Pathogens in Ixodes persulcatus and Ixodes ricinus ticks (Acari, Ixodidae) in Karelia (Russia).

Ixodid ticks (Acarina, Ixodidae) are vectors of dangerous human infections. The main tick species that determine the epidemiological situation for tick-borne diseases in northern Europe are Ixodes ricinus and Ixodes persulcatus. In recent years, significant changes in the number and distribution of these species have been observed, accompanied by an expansion of the sympatric range. This work summarizes the data of long-term studies carried out in Karelia since 2007 on the infection of I. persulcatus and I. ricinus ticks with various pathogens, including new viruses with unclear pathogenic potential. As a result, tick-borne encephalitis virus (TBEV, Siberian genotype), Alongshan virus, several representatives of the family Phenuiviridae, Borrelia afzelii, Borrelia garinii, Ehrlichia muris, Candidatus Rickettsia tarasevichiae and Candidatus Lariskella arthropodarum were identified. Data were obtained on the geographical and temporal variability of tick infection rates with these main pathogens. The average infection rates of I. persulcatus with TBEV and Borrelia burgdorferi sensu lato were 4.4% and 23.4% and those of I. ricinus were 1.1% and 11.9%, respectively. We did not find a correlation between the infection rate of ticks with TBEV, B. burgdorferi s.l. and Ehrlichia muris/chaffeensis with the sex of the vector. In general, the peculiarities of the epidemiological situation in Karelia are determined by the wide distribution and high abundance of I. persulcatus ticks and by their relatively high infection rate with TBEV and B. burgdorferi s.l. in most of the territory, including the periphery of the range.

ON MODERN APPROACHES TO CREATION OF A SINGLE-CYCLE VACCINE AGAINST TICK-BORNE ENCEPHALITIS.

In Russia, about 2000 people get tick-borne encephalitis (TBE) every year. Almost none of them are vaccinated. For the prevention of TBE, inactivated vaccines (IVTBE) are used. IVTBE are safe and protect from TBE not less than 95% of vaccinated. The disadvantages of IVTBE are the need for numerous intramuscular injections by medical personnel, the high cost of vaccination and the vaccination refusals. A new vaccine against TBE should not be inferior to IVTBE in its safety and efficacy, should cause long-term immunity after a single application, and, preferably, be effective after oral administration. Currently, genetic engineering methods for producing replication-defective (single-cycle) flaviviruses that can serve as the basis for creating new types of safe vaccines similar in many characteristics to classic live vaccines based on attenuated strains of viruses have been proposed. The possibility of infecting humans with TBE by the use of milk of naturally infected animals, as well as the experience of using experimental live TBE vaccines, are prerequisites for the creation of a safe oral single-dose TBE vaccine.

COMPARISON OF RESULTS OBTAINED BY ELISA AND NEUTRALIZATION TEST IN ASSESSING THE PROTECTION OF POPULATION FROM TICK-BORNE ENCEPHALITIS.

The enzyme-linked immunosorbent assay (ELISA) and the neutralization test (NT) are often used to determine the level of seropositive population and to evaluate the immunogenicity of vaccines. ELISA provides information on the total pool of antiviral antibodies, while NT allows the antiviral protection level of a person to be estimated. It is assumed that the 1:100 titer in ELISA and the 1:10 titer in NT are protective. Obviously, the ratio of the total pool and virus neutralizing antibodies can vary as a result of natural immunization or vaccination. In this study, two methods were used to study the blood serum samples taken in a group of inhabitants of the Sverdlovsk region aged from 1 to 60 years. The samples were collected before immunization and 30 days after two immunizations with inactivated vaccines against tick-borne encephalitis of different manufacturers. Immunizations were performed either according to a standard scheme (30-day interval between immunizations), or according to an emergency scheme (14-day interval). It was shown that the data on the presence of antiviral antibodies in protective titers obtained by ELISA and NT were consistent in more than 85% of cases. The discrepancies between the data are due, in the first place, to the difference in the sensitivities of the two methods. The proportion of seropositive people according to NT data is always greater than that according to the results of ELISA. Nevertheless, among 174 children, about 5% of recipients after a double immunization were seropositive according to ELISA, but did not have neutralizing antibodies in protective titers.

Immunogenicity and safety of the adult tbe vaccine «tick-e-vac¼.

