SMGr up Molecular-Genetic Characteristics and Pathogenicity of Tick-Borne Encephalitis Virus Strains Isolated in the Far East
Leonova GN*1, Somova LM1 and Belikov SI2 1Institute of Epidemiology and Microbiology, Siberian Branch of Russian Academy of Medical Sciences, Russian Federation 2Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Russian Federa- tion *Corresponding author: Leonova GN, Institute of Epidemiology and Microbiology, Siberian Branch of Russian Academy of Medical Sciences, Selskaya St, Vladivostok, Russian Federa- tion, Email: [email protected]
Published Date: September 04, 2017
ABSTRACT In 2017, the 80th anniversary of the discovery in the Far East of Russia of a new neuroviral infection caused by tick-borne encephalitis virus (TBEV). The full-genomic sequencing of the 63rd
TBEV strains of the Far Eastern subtype determined their location in three clusters (Sofjin-, strains with a certain molecular genetic characteristic. Senjang- and Oshima-like isolates) indicated the territorial attachment of individual groups of The marked difference in biological characteristics is shown by the example of the Dalnegorsk and Primorye-437 strains belonging to two different clusters of the virus of the Far Eastern
6th non-synonymous amino acid substitutions in the polyprotein. Besides, in the Primorye-437 subtype Sofjin-like and Oshima-like. This difference can be associated with 34 synonymous and strain, as well as in other TBEV studied strains that did not cause the manifest forms of infection, a typical deletion of one amino acid was determined in the capsid protein. The revealed features of such strains predetermine their reduced pathogenicity and the relative safety for humans.
Encephalitis | www.smgebooks.com 1 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. However, the Far East should be considered as an epidemic unfavorable territory. According to the TBE morbidity for the entire period, only in Primorsky Krai the mortality was registered on average in 17% of cases. The pathogenic features of the infectious process, obtained on the informative monkeys model infected with the highly virulent Far Eastern TBEV strains (Sofjin the morphogenesis of changes in the central nervous system (CNS) in focal forms of tick-borne and Khabarovsk-17), isolated from the brain of dead patients, expanded the representation about the unequal degree of morphological changes in these or other formations of the brain and spinal encephalitis in humans. The diffusivity of destructive-inflammatory changes in the CNS is shown cord, which is the basis of the variety of clinical manifestations of tick-borne encephalitis, noted by many researchers.
Key words: Molecular genetic characteristic; Pathogenicity; Tick-borne encephalitis virus (TBEV) strains; Far east INTRODUCTION In 2017, the 80th anniversary of the discovery in the Far East of Russia of a new neuroviral infection caused by tick-borne encephalitis virus (TBEV). Up to now, in the different regions of the Eurasian continent, the annual approach of the spring-summer period reminds that this problem does not lose its relevance. As early as 1937, the pioneer of the TBE LA Zilber [1] understood pathogen. During the entire study period, large collections of TBEV strains have been created in that the main evidence of the verification of cases of an unknown disease is the isolation of the various virology laboratories, numerous studies have been carried out to study this pathogen.
Based on the results of studying the antigenic characteristic, the TBEV was identified as the main representative of the serological group of the TBE complex viruses, which according to Casals, J [2].In Clarke, a paper DH [4] [3] dealing also included investigating 6 antigenically the antigenic similar characteristics viruses. of TBEV by cross-neutralization, the range of Flavivirus representatives of TBE antigenic complex included 12 viruses. According to
(GGYV); Kyasanur the classification [5], this complex list has changed and the following virus groups were included Forest disease virus (KFDV); Langat virus (LGTV); Louping ill virus (LIV) into the genus Flavivirus (mammalian tick-borne viruses): Gadgets Gully virus fever virus (OHFV); Powassan virus (POWV); Royal Farm virus (RFV); Tick-borne encephalitis ; Omsk hemorrhagic virus (TBEV); Meaban virus (MEAV); Saumarez Reef virus (SREV); Tyuleniy virus (TYUV); Kadam virus (KADV). A large collection of the TBEV strains isolated in different regions of the Eurasian continent basis of nucleotide sequencing (ICTV) [6]. The determination of the nucleotide sequence is an required an objective evaluation of their characteristics, which can be obtained only on the indispensable basis both for further study of the currently recognized virus species, and for the
Taxonomy of Viruses (ICTV) [6], the TBEV belongs to the Flaviviridae family, the Flavivirus genus. identification and characterization of new viruses. According to the International Committee on Encephalitis | www.smgebooks.com 2 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. In 1990, the complete genome of the NBE virus was first read [7], for which the Sofjin strain became a classic reference for all strains of the TBEV, and then, as data on molecular genetic isolated in 1937 from the brain of the dead patient (the Far East of Russia). The Sofjin strain studies of strains accumulated in different territories of the Eurasian continent was accumulated, it became a reference for a group of TBEV strains of the Far Eastern subtype. In Russia, a vaccine againstIt has TBE been is producedestablished on that the basisthe TBEV of the contains Sofjin strain a positive-polarity [8]. RNA genome with a length of about 11,000 bases, which encodes a single protein, a polyprotein with a length of 3414 amino acid residues. During maturation, the polyprotein is cleaved by viral and cellular proteases with the formation of 10 proteins, three of which are structural (M,C,E), the rest (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) are non-structural. According to the Report of the International Committee on Taxonomy of Viruses [5], based on the biological and molecular genetic features of the TBEV strains, three subtypes were identified (I-Far Eastern, II-European and III-Siberian), which, contain the useful descriptive information about these groups of strains that dominate on the according to [6], do not have a formally recognized taxonomic significance. However, they can different territories of the continent.
