passing from one generation to another) is assumed the major way of virus survival. In TBE non-endemic areas, the co-infestation of small mammals with nymphs and larvae was found to be very rare compared to a TBE endemic focus.

3.5 Prevalence rate of tick-borne encephalitis virus in ticks The average prevalence rate of TBEV in I. ricinus ticks does not usually exceed 2 % although occasionally numbers as high as 14 % were recorded (Casati et al., 2008). In a meta- analysis concerning prevalence reports for Scandinavia, an average MIR (minimum prevalence rate) of 0.28 % was obtained (Pettersson et al., 2014). As suggested the differences may be 17 induced by the difference of sampling area and area of TBEV natural focus – sampling out of the focus of TBEV circulation may result in inclusion of low TBEV infected tick populations and hence lower prevalence rate. Interestingly, much higher prevalence rate (about 4 times higher) was recorded in partially engorged ticks collected from humans than in questing ticks collected on vegetation (Bormane et al., 2004). Proposed causes of the difference are: virus amplification in infected ticks after obtaining another blood-meal and hence increased probability of successful detection (Suss et al., 2004) and/or, increased host-seeking activity of infected ticks.

MATERIALS AND METHODS

1. Collection of questing ticks Materials used: • 1.5 ml Safelock microcentrifuge tubes (Eppendorf) • Light coloured cotton blanket, 1 m x 1 m • Forceps The ticks were collected at two locations of Belarus in south and north of Minsk region during April-June 2019. (Table, Figure). The collection sites are characterised as mixed and pine forests, and are known as high-risk areas of I. ricinus and D. reticulatus ticks. The collection of questing ticks by cloth dragging was first used by Macleod (1932) and has been described by Hillyard (1996). Questing nymphal and adult ticks were collected by dragging a white cotton blanket of 1 m2, over the vegetation. The blanket was checked for attached ticks after every 20 steps (ca. 10 m). Ticks were removed with forceps and placed in a 1.5 ml Eppendorf tube. The date, time, location and weather conditions were recorded each session, as were temperature and vegetation cover. Ticks were maintained alive until later identification. In the laboratory, they were sorted into larvae, nymphs, females, and males using a microscope, and stored at -80°C. Ticks were pooled into groups according to the sampling site, the development stage and sex by five males or females per pool and 10 nymphs per pool.

2. Identification of ticks Questing ticks and were identified by morphological criteria using the classification keys provided by Hillyard (1996). It can be assumed that due to their ecology the vast majority of all ticks collected by blanket dragging are I. ricinus and D. reticulatus. First of all, Ixodes ticks were 18 checked to exclude possible individuals of I. persulcatus among I. ricinus by the presence of lateral groove along all the alloscotum in I. persulcatus females, while in I. ricinus females it is visible only on the side parts. In males, the internal spur of I. persulcatus ticks is bigger than in I. ricinus and covers the coxa of the second appendages pair (Figure 6).

Fig. 6. Differentiation of I. ricinus and I. persulcatus.

After that, both I. ricinus and D. reticulatus were classified according to stage, i.e. larva, nymph or adult. Adult ticks were further differentiated into males and females by the size of the dark scutum, which covers almost the entire dorsal surface in males, while it is confined to the anterior part of the idiosoma in females and immature stages, leaving the alloscutum clearly visible (Figure 7).

Fig. 7. Males, females and developing stages of I. ricinus and D. reticulatus.

3. RNA extraction from tick samples Materials used: DNA from I. ricinus and D. reticulatus nymphs and adult ticks was extracted by ISOLATE RNA Mini Kit (Bioline). Equipment and reagents to be supplied: • β-mercaptoethanol (8-ME)* (for Lysis Buffer RLY) • 70% ethanol; (to adjust RNA binding conditions) • 96-100% ethanol; (for Wash Buffer RW2) • Equipment for sample disruption and homogenization (Mortar and pestle with Liquid nitrogen) • Microcentrifuge tubes (1.5 mL) • Sterile RNase-free tips • Pipettes • Microcentrifuge (capable of 11,000 x g)

Disruption using a mortar and pestle An RNase-free mortar and pestle was used in combination with liquid nitrogen to disrupt and lyse frozen ticks. Grind the frozen tissue into a fine powder and add liquid nitrogen as necessary. It is important to ensure the sample does not thaw during or after grinding. Then transfer tissue powder into the liquid nitrogen cooled tube and allow liquid nitrogen to evaporate.