About 3,000 cases of TBE are registered annually in the Russian Federation. Vaccination is the main way to prevent the tick-borne encephalitis disease. Comparative study of the reactogenicity and immunogenicity of a new vaccine «Tick-E-Vac¼ was held. Volunteers aged from 16 years old were twice immunized with the vaccines «Tick-E-Vac¼ or «Encevir¼ derived from strains of Far East subtype of TBE virus, according to standard and emergency schemes. The clinical study was randomized, comparative, blind, and controlled. The frequency, intensity, time of occurrence, and duration of local and general reactions had been recorded. The titers of antiviral antibodies in ELISA had been determined to assess the immunological efficacy of vaccination. According to the results of the clinical study, the severity of local and general reactions in initial seronegative recipients was weak or moderate. The symptoms were usually manifested within 1-2 days after injection and persisted for not more than 4 days, after which time the symptoms disappeared. There was no statistically significant difference in the reactogenicity of the vaccines after the first and after the second injection. The reactogenicity also did not depend on the gender of recipients. After the first immunization, the level of seroprotection was not less than 43%; the average geometric titer of antibodies (GTA), not less than 1:200. After the second injection, the level of seroprotection reached 90-100%; GTA, not less than 1:500. The data on the reactogenicity and immunogenicity to the original seropositive recipients is not significantly different from the data for the initial seronegative recipients. The data indicate weak reactogenicity of the vaccines «Tick-E-Vac¼ and «Encevir¼. Double vaccination with an interval of 14 or 30 days leads to the formation of expressed immune response. Thus, differences in the level of seroprotection and in antiviral titers in the cases of the standard and emergency vaccination schedules are not statistically significant. The correlation between the development in recipients of local and general symptoms and the immunological efficacy of the vaccines has not been identified.

[Nonstructural protein 1 of tick-borne encephalitis virus activates the expression of immunoproteasome subunits].

The interaction of viral proteins with host cell components plays an important role in antiviral immune response. One of the key steps of antiviral defense is the formation of immunoproteasomes. The effect of nonstructural protein 1 (NS1) of tick-borne encephalitis virus on the immunoproteasome formation was studied. It was shown that cell expression of NS1 does not reduce the efficacy of the immunoproteasome generation in response to interferon-γ stimulation and even increases the content of the immunoproteasome subunits without the interferon-γ treatment. Thus, NS1 of tick-borne encephalitis virus activates, rather than blocks the mechanisms of immune defense in the cell.

Protective immunity spectrum induced by immunization with a vaccine from the TBEV strain Sofjin.

Tick-borne encephalitis (TBE) circulates widely in the territory of Eurasia with up to 10,000 cases registered annually. The TBE virus (TBEV) includes three main subtypes: European, Siberian and Far-Eastern, and two new Asiatic variants, phylogenetically distant from the others. The inactivated antigen of European or Far-Eastern strains is used in commercial TBE vaccines. A set of 14 TBEV strains, isolated in 1937-2008, with different passage histories, representing all subtypes and variants, was used in this work. The chosen set covers almost all the TBE area. Sera of mice, immunized with the TBE vaccine Moscow, prepared from the TBEV strain Sofjin, were studied in a plaque neutralization test against the set of TBEV strains. The vaccine induced antibodies at a protective titer against all TBEV strains and Omsk hemorrhagic fever virus (OHFV) with Е protein amino acid distances of 0.008-0.069, but not against Powassan virus. We showed that after a course of two immunizations, factors such as the period between vaccinations (1-4 weeks), the challenging virus dose (30-1000 LD50) and terms of challenge (1-4 weeks after the last immunization) did not significantly affect the assessment of protective efficacy of the vaccine in vivo. The protective effect of the TBE vaccine Moscow against the set of TBEV strains and the OHFV was demonstrated in in vivo experiments. TBE vaccine Moscow did not protect mice against 10 LD50 of the Powassan virus. We showed that this range of Е protein amino acid distances between the vaccine strain and challenging virus do not have a decisive impact on the TBE vaccine protective effect in vitro and in vivo. Moreover, the TBE vaccine Moscow induces an immune response protective against a wide range of TBEV variants.

[Vaccines based on the Far-Eastern and European strains induce the neutralizing antibodies against all known tick-borne encephalitis virus subtypes].

Serum of children aged 1 to 16 obtained in the course of clinical trials conducted in the sverdlovsk region in 2011 was used to study the post-vaccination immunity. Children were immunized twice with vaccines against the tick-borne encephalitis (TBE) Tick-E-Vak on the basis of the strain sofjin of the Far-Eastern subtype and FSME-IMMUN Junior based on the neudorfl strain of the european subtype. According to the plaque reduction neutralization test (PRNT), both vaccines have a high immunogenicity: after 30 days since two-time vaccination in the sera of 100% of children immunized with the vaccine Tick-E-Vak and in the 95% of children immunized with the vaccine FSME-IMMUN Junior antibodies (AT) against strain sofjin were identified in protective titers, whereas 24.5% and 21.4% of children, respectively, had antibody titers higher than 1:10000. selected sera of recipients with titers from 1:25 to 1:1000 were examined in the PRNT in a single experiment using the sofjin (Far-Eastern subtype), absettarov (European subtype) and Vasilchenko (Siberian subtype) strains. The two vaccines induced AT against the representatives of all three subtypes.

Genetic description of a tick-borne encephalitis virus strain Sofjin with the longest history as a vaccine strain.

Vaccines based on the strain Sofjin of the Far-Eastern tick-borne encephalitis virus (TBEV) subtype have been used for TBE prophylaxis for over 50 years in Russia and neighboring countries. On the wide territory, where all known TBEV subtypes are circulating, the cultural, purified, concentrated, inactivated TBE vaccine Moscow has been shown to be safe and efficacious in a massive immunization. In the present work, we describe the genome of the vaccine strain Sofjin. We have shown that it differs from TBEV strains previously published with the name "Sofjin". Moreover, we have shown the stability of the virus during the vaccine manufacturing process on the molecular level.