In this report, on the basis of full genome sequencing, biological properties and pathogenic potential of the strains, we present a characteristic of the TBEV population which is widespread common in the Far East. STUDY OF TICK-BORNE ENCEPHALITIS VIRUS STRAINS ISOLATED FROM SICK PEOPLE IN THE FAR EAST The analysis of morbidity in the Primorsky Territory, located on the southern and middle spurs of the Sikhote-Alin, gives an idea of the activity of the epidemic process of TBE in the Far
East, where for the first time 80 years ago a new neuroviral disease was discovered, called the spring-summer tick-borne encephalitis. During this period, 5,202 cases of TBE have been officially registered only in Primorsky Krai, of which 852 patients died (mean annual mortality rate was given for the entire period of the study of the disease, the indices of which varied according to 17%). In (Figure 1) the dynamics of TBE morbidity in the Primorsky Territory of the Far East is years from 0.85 to 15.2 per 100 thousand of the population (official statistics on the Primorsky Territory).
Encephalitis | www.smgebooks.com 3 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. Figure 1: The incidence of tick-borne encephalitis in the Primorsky Territory of the Far
The basic conceptsEastern of the Far region Eastern over populationan 80-year periodof the TBEV, (1937-2016). capable of causing from mild to severe forms with lethal outcomes, can be obtained by studying their antigenic and biological characteristics. However, only the full genomic sequencing of a large number of different strains population circulating in certain focal areas. can give a full-fledged representation of the molecular-genetic characteristics of the viral Molecular-Genetic Characteristics of Strains Iisolated in the Far East Phylogenetic analysis of the complete genomes of 63 strains of the TBEV isolated in the Far
Eastern population of the TBEV and made it possible to distinguish three main clusters, which East of Russia was carried out (Table 1). The analysis showed a significant variability in the Far included strains close to the Sofjin strain isolated from the brain of a dead patient in 1937 in the Primorye Territory [1], to the Senzhang strain, isolated in 1953 in the Heilongjiang province of China [9] and to the Oshima strain, first isolated in 1997 in the territory of Hokkaido Island of Japan [10]. (Figure 2) shows the phylogenetic tree of the Far Eastern strains, in which cluster-forming strains are shown in bold type. To simplify the scheme, only one variant of the Sofjin-1953 strain the world and described by us earlier [11], we did not consider. Also in our scheme, only one strain is taken into the analysis, other variants of the Sofjin strains available in different laboratories of of these groups of strains. As an outgroup, the Aina strain of the TBEV of the Siberian subtype was from China (Senzhang) and from Japan (Oshima 5-10) [12] are shown as typical representatives selected, which was isolated in 1963 in the Irkutsk region.
Encephalitis | www.smgebooks.com 4 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. Table1: The tick-borne encephalitis virus strains that had been isolated in the Far East of Russia.