Sample homogenization Up to 5 x 10 eukaryotic cultured cells can be collected by centrifugation and directly lysed by adding Lysis Buffer RLY. Disrupt up to 30 mg of tissue. Cell lysis Add 350 µL Lysis Buffer RLY and 3.5 ml B-ME to the cell pellet or to ground tissue and vortex vigorously. Filter lysate Place ISOLATE II Filter (violet) in a Collection Tube (2 mL), load the lysate and centrifuge for 1 min at 11,000 x g. This step helps reduce viscosity and clears the lysate. If there is a visible pellet formed, depending on sample amounts and properties, transfer supernatant avoiding any pellet to a new 1.5 mL microcentrifuge tube. Alternatively, pass lysate 5-10 times through a nuclease-free 20 gauge (0.9 mm) needle and syringe. Adjust RNA binding conditions Discard ISOLATE II Filter and add 350 µL of ethanol (70%) to the homogenized lysate. Mix by pipetting up and down 5 times. Alternatively, transfer the flow-through into a new 1.5mL microcentrifuge tube, add 350 µL of ethanol (70%) and mix by vortexing (2 x 5s). Bind RNA For each preparation place one ISOLATE II RNA Mini Column (blue) in a Collection Tube (2 mL). Pipette lysate up and down 2-3 times and load lysate onto the column. Ensure al lof the lysate is loaded on the column. Centrifuge for 30s at 11,000 x g. Place column in a new Collection Tube (2 mL). Desalt silica membrane Add 350 µL Membrane Desalting Buffer (MEM) and centrifuge at 11,000 x g for 1 min to dry the membrane. Digest DNA Prepare a DNase l reaction mixture in a sterile 1.5 mL microcentrifuge tube. For each isolation, add 10 µL reconstituted DNase to 90 µL Reaction Buffer for DNase | (RDN). Mix by gently flicking the tube. Apply 95 µL DNase l reaction mixture directly onto center of silica membrane. Incubate at room temperature for 15 min. Wash and dry silica membrane.  Add 200 µL Wash Buffer RW1 to the ISOLATE II RNA Mini Column. Centrifuge for30s at 11,000 x g. Place the column into a new Collection Tube (2 mL).  Add 600 µL Wash Buffer RW2 to the ISOLATE II RNA Mini Column. Centrifuge for30s at 11,000 x g. Discard flow-through and place the column back into the Collection Tube. 21

 Add 250 µL Wash Buffer RW2 to the ISOLATE II RNA Mini Column. Centrifuge for 2 min at 11,000 x g to dry the membrane completely. Place the column into a nuclease-free 1.5 mL Collection Tube. Elute RNA Elute the RNA with 60 µL RNase-free water and centrifuge at 11,000 x g for 1 min.

4. Real-time reverse transcription-PCR Materials used: • Microcentrifuge tubes (1.5 mL) • Sterile RNase-free tips • Pipettes • Microcentrifuge (capable of 11,000 x g) • PCR RNase-free tubes (0,2mL) • StepOnePlus™ Real Time PCR system •

PCR can be applied to the diagnosis of any disease in which nucleic acids (e.g., DNA and RNA) or their expression as mRNA plays a role. PCR techniques allow for the in vitro synthesis of millions of copies of a specific gene segment of interest, allowing the rapid detection of as few as 1 to 10 copies of target RNA from the original sample. PCR is widely used for detection of both DNA and RNA viruses in ticks samples. For the identification of TBEV-specific RNA, F-TBE1 (5’-GGG CGG TTC TTG TTC TCC- 3’) and R-TBE1 (5’-ACA CAT CAC CTC CTT GTC AGA CT-3’) primers and a TBE- Probe- WT (FAM-TGA GCC ACC ATC ACC CAG ACA CA-BHQ1/TAMRA) of a quantitative real-time reverse transcription-PCR (RT-PCR) protocol according to Schwaiger and Cassinotti (2003) were used. This method targets a part of the 3” noncoding region of the TBEV genome that is conserved in essentially all TBEV subtypes and the amplicon is located at nt 11054–11123 of the European subtype of TBEV (accession number U27495). RNAs of the TBEV genome-positive samples were used for a one step RT-PCR as positive control. Samples were screened for the presence of specific TBEV RNA by quantitative real-time PCR using primers: F-TBE1 and R-TBE1. TBEV RNA was amplified in a 16 µl reaction mixture containing of 5 µl of sample RNA, 10 µl of 2X Reaction Mix, 0.25 µl of probe, 0,4 µl RNAinh and 0,2 µl RT. The cycling conditions comprised 30 min of reverse transcription at

47ºC, denaturation for 2 min at 97ºC, followed by 45 cycles for 15 sec. at 95ºC and 1 min at 57ºC.

5. Pooled prevalence calculations The TBEV prevalence in ticks was calculated as a minimum infection rate (MIR). MIR is a widely used method for estimating the proportion of infected individuals from pooled samples. It is calculated as the ratio of the number of positive pools to the total number of ticks tested. The underlying assumption of the MIR is that only one infected individual exists in a positive pool (Gu et al., 2003). Minimum Infection Rate (MIR): MIR = (x/(mk))*100% k = pool size, m = the number of pools tested, x = the number of positive pool.

RESULTS AND DISCUSSION

All collected ticks were identified as I. ricinus and D. reticulatus. A total of 52 ticks (27 of D. reticulatus and 25 of I. ricinus) were pooled with five male or female adults or ten nymphs in each (10 pools). TBEV RNA was not detected in these pools (Table 3).

Table 3. Np – number of pools, np – number of positive pools. Location I. ricinus Female Np/np Male Np/np Nymphs Np/np Total Mir, Grodno region 8 2/0 6 1/0 1 1/0 15/0

N – 53°467336’; E – 26°46.8001’ Pelika, Minsk region 4 1/0 3 1/0 3 1/0 10/0 N – 53°9838032’; E – 28°2849291’ D. reticulatus Mir, Grodno region 3 1/0 4 1/0 0 - 7/0

N – 53°467336’; E – 26°46.8001’ Pelika, Minsk region 10 2/0 10 2/0 0 - 20/0 N – 53°9838032’; E – 28°2849291’

In Belarus, tick-borne encephalitis began to be studied from the beginning of the 40s of the last century, when the virus was the first tome isolated from I. ricinus in the territory of Belovezhskaya Pushcha. 23

Unlike the eastern tick-borne encephalitis, where the virus circulation is found in at least four types of ticks, it was believed that the circulation of the TBEV in Belarus is carried out by only one species, while the role of the second mass species, D. reticulatus, remained questionable. For the first time, TBEV from D. reticulatus ticks was isolated in the Gomel region in 1985 [8]. In the 90s TBEV in D. reticulatus ticks, has already been found in the territory of four regions - Brest, Minsk, Grodno and Gomel. In some areas of Belarus, D. reticulatus was found to be the dominant species of ixodid ticks. But in the foci of tick-borne encephalitis I. ricinus dominated, accounting for 97.3% of the number of ticks, collected for over 30 years in all natural zones of Belarus. The general trend of significant activation of tick-borne encephalitis foci emerging in the early 90s coincided with an increase in the number of actively attacking ticks mainly due to D. reticulatus. In the country as a whole, the share of this species was 38.5%, while in the Gomel region it exceeded the number of I. ricinus collected, making 74.7% (Table 4).