Year of Region of Strains Source of isolation Accession isolation isolation Sofjin-1953 1937 The brain dead patient PrimorskyKrai KU761576
Primorye-89 1987 Thebraindeadpatient PrimorskyKrai FJ906622
Primorye-87 1987 Thebraindeadpatient PrimorskyKrai JQ825149
Primorye-739 1992 The brain dead patient PrimorskyKrai JQ825156
Dalnegorsk 1973 The brain dead patient PrimorskyKrai FJ402886
Primorye-1153 2009 The blood patient PrimorskyKrai HQ901366
Primorye-501 2010 The blood patient PrimorskyKrai HQ901367
Primorye-2239 1985 I.persulcatus PrimorskyKrai HM859895
Primorye-92 1992 The brain dead patient PrimorskyKrai HQ201303
Primorye-94 1994 Thebloodpatient PrimorskyKrai EU816454
Spassk-72 1972 The brain dead patient PrimorskyKrai JQ825151
Primorye-91 1991 The brain dead patient PrimorskyKrai JQ825150
Chichagovka 1222 2012 I.persulcatus KhabarovskyKrai KP844724
Khekhtzir 17-13 2013 I.persulcatus KhabarovskyKrai KT001072
Khekhtzir 9-13 2013 I.persulcatus KhabarovskyKrai KT001070
Khekhtzir 10-13 2013 I.persulcatus KhabarovskyKrai KT001071
Birobidzhan 1354 2013 I.persulcatus KhabarovskyKrai KP844726
Malishevo 1978 Aedesvexansnipponii KhabarovskyKrai KJ744034
1230 2012 I.persulcatus KhabarovskyKrai KF880805
Primorye-1001 1958 I.persulcatus PrimorskyKrai KU761571
Primorye-696 1960 The brain dead patient PrimorskyKrai KU761569
8696 1986 I.persulcatus KhabarovskyKrai KF880804
Nikolaevsk 855 1985 I.persulcatus KhabarovskyKrai KP869172
Primorye-1285 1958 The blood patient PrimorskyKrai KU761575
Primorye-1056 1958 I.persulcatus PrimorskyKrai KU761573
Primorye-949 1958 Haemaphysalisjaponica PrimorskyKrai KU761570
Primorye-1035 1958 The brain dead patient PrimorskyKrai KU761572
9024 1990 Haemaphysalisconcinna KhabarovskyKrai KF880803
205 1973 I.persulcatus KhabarovskyKrai DQ989336
Primorye-196 2000 Thebloodpatient PrimorskyKrai JQ825155
Primorye-52 1999 The blood patient PrimorskyKrai JQ825154
Glubinnoe 2004 The brain dead patient PrimorskyKrai DQ862460
Svetlogorie 2008 Thebraindeadpatient PrimorskyKrai GU121642
Primorye-512 1959 Thebraindeadpatient PrimorskyKrai KU761568
Senzhang 1953 Thebraindeadpatient China JQ650523
Kavalerovo 1985 Thebraindeadpatient PrimorskyKrai FJ402885
Encephalitis | www.smgebooks.com 5 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. Zabaikalye 30-00 2000 Thebraindeadpatient Zabaikalye KC422667
Zabaikalye-1-98 1998 Thebraindeadpatient Zabaikalye JX968560
Primorye-895 2000 Thebloodpatient PrimorskyKrai JQ825145
Primorye-274 1999 Thebloodpatient PrimorskyKrai JQ825159
Primorye-75 1999 Thebloodpatient PrimorskyKrai JQ825152
Primorye-750 1998 Thebloodpatient PrimorskyKrai JQ825163
Primorye-823 2000 Thebloodpatient PrimorskyKrai JQ825164
Primorye-828 1998 Thebloodpatient PrimorskyKrai JQ825144
Primorye-345 1999 Thebloodpatient PrimorskyKrai JQ825161
Primorye-183 1991 Thebloodpatient PrimorskyKrai JQ825153
Primorye-86 1986 Thebloodpatient PrimorskyKrai EU816455
Primorye-320 1999 Thebloodpatient PrimorskyKrai JQ825160
Primorye-208 1991 Thebloodpatient PrimorskyKrai JQ825158
Primorye-90 1990 Thebloodpatient PrimorskyKrai FJ997899
Primorye-437 1999 Thebloodpatient PrimorskyKrai JQ825162
Primorye-18 1997 Thebloodpatient PrimorskyKrai GQ228395
Primorye-202 1997 Thebloodpatient PrimorskyKrai JQ825157
Primorye-253 1991 Thebloodpatient PrimorskyKrai EU816451
Primorye-270 1991 Thebloodpatient PrimorskyKrai EU816452
Primorye-332 1991 Thebloodpatient PrimorskyKrai AY169390
Primorye-212 1991 The bloodpatient PrimorskyKrai EU816450
Primorye-82 1982 Thebloodpatient PrimorskyKrai JQ825148
Kiparis-94 1994 Thebloodpatient PrimorskyKrai JQ825146
Primorye-1284 1958 Thebloodpatient PrimorskyKrai KU761574
Primorye-69 2000 Thebloodpatient PrimorskyKrai EU816453
Oshima 5-10 1999 Theblooddog Hokkaido AB062063
Shkotovo-94 1994 Thebloodpatient PrimorskyKrai JQ825147
Aina 1963 Thecerebrospinal fluidpatietn Irkutsk JN003206
Encephalitis | www.smgebooks.com 6 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. Figure 2: The tree was based on the complete genomes of 63-rd strains of virus, isolated in the Far NJ (distance method neighbor-joining) [27] phylogenetic tree of TBEV strains. East. Prototype strains in each of the three clusters are shown in capital letters. The strains taken in the pathogenicity test are shown in bold lowercase letters. The strains isolated in the Khabarovsk Territory are in italics.