Table 4 - The of the number of I. ricinus and D. reticulatus ticks collected from some regions of Belarus in 1985 – 1994. Region I. ricinus D. reticulatus Number % Number % Vitebsk 3551 95,2 180 4,8 Minsk 4542 65,2 2420 34,8 Brest 14327 78,2 4005 21,8 Gomel 3063 25,2 9021 74,7 Total 34167 61,5 21421 38,5

As a result of large-scale virological studies in 1985-1994 in the territory of the Republic of Belarus 244 strains of TBEV were isolated from various sources. The greatest number of virus strains was isolated in Gomel - 36% and Brest - 18.3% regions. As can be seen from table 2, the TBEV was isolated not only from I. ricinus ticks (3.8% on country average), but also from D. reticulatus (3.4%). Of the 97 strains of TBE virus isolated from ticks, 65 were isolated from I. ricinus and 32 from D. reticulatus (Table 5).

Table 5 - TBEV isolation frequency from I. ricinus and D. reticulatus ticks in Belarus (1985– 1994) Ticks Pools assayed N of positive % of positive pools I. ricinus 1692 65 3,8 D. reticulatus 925 32 3,4

In recent years, there has been a tendency to an increase in the number of vectors, an increase in the level of their natural infection, and an expansion of the distribution areas of infected ticks throughout the Republic of Belarus. To assess the epidemiological situation and study the patterns of virus circulation in the natural foci of the Republic of Belarus, studies of tick for their encephalitis infection held annually. In addition to our research, TBEV prevalence study in 2019 was also held by Belarus Research and Practical Centre for Epidemiology and Microbiology in the territories of Minsk, Gomel and Mogilev regions, where the virus is detected annually in carriers (Samoilova T et al., 2019). Collection site of the both studies presented on the Figure 8.

collection sites of our study collection sites of Samoilova et. al., no TBEV found collection sites of Samoilova et. al. with TBEV positive pools

Figure 8. Ixodes tick collection sites in Belarus in 2019 25

In total seven positive pools of I. ricinus and D. reticulatus from four different places were detected (Table 6).

Table 6. TBEV prevalence study results held in 2019 in Belarus (Samoilova T et al., 2019). Regions Collection I.ricinus D.reticulatus sites Number of Positive Number of Positive ticks/pools ticks/pools Minsk Gonoles 25/5 38/7 1 Maladzyechna 10/2 Staryya Darohi 52/8 2 26/5 1 Cervien 40/5 Gomel Svietlahorsk 15/3 1 Yelsk 28/4 Kalinkavichy 10/2 Mogilev Babruysk 20/3 Kirawsk 20/3 Cherykaw 28/5 2 Bychau 16/2 10/2 Total 224/37 5 114/19 2

In the present study TBEV was not detected in ticks collected from two places in Belarus. The number of places planned for this research was significantly reduced due to the coronavirus pandemia and border closure. However, similar studies conducted in Belarus during the same period indicate the presence of encephalitis in varios locations of the country. The TBEV prevalence (MIR) was 2.23% in I. ricinus and 1,75% in D. reticulatus. It should be noted that the current epidemiological situation in Belarus, as well as the situation for the near future, is estimated as unstable, depending on climatic and geographical conditions, the number of carriers and their virus prevalence, as well as on the population visiting tick habitats. More contacts between humans and ticks, abundance of ticks and their hosts may play a significant role in local spread of TBE and other tick-borne diseases.

CONCLUSIONS Encephalitis virus RNA was not detected in 52 I. ricinus and D. reticulatus ticks collected in 2019 from two different places in Belarus. Parallel study held at the same period by Belarus Research and Practical Centre for Epidemiology and Microbiology detected seven positive samples of I. ricinus and D. reticulatus from four collection sites. The TBEV prevalence (MIR) was estimated as 2.23% in I. ricinus and 1,75% in D. reticulatus. 26

Thus, encephalitis continues to be a priority tick-borne infection in Belarus. In the recent years a steady increase in the number of ticks in natural biotopes, an increase in their level of infection, and expansion of the distribution ranges of infected vectors were observed.

REFERENCES

1. Biernat, B., Karbowiak, G., Werszko, J., Stańczak, J., 2014. Prevalence of tick-borne encephalitis virus (TBEV) RNA in Dermacentor reticulatus ticks from natural and urban environment, Poland. Exp. Appl. Acarol. 64, 543–551. 2. Bormane, A., Lucenko, I., Duks, A., Mavtchoutko, V., Ranka, R., Salmina, K., Baumanis, V., 2004. Vectors of tick-borne diseases and epidemiological situation in Latvia 1993-2002. Int. J. Med. Microbiol. 293 (Suppl 37), 36-47. 3. Burgdorfer, W., Barbour, A. G., Hayes, S. F., Benach, J. L., Grunwaldt, E., & Davis, J. P. (1982). Lyme disease - a tick-borne spirochetosis? Science, 216, 1317-1319. 4. Casati, S., Bernasconi, M.V., Gern, L., Piffaretti, J.-C., 2008. Assessment of intraspecific mtDNA variability of European Ixodes ricinus sensu stricto (Acari: Ixodidae). Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis. 8, 152–158. 5. Chambers, T.J., Hahn, C.S., Galler, R., Rice, C.M., 1990. Flavivirus genome organization, expression, and replication. Annu. Rev. Microbiol. 44, 649–688. 6. Charrel, R.N., Attoui, H., Butenko, A.M., Clegg, J.C., Deubel, V., Frolova, T.V., Gould, E.A., Gritsun, T.S., Heinz, F.X., Labuda, M., Lashkevich, V.A., Loktev, V., Lundkvist, A., Lvov, D.V., Mandl, C.W., Niedrig, M., Papa, A., Petrov, V.S., Plyusnin, A., Randolph, S., Süss, J., Zlobin, V.I., de Lamballerie, X., 2004. Tick-borne virus diseases of human interest in Europe. Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect. Dis. 10, 1040–1055. 7. Daniel, M., Materna, J., Honig, V., Metelka, L., Danielova, V., Harcarik, J., Kliegrova, S., Grubhoffer, L., 2009. Vertical distribution of the tick Ixodes ricinus and tickborne pathogens in the northern Moravian mountains correlated with climate warming. Cent. Eur. J. Public Health 17, 139–145. 8. Dinnis, R.E., Seelig, F., Bormane, A., Donaghy, M., Vollmer, S.A., Feil, E.J., Kurtenbach, K., Margos, G., 2014. Multilocus sequence typing using mitochondrial genes (mtMLST) reveals geographic population structure of Ixodes ricinus ticks. Ticks Tick-Borne Dis. 5, 152–160. 9. ECDC meeting report: Second expert consultation on tick-borne diseases with emphasis on Lyme borreliosis and tick-borne encephalitis, 2012. European Centre for Disease Prevention and Control, Stockholm, Sweden. p: 10. 10. 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 (Pt 1), 179–185. 28

11. Filippova, N. A. (2002). Forms of sympatry and possible ways of microevolution of closely related species of the group Ixodes ricinus persulcatus (Ixodidae). Acta Zoologica Lituanica, 12, 215- 227. 12. Franke J, Fritzsch J, Tomaso H, Straube E, Dorn W, et al. (2010) Coexistence of pathogens in host-seeking and feeding ticks within a single natural habitat in Central Germany. Appl Environ Microbiol 76: 6829-6836. 13. Gaunt, M.W., Sall, A.A., de Lamballerie, X., Falconar, A.K., Dzhivanian, T.I., Gould, E.A., 2001. Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography. J. Gen. Virol. 82, 1867–1876. 14. Geller J., Epshtein J., Zygutiene M., Bormane A. (2018). Tick-borne infections in Baltic states. Proceedings of international scientific and practical conference “Molecular diagnostics 2018”. Minsk. P 441. 15. Golovljova, I., Vene, S., Sjolander, K.B., Vasilenko, V., Plyusnin, A., Lundkvist, A., 2004. Characterization of tick-borne encephalitis virus from Estonia. J. Med. Virol. 74, 580–588. 16. Gray, J. S. (2002). Biology of Ixodes species ticks in relation to tick-borne zoonoses. Wien Klin Wochenschr, 114, 473-478. 17. Gray, J.S., Dautel, H., Estrada-Pena, A., Kahl, O., Lindgren, E., 2009. Effects of Climate Change on Ticks and Tick-Borne Diseases in Europe. Interdiscip. Perspect. Infect. Dis. 2009, e593232. 18. Gritsun, T.S., Lashkevich, V.A., Gould, E.A., 2003. Tick-borne encephalitis. Antiviral Res. 57, 129–146. 19. Grzeszczuk A, Stanczak J, Kubica-Biernat B, Racewicz M, Kruminis-LozowskaW, et al. (2004) Human anaplasmosis in north-eastern Poland: seroprevalence in humans and