The cluster of Sofjin-like strains showed the pronounced heterogeneity, in which two large groups of the TBEV strains were formed: a group of strains from the Primorye Territory, previously described in detail by us [13] and a separate branch of strains isolated in the Khabarovsk Territory. from Khabarovsk strains. In addition, in this cluster, 4 more branches were defined, consisting either from Primorye or Encephalitis | www.smgebooks.com 7 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. The second Senzhang-like cluster includes strains isolated both in Primorsky and in the Khabarovsk Territory, which indicates the widespread distribution of this cluster strains in the
FarThe East, third including cluster the ofnorthern Oshima-like territory strains of China. is represented mainly by strains isolated from patients with an inapparent infection in the southern focal area of Primorsky Krai. Strains from this group according to the molecular genetic structure are divided into two subgroups and differ significantlyThus, the from population the rest ofFar the Eastern TBEV strains of the out Far other Eastern clusters. subtype is widely distributed from south to north in the territories of Primorsky and Khabarovsk territories of Russia, as well as in the range of the TBEV of the Far Eastern subtype was not included in the task of our investigations. Heilongjiang Province of China and in Hokkaido Japan. The definition of the western borders of Biological Characteristics of Strains with High and Low Pathogenicity differences can be assessed by comparing the biological properties of strains belonging to The specific features of biodiversity of a heterogeneous population of strains and their different clusters of the Far Eastern subtype TBEV. The detailed properties of these strains have been characterized by us in several publications [11,13,14].
A clearer connection between the biological characteristics and the molecular genetic properties of the causative agent can be traced to the example of a comparative study of the experimental infection of two diametrically different strains of the TBEV. Figure 2 shows the strain Dal’negorsk (Dal’), isolated in 1973 from the brain of a died patient with a focal shape of the TBE, infected in a clinically healthy man after a tick bite in the suburban forests of Vladivostok. Both strains were the Dalnegorsky region, and the Primorye-437 (P-437) strain, isolated in 1999 from the blood of compared after 8-fold passages with intracerebral infection of 2-day white mice. The Dal ‘ strain is characterized by molecular genetic properties as a typical representative of Sofjin-like ones, and the TheP-437 initial strain titers - Oshima-like of the virus strains on of the the modelTBEV of of the noninbred Far Eastern mice subtype. weighing 10-12 g with intracerebral (ic) and subcutaneous (sc) /ml
infection for the Dal’strain were 9.8 and 9.5 lg LD50 on porcine kidney (PK) cell culture also occurred in different ways: for the Dal’ strain - up to 8.0 (respectively), for P-437 strain - 7.7 and 5.5 lg LD50/ml (respectively). The accumulation of virus /ml. lg TCDVirulence50/ml, for of theP-437 strains - up onto 5.0the lgmodel TCD 50of adult white mice was shown in two variations: by way of ic and sc infection with different doses (10 and 1000 LD A high degree of virulence was shown when the mice are infected with both strains by ic way. 50) or one dose (100 LD50) of the virus. Differences in virulence were obtained only with sc inoculation of the virus. The greatest activity was demonstrated by the Dal’ strain, the average life duration (ALD) was: 7.1 days - with the introduction of 1000 LD and 14.7 days - at 100 LD . The less active was the P-437 strain (ALD
50 50
Encephalitis- 25.1 and 22.1 | www.smgebooks.com days, respectively). 8 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. A comparative study of haemagglutinating activity (HA) of these TBE strains showed their
Dal’ strain, which caused focal infection. The HA activity indicators for P-437 strain, which caused differences at three pH values (5.4, 6.2, 6.8). The greatest HA activity (1: 120, 1: 128, 1: 182) had a the Theinapparent different form rate of of TBE, accumulation were lower of (1:84,these 1:79,TBEV 1:79). strains in the culture medium of infected of PK cells, while studying hemagglutination, plaque-forming activity and K antigen values in ELISA plaque, and the P-437 strain as small-plaque, retained their properties regardless of passages, indicated differences in their replicative activity [15]. The Dal’ strain, characterized as large- these strains on the accumulation of hemagglutinins in the culture medium of infected PK cells. which indicated the stability of this genetic characteristic. In Table 2, one can find differences in The maximum HA values for both strains reached only on the 6th and 7th days post infection, but for the Primorye-437 strain, a delayed and less active accumulation of HA was observed.
The indicators of antigen accumulation of the TBEV in infected PK cells in ELISA revealed a characteristic feature for the P-437 strain: in the supernatant, from 3 hours post infection, its constant presence was determined. The antigen of the Dal’ strain in the supernatant of the infected PK cells was determined only after 2 days (K = 9.3). The accumulation of the antigen of the P-437 strain was significantly lagging behind (K = 2.9). Similar data were observed when a virus was detected in the supernatant of infected PK cells using a plaque-forming test (Figure 3). defective virus particles that are unable to penetrate the cell quickly, like the Dal’ strain. However, At first sight, one can think on the presence of the antigen of P-437 strain in the form of according to the results of the plaque-forming test, it can be seen that in viable samples of the not penetrate into the cell. Apparently, the receptor communications of virus particles to cells was culture fluid of PK cells infected with the P-437 TBEV strain, there are viable viral particles that do not strong. Throughout the experiment, they could easily return to the culture medium, where did not observe such a phenomenon in the Dal’ strain - in the culture liquid up to 48 hours of we detected a specific antigen in ELISA, as well as viral particles with a plaque-forming test. We incubation, neither the virus nor the antigen was detected, indicating the penetration of all viral particles into PK cells (Figure 3 and Table 2).
Encephalitis | www.smgebooks.com 9 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. Table 2: Dynamics of accumulation of the antigen of the TBE virus in the supernatant of PK cells infected with Dal’negorsk and Primorye-437 strains.
Infecting virus titer Observation time after infection PK cell (h – hour; d – day) Strains Cell infection rate (lgTCID ml) 50/ 3 h 6 h 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d
HA titers 0 0 0 0 0 0 2 8 32 64 Dal´negorsk 2.5 0 K- factor 0 0 0 9.3 9.5 н.и н.и н.и н.и
HA titers 0 0 0 0 0 0 0 8 16 16 Рrimorye-437 3.0 K- factor 1.6 1.5 0.9 1.2 2.9 3.1 н.и н.и н.и н.и
The specific antigen TBEV was determined by haemagglutinating test (HA titers ) and ELISA (K- factor; positive sample at K of more than 1.0).
Figure 3:
Accumulation dynamics in the supernatant of PK cell culture injected with TBEV strains from patients with focal (Dal’negosk) and inapparent (Primorye-437) forms of infection. The strain that caused severe focal forms of infection showed virus accumulation only after Observations were performed by PFU teste up to 6 days after infection of the cells of TBEV.
24 h. The inapparent strain was observed in culture fluid over the whole experiment 1 h after injection. Encephalitis | www.smgebooks.com 10 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. The revealed features of biological properties of the different pathogenicity and molecular- genetic characteristics TBEV strains help to understand how the events of the infectious process develop differently at the early stage of virus penetration into the cell. The low pathogenic P-437 strain, which slowly penetrates into the cell, promotes a quick and more active reaction of the body’s immune system [16], as a result of which prerequisites develop for eliminating it from the body at an early stage of the infectious process. Using the example of the Dal’ strain, the highly pathogenic strains actively penetrate the cell and in the initial stage become inaccessible to the action of the immune system, which determines the further unfavorable course of the the characteristics of the pathological process in humans when infected with highly pathogenic infectious process. Identified in the experiment, the properties of the virus help understand strains of the TBEV.
Khabarovsky-17 strains, isolated from the brain of dead patients, is an important addition to the The pathomorphology of the central nervous system caused by highly virulent Sofjin and complex characterization of the Far Eastern population of TBEV. Features of the Pathology Caused by Highly Virulent Strains of Tick-borne Encephalitis Virus The tick-borne encephalitis in humans in the Far East manifests itself in a variety of clinical syndromes of the disease, indicating a multi-level involvement in the pathological process of the central nervous system (CNS) brain [17,18,19]. . This is confirmed by morphological studies of the dead patient’s The priority of detailed disclosure of pathomorphogenesis of tick-borne encephalitis in the informative monkey’s model belongs to M.P. Frolova. As the infectious agents, the reference strain
Sofjin (Primorsky Krai) and the Khabarovsky-17 strain were used, which is most closely grouped with the Nikolaevsk 855, Chichagovka 1222, 8696 and 205 strains for a short fragment (201 nucleotide, Acc. No AY363846) of the E gene. The intracerebral infection of rhesus monkeys was of mice (108 LD performed in the state of hexenal anesthesia by injecting of the virus-containing brain suspension from 3 to 7 days. Morphological studies were conducted at different times of the disease on days 50/ml) into the optical tubers of 0.5 ml on both sides. The incubation period was
1, 3, 5, and 6 days of the clinical manifestations.
The detailed microscopic examination of monkeys CNS infected in the brain with Sofjin (No. 315 and 316) and Khabarovsky-17 (No. 820, 823 and 824) strains showed the presence of destructive-inflammatory changes of various severity in all animals that was in direct connection with the duration course of infection. Initial inflammatory changes were detected already in encephalitis, with the emergence of meningeal phenomena, convulsions, paresis of limbs in the incubation period of the disease. With the development of clinical symptoms of tick-borne infected monkeys patients at microscopic examination there were found the same, diffuse
Encephalitisthroughout |the www.smgebooks.com long CNS, but not the same in intensity changes. 11 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. The severity of pathomorphological changes in the brain was assessed on a 4-point scale developed by M.P. Frolova [20]: ± - single vascular infiltrates; + - small-focal changes (lesion and prolapse of single, less than 1/3 neurons, individual vascular infiltrates, small glial nodules); ++ - focal changes (death and prolapse less than 1/2 neurons, moderate inflammatory reaction); +++ - diffuse massive changes (prolapse more than half, up to 2/3 neurons, intensive inflammatory reaction; ++++ - massive, merging changes (prolapse more than 2/3 neurons, pronounced inflammatoryIn the early reaction). stages of acute tick-borne encephalitis, the morphological changes differed focality, a clear limitation and low prevalence. Increasing the duration of the process was accompanied foci of lesion, encompassing various parts of the brain. In all studied monkeys, the pathological by an increase in the severity of the morphological changes, the presence of extensive confluent process was characterized as the diffuse meningoencephalomyelitis.