prevalence in Ixodes ricinus ticks. Ann Agric Environ Med 11: 99-103. 20. Gu W, Lampman R, Novak RJ. Problems in estimating mosquito infection rates using minimum infection rate. J Med Entomol. 2003; 40(5): 595–596. 21. Guglielmone, A. A., Robbins, R. G., Apanaskevich, D. A., Petney, T. N., Estrada-Peсa, A., Horak, I. G., et al. (2010). The Argasidae, Ixodidae and Nuttalliellidae (Acari: Ixodida) of the world: a list of valid species names. Zootaxa, 2528, 1 - 28. 22. Jongejan, F., Uilenberg, G. (2004). The global importance of ticks. Parasitology, 129 Suppl, S3-14. 23. Keirans, J., Needham, G., & Oliver Jr., J. H. (1999). The Ixodes ricinus complex worldwide: Diagnosis of the species in the complex, hosts and distribution. In G. Needham (Ed.), Acarology IX: Symposia (pp. 341-346). 29

24. Kurtenbach, K., Hanincova, K., Tsao, J. I., Margos, G., Fish, D., & Ogden, N. H. (2006). Fundamental processes in the evolutionary ecology of Lyme borreliosis. Nat Rev Microbiol, 4, 660-669. 25. Labuda, M., Kozuch, O., Zuffova, E., Eleckova, E., Hails, R.S., Nuttall, P.A., 1997. Tick- borne encephalitis virus transmission between ticks cofeeding on specific immune natural rodent hosts. Virology 235, 138–143. 26. Mandl, C.W., 2005. Steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis. Virus Res., Replication strategies of neurotropic viruses and influence on cellular functions 111, 161–174. 27. Nava, S., Guglielmone, A.A., Mangold, A.J., 2009. An overview of systematics and evolution of ticks. Front. Biosci. Landmark Ed. 14, 2857–2877. 28. Noureddine, R., Chauvin, A., Plantard, O., 2011. Lack of genetic structure among Eurasian populations of the tick Ixodesricinus contrasts with marked divergence from north-African populations. Int. J. Parasitol. 41, 183–192. 29. Olsen, B., Jaenson, T. G., Noppa, L., Bunikis, J., & Bergstrom, S. (1993). A Lyme borreliosis cyclein seabirds and Ixodes uriae ticks. Nature, 362, 340-342. 30. Parola, P., & Raoult, D. (2001). Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin Infect Dis, 32, 897-928. 31. Parola, P., Paddock, C.D., Socolovschi, C., Labruna, M.B., Mediannikov, O., Kernif, T., Abdad, M.Y., Stenos, J., Bitam, I., Fournier, P.-E., Raoult, D., 2013. Updateon Tick- Borne Rickettsioses around the World: a Geographic Approach. Clin. Microbiol. Rev. 26, 657–702. 32. Paulauskas A, Galdikaitė-Brazienė E, Radzijevskaja J, Aleksandravičienė A, Galdikas M. Genetic diversity of Ixodes ricinus (Ixodida: Ixodidae) ticks in sympatric and allopatric zones in Baltic countries. Journal of Vector Ecology. 2016;41(2):244–53. 33. Pettersson, J.H.-O., Golovljova, I., Vene, S., Jaenson, T.G., 2014. Prevalence of tickborne encephalitis virus in Ixodes ricinus ticks in northern Europe with particular reference to Southern Sweden. Parasit. Vectors 7, 102. 34. Podsiadly E, Karbowiak G, Tylewska-Wierzbanowska S (2009) Presence of Bartonella spp. in Ixodidae ticks. Clin Microbiol Infect 15 Suppl 2: 120-121. 35. Radzijevskaja J, Paulauskas A, Rosef O (2008) Prevalence of Anaplasma phagocytophilum and Babesia divergens in Ixodes ricinus ticks from Lithuania and Norway. Int J Med Microbiol 298: 218-221.