th
In monkey No. 315, on the third day of the clinic (8 day after infection with the Sofjin strain), with moderate changes (+ / ++) in the optical tubers, subcortical nodes, middle and oblong brain, and oligodendroglia and the formation of loose glial nodules near the vessels. In the middle brain, individual perivascular infiltrates were found accompanied by proliferative microglia reaction the changes were localized in the red nucleus and black matter, where dystrophic changes in of microglia and its participation in the formation of neuronophagous nodules were detected. part of the neurons, death and prolapse of a significant number of cells, pronounced proliferation medulla oblongata. In the lower olives, there were small, loose glial nodules at the site of neuronal Vascular infiltrates, loose glial nodules were found in the nuclei of the varoliyev pons and the prolapse. In the spinal cord, focal changes were spread along its whole length. Dystrophic changes in motoneurons were observed: partial and total chromatolysis, neuronophagia, accompanied by single perivascular “couplings”, vasculitis, diffuse and focal reaction of glia. Expressed changes were observed in the cerebellum: loss of Purkinje cells (++), pronounced proliferation of microglia soft meninges. and Bergman glia, the damage of the internal formation nuclei of the cerebellum, infiltration of the
At the same time, another monkey (No. 316) infected with the Sofjin strain showed the intensive of the brain and spinal cord. In the oblong and spinal cord, a large number of polymorphonuclear inflammatory-degenerative changes with severity of ++ / +++, diffusely spread in various parts process. The destructive changes in neurons were combined with a pronounced diffuse and leukocytes were detected in the infiltrates, which indicated the severity of the pathological focal glial reaction, diffuse infiltration of brain matter by hematogenous elements (Figure 4 and 5). As in the first case, the most pronounced changes were in the cerebellum, which manifested itself in the form of diffuse lympho-histiocyte infiltration of the soft meninges, a large number of cells and formation in their place a shaft from hyperplastic glial cells (microglia, oligodendroglia, vascular infiltrates and multiple-row perivascular “couplings”. The subtotal prolapse of Purkinje the cerebellum. astrocytes) was observed. There was a rapid proliferation of microglia in the molecular layer of Encephalitis | www.smgebooks.com 12 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. Figure 4
: Monkey No. 316. Sofjin strain, the 8th day postinfection. Spinal cord. Diffuse inflammatory reaction in the anterior horn of the L4 segment. Prolapse of motoneurons, neuronophagic nodules. x 112.5. Nissl staining.
Figure 5:
Monkey No. 316. Sofjin strain, the 8th day postinfection. Changes in spinal cord motoneurons. Diffuse infiltration of brain tissue with hematogenous (monocytes / macrophages,
Encephalitis | www.smgebooks.complasmocytes) cells. x 500. Nissl staining. 13 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. Khabarovsky-17 strain also indicated a marked increase in the degree of brain damage The study of pathomorphological changes in the CNS of monkeys infected with the stages of the pathological process, including the depth of neuronal damage and the growth of depending on the duration of the clinical course of the infection, which reflected the various immunopathological changes. Analysis of the pathological process of tick-borne encephalitis in monkeys allowed us to conclude that the severity of the morphological changes in the central nervous system is due to the biological activity and neurovirulence of the Khabarovsky-17 strain, its pronounced ability to reproduce in the brain tissue.
In monkey No. 820, taken for examination on 3rd day of the disease clinic, 10th day after intra parts of the upper and lower limbs, there were changes similar to those described in monkeys cerebral infection (incubation period of 7 days), in the presence of weakness of the proximal
No. 315 and 316. Inflammatory-destructive changes were present in all parts of the brain and spinal cord, the most pronounced (up to +++) in black matter, in the meningeal coverage and lysis, diffuse proliferation of glia, massive perivascular “couplings” from mononuclear cells were cortex of the cerebellum (Figure 6 and 7). The loss of a large number of nerve cells, mainly due to observed. In the cortex of the cerebellum there was a considerable prolapse of the Purkinje cells meningeal coverage, wherein the changes in the nuclei of the internal cerebellar formation were with a powerful growth of bergman glia in their place, deformation and diffuse infiltration of the weakly expressed.
Figure 6: Monkey No. 820. Khabarovsky-17 strain, 10th day postinfection. The damage of
neurons, the glial reaction, perivascular infiltrates in the black substance of the middle brain. x Encephalitis | www.smgebooks.com 112.5. Nissl staining. 14 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. Figure 7:
Monkey No. 820. Khabarovsky-17 strain, 10th day postinfection. Cerebellum. Prolapse of Purkinje cells, proliferative reaction of glia. Inflammatory infiltration of soft meninges. x 112.5. Nissl staining. staining. proliferative reaction of glia. Inflammatory infiltration of soft meninges. x 112.5. Nissl Further analysis of the pathological process in the central nervous system caused by the Khabarovsky-17 strain was carried out in the study of monkeys No. 824 and 823 who dead on the 12th th and 7th days of clinical
day after infection (incubation period of 6 and 5 days, 6 degree of severity and prevalence of changes in monkey No. 824 was less than in monkey No. 823. manifestations of the disease, respectively ). Both monkeys had the same CNS lesions, but the the large hemispheres and ending with the spinal cord. Despite the varying severity of the lesion, The diffuse inflammatory process has embraced all parts of CNS, beginning from the cortex of prescription of the pathological process in terms of the extent and depth of brain tissue damage. a similar morphological pattern was observed in the CNS of both monkeys, reflecting the same In monkey No. 823, in which right-sided hemiparesis and left-sided hemiplegia were developed, the heavy changes were found in the brain and spinal cord with a pronounced destructive component, a clear inflammatory response, and a diffuse spread of the process. The severity of the lesion in the subcortical formations, cerebellum, oblong and spinal cord reached +++ / ++++ scores (Figure 8 and 9). In the spinal cord, at the level of the cervical and thoracic areas, the most several segments, representing a merging necrotic focus in the brain parenchyma. In the lumbar severe changes were observed. Significant foci of motoneurons loss in the anterior horns seized Encephalitis | www.smgebooks.com 15 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. spinal cord lesions were less expressed, but many surviving neurons had sharp changes in the form of wrinkling and hyperchromatosis, indistinctness of the nucleus contours.
Figure 8: Monkey No. 823. Khabarovsky-17 strain, died on the 12th day postinfection.
Destructive-inflammatory changes in the medulla oblongata in the nuclei of gentle and wedge- shaped cords, x 43.75. Nissl staining.
Figure 9:
Monkey No. 823. Khabarovsky-17 strain, died on the 12th postinfection. Changes in the medulla oblongata: lower olive, a site of neurons prolapse with focal inflammatory Encephalitis | www.smgebooks.com infiltration, x 112.5. Nissl staining. 16 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. strains, depending on the duration of the pathological process, the differences in morphological In the dynamics of tick-borne encephalitis caused in monkeys by the Sofjin and Khabarovsky-17 the early stages and a rapid pace of its development. In one and the same monkey it was possible changes in CNS were traced. Leukocyte infiltration has always been a sign of an acute process in to observe lesions of different prescription, judging by the composition of the inflammatory different prescription in the central nervous system in tick-borne encephalitis in humans was infiltrates and the nature of the lesion of the brain parenchyma. The presence of lesions with noted by a number of researchers [17,21].
Thus, the presented material on the CNS pathomorphology of monkeys infected with Far process in the brain and spinal cord with the predominant lesion of gray matter. At various Eastern Sofjin and Khabarovsky-17 strains of TBEV, indicated a pronounced diffusion of the times after infection with the TBEV, it was possible to identify a certain sequence of inclusion of CNS structural formations into the pathological process and to note its clear systemic nature, manifestations. There was constant involvement in the process of soft meninges and white matter some strain-specific histological differences in the intensity of the experimental infection same animal in different parts of the brain and spinal cord. The most pronounced meningeal of the brain. The intensity of the soft meninges infiltration varied to a great extent even in the and degenerative changes in the brain matter. response was observed in those parts of the brain where there were more severe inflammatory
It should be noted that the greatest destructive-inflammatory lesion affected the black matter of Sommering, which was observed in all monkeys (No. 315, 316, 820, 823) and had a greater of black matter in the middle brain is a constant phenomenon in tick-borne encephalitis. In the severity, compared with other formations, even in cases of moderate CNS damage. The damage cerebellum,All explored all the monkeys monkeys in alsobrain showed matter pronounced had proliferative inflammatory-degenerative phenomena of cellular changes. elements of the vascular wall in the form of endotheliocytes hyperplasia, proliferation of adventitial cells - spread in the form of strands to the surrounding brain substance. The hematogenous elements pericytes and histiocytes. The vascular infiltration was not limited to the periadventivial space and diffusely infiltrated it, surrounded the damaged nerve cells and participated in the formation of neuronophagous nodules. The cellular composition of tissue infiltrates was not the same in differentAlthough monkeys there andwas changed a clear correlationin dynamics, between depending the on intensity the stage of of the the central inflammatory nervous process. system damage in different monkeys and the duration of the pathological process, it is nevertheless noteworthy that under the conditions of the same experiment, when one and the same virulent strain of tick-borne encephalitis virus, with the same observation periods, there were small variations in the severity and spread of morphological changes. This can be explained by the individual characteristics of the animal, its physiological condition, especially immunoreactivity of organism.