36. Reye AL, Stegniy V, Mishaeva NP, Velhin S, Hu¨ bschen JM, et al. (2013) Prevalence of Tick-Borne Pathogens in Ixodes ricinus and Dermacentor reticulates Ticks from Different Geographical Locations in Belarus. PLoS ONE 8(1): e54476. 37. Rizzoli, A., Hauffe, H., Carpi, G., Vourc H, G., Neteler, M., Rosa, R., (2011). Lymeborreliosis in Europe. Euro Surveill. 16, pii: 19906. 38. Ruzek, D., Salat, J., Palus, M., Gritsun, T.S., Gould, E.A., Dykova, I., Skallova, A., Jelinek, J., Kopecky, J., Grubhoffer, L., 2009. CD8+ T-cells mediate immunopathology in tick-borne encephalitis. Virology 384, 1–6. 39. Samoilova T.I., Krasko A.G., Yashkova S.E., Shypul V.N., Zaleuskaya O.S., Klimovich O.V., Tsvirko L.S., Drakina S.A., 2019. Iinvestigation of ixodoidea tick's infectiousness by tick-borne encephlitis virus in 2019 year. Proceedings of international scientific and practical conference “Molecular diagnostics 2019”. Minsk. P 171-173. 40. Schwaiger M, Cassinotti P. Development of a quantitative real-time RT-PCR assay with internal control for the laboratory detection of tick-borne encephalitis virus (TBEV) RNA. J Clin Virol. 2003; 27: 136–45. 41. Sprong H, Tijsse-Klasen E, Langelaar M, De Bruin A, Fonville M, et al. (2011) Prevalence of Coxiella Burnetii in Ticks After a Large Outbreak of Q Fever. Zoonoses Public Health. 42. Stefanoff, P., Polkowska, A., Giambi, C., Levy-Bruhl, D., O’Flanagan, D., Dematte, L., Lopalco, P.L., Mereckiene, J., Johansen, K., D’Ancona, F., 2011. Reliable surveillance of tick-borne encephalitis in European countries is necessary to improve the quality of vaccine recommendations. Vaccine 29, 1283–1288. 43. Suss, J., Schrader, C., Falk, U., Wohanka, N., 2004. Tick-borne encephalitis (TBE) in Germany--epidemiological data, development of risk areas and virus prevalence in field- collected ticks and in ticks removed from humans. Int. J. Med. Microbiol. 293 (Suppl 37), 69–79. 44. Swanson, S. J., Neitzel, D., Reed, K. D., & Belongia, E. A. (2006). Coinfections acquired from ixodes ticks. Clin Microbiol Rev, 19, 708-727. 45. Waldenström, J., Lundkvist, Å., Falk, K.I., Garpmo, U., Bergström, S., Lindegren, G., Sjöstedt, A., Mejlon, H., Fransson, T., Haemig, P.D., Olsen, B., 2007. Migrating Birds and Tickborne Encephalitis Virus. Emerg. Infect. Dis. 13, 1215–1218. 46. Wojcik-Fatla A, Szymanska J, Wdowiak L, Buczek A, Dutkiewicz J (2009) Coincidence of three pathogens (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti) in Ixodes ricinus ticks in the Lublin macroregion. Ann Agric Environ Med 16: 151-158. 31

47. Красько А.Г., Князева О.Р., Погоцкая Ю.В., Рустамова Л.М., Яшкова С.Е., Пашкович В.В. (2018). Выявление патогенных для человека микроорганизмов в иксодовых клещах, отловленных на территории республики Беларусь в сезон 2017 Г. Proceedings of international scientific and practical conference “Molecular diagnostics 2018”. Minsk. P 443-444.