Encephalitis | www.smgebooks.com 17 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. CONCLUSION Thus, it was established that at the expressed heterogeneity of the molecular-genetic structure of the analyzed strains, all 63 isolates took positions in the group of the TBEV strains only in the Far Eastern subtype. No strain from other subtypes of the TBEV in the Far East has been and the TBEV is considered a single species, at the same time, the data we obtained on the full determined. Although the ICTV Committee [6] does not attach a taxonomic value to the subtypes, genome sequencing of a large number representatives of the Far Eastern subtype testify about the territorial attachment of individual groups of strains with a certain molecular genetic characteristic. Independent groups of strains isolated in the territories of only the southern spurs of the Sikhote-Alin (Primorsky Krai) and certain groups of strains-only in the more northern focal areas of the Far East (the Khabarovsk Krai) were identified in the cluster of Sofjin-like strains. The cluster of Senjang-like strains is represented by a mixture of strains isolated in China and throughout the Far East. The cluster of Oshima-like isolates, except strains from Japan, consists fromThe strains obtained isolated results only indicate in the south that, ofin thecomparison Far East (inwith the other Primorsky regions, Territory). the biodiversity of the viral population is more pronounced in the southern focal areas of the Far East. Here a group
Primorye-437 strain isolated from the blood of a clinically healthy patient after a tick bite, the of strains incapable of causing the manifest forms of TBE are identified. As an example of the reduced biological activity of the studied virus is shown in different experimental models. The marked difference in biological characteristics is shown by the example of the Dalnegorsk and Primorye-437 strains, belonging to two different clusters of the virus of the Far Eastern subtype
Sofjin-like and Oshima-like. Earlier in our publications, it was shown that the differences in the biological characteristics of strains from these clusters depend, first of all, on the revealed changes in aminoIt is also acids shown in the that polyprotein in the polyprotein [14,15,22]. of these two strains from 3414 amino acid residues, 3374 coinciding, of which 34 amino acid substitutions are synonymous and only 6 amino acid substitutions are non-synonymous. Besides, in the Primorye-437 strain, as well as in other studied TBEV strains that did not cause the manifest forms of infection, a typical deletion of one amino acid was determined in the capsid protein [13]. The revealed features of such strains predetermine their reduced pathogenicity and relative safety for humans.
At the same time, the Far East should be considered as an epidemic unfavorable territory. According to the TBE morbidity for 80-year period, only in the Primorsky Territory mortality registered on average 17% of cases. The pathogenetic features of infectious process identified on the informative monkeys model infected with highly virulent Far Eastern TBEV strains (Sofjin and Khabarovsk-17), isolated from brain of dead patients, have expanded the representation about differential criteria for a pathoanatomical diagnosis of the disease. the morphogenesis of changes in the CNS at focal forms of tick-borne encephalitis in humans and Encephalitis | www.smgebooks.com 18 Copyright Leonova GN.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for com- mercial purposes, as long as the author and publisher are properly credited. of the Far Eastern TBEV strains, then for the Khabarovsk-17 strain with the previously read And if the Sofjin strain isolated in the Primorsky Territory is a reference for the whole group small fragment (201 nucleotides) of the E protein gene the position in the phylogenetic tree is protein of Khabarovsk-17 strain and the 63rd studied strains, it was found that it is close only to not defined. At the same time, based on a comparative analysis of this fragment of the gene E Khabarovsk isolates.
The revealed localization of morphological changes in monkeys infected with strains of the Far Eastern subtype virus of tick-borne encephalitis is identical to the topography of lesions, dead patients in the Far East. The most affected formations are the anterior horns of the spinal first described in detail by I.A. Robinson and Yu.S. Sergeeva [23,24] on the sectional material of cerebellum, the black substance, the vestibular nuclei, the reticular formation, the lower olives, cord, especially in the cervical region, the cortex (Purkinje cells) and the proper nuclei of the the optical tubers and the sub cortical nodes [20,25,26]. The diffuseness of destructive and different cases, in these or other formations of the brain and spinal cord, is the basis of the variety inflammatory changes in the central nervous system, unequal degree of morphological changes in of clinical manifestations of tick-borne encephalitis, noted by many researchers.
This message is the result of an 80-year study of the molecular genetic structure and pathogenicity of the TBEV population in the Far East, where the discovery of this severe
FUNDINGneuroinfection was first made.
This work was supported by grants 0545-2014-0011 and 0345-2014-0002 from the Federal analysis, decision to publish, or preparation of the manuscript. Scientific Organizations Agency. The funders had no role in study design, data collection and References
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