Prevalence of Kemerovo virus in ixodid ticks from the Russian Federation.

The prevalence of Kemerovo virus in ixodid ticks collected in 2008-2012 from 11 regions of the Russian Federation was investigated by real-time reverse-transcription PCR (RT-PCR). The presence of Kemerovo virus in Ixodes persulcatus, Ixodes ricinus, and Dermacentor reticulatus was confirmed. Virus prevalence depended on the region and varied from zero to 10.1%.

[The tick-borne encephalitis virus detection efficiency in the Ixodid ticks (Acari: Ixodidae) with ELISA and real-time PCR].

According to the data of the Federal Service on Customers' Rights Protection and Human Well-being Surveillance, the enzyme-linked immunosorbent assay (ELISA) detects the tick-borne encephalitis virus (TBEV) in ticks more often than the polymerase chain reaction (PCR). The goal of this work was to compare TBEV detection efficiency in the ixodid ticks of different species with the commercial kits based on ELISA and real-time PCR. Ticks of five species were parenterally infected with 2-6 IgPFU of the European or Siberian TBEV subtypes. We formed randomized and encoded series of infected and intact ticks of different species, and in "blind" experiment analyzed the ticks on the TBEV presence with the kits based on ELISA and real-time PCR. ELISA and real-time PCR effectiveness of the TBEV detection in ticks was not affected by gender, species of ticks or presence of blood meal. The kits based on ELISA were less sensitive than those based on real-time PCR. ELISA effectiveness depended on the TBEV subtype. The presence of the false positive reactions and sensitivity of ELISA were affected by the protocols of reaction. The problem of the different TBEV prevalence in the field-collected ticks obtained with various methods remains to be studied.

GAG-binding variants of tick-borne encephalitis virus.

Previously different authors described various flavivirus mutants with high affinity to cell glycosaminoglycans and low neuroinvasiveness in mice that were obtained consequently passages in cell cultures or in ticks. In present study the analysis of TBEV isolates has shown existence of GAG-binding variants in natural virus population. Affinity to GAG has been evaluated by sorption on heparin-Sepharose. GAG-binding phenotype corresponds to such virus properties, like small plaque phenotype in PEK cells, absence of hemagglutination at pH 6.4, and low neuroinvasiveness in mice. Mutations increasing charge of E protein were necessary but not sufficient for acquisition of GAG-binding phenotype. Molecular modeling and molecular dynamics simulation have shown that the flexibility of E protein molecule could bear influence on the phenotypic manifestation of substitutions increasing charge of the virions.

[Prevention of tick-borne encephalitis: current aspects].

The authors consider the present tick-borne encephalitis situation in our country and prospects for this infection control.

[Evidence for the absence of tick-borne encephalitis virus RNA in bioassays].

Reverse transcription-polymerase chain reaction was used to develop a procedure with internal monitoring that may provide evidence for the absence of tick-borne encephalitis virus RNA in bioassays. The proposed procedure is useful in evaluating the safety and effectiveness of a tick-borne encephalitis vaccine.

[Change in the antigenic structure of surface glycoprotein E of tick-borne encephalitis virus during its adaptation to ticks and mammals].

Enzyme immunoassay and immunoprecipitation test of viral proteins, by applying a tick-borne encephalitis (TBE) viral protein E monoclonal antibody kit, have shown that TBE adaptation to ticks and mammals may cause a considerable change in the antigenic structure of surface glycoprotein E, by involving different antigenic domains, including a neutralizing epitope that seems to participate at the early stages of interaction of virions with the cell membrane.

[Residual neurovirulence of the chimera of Langat and Denge-4 flaviviruses in intracerebral infection of monkeys].

The residual neurovirulence of the previously obtained chimeric virus Tp21/DEN4, containing the RNA region encoding the pre M and E structural enzymes, strain Tp21, virus Langat, and the remaining part of the genome from the Denge 4 virus, was studied in experiments with monkeys Cercopithecus aethiops involving the intracerebral administration of the virus. The tick-borne encephalitis virus, strain Absettarov, was used as positive control. A comparative analysis of the experimental and published data showed the chimeric virus to be less virulent by its degree of morphological affection in the CNS zones, its spread into the CNS and by a percentage share of animals with viremia versus the Tp21 parent strain and Elantsev strain of the Langat virus.

Molecular epidemiology of enteroviruses causing uveitis and multisystem hemorrhagic disease of infants.

We studied molecular epidemiology of highly virulent echovirus 11 and 19 strains that were isolated during five outbreaks of enterovirus uveitis (EU) in Siberia in 1980-1989, and three outbreaks of multisystem hemorrhagic disease of infants (MHD) in 1988-1991. Three genome regions, 5'NTR, VP1-2A junction, and a fragment of 3D polymerase, were analyzed. Phylogenetic grouping in the VP1-2A region correlated with serotyping results. All studied EV11 and EV19 strains, including the prototype EV11 and EV19, formed a major phylogenetic group in VP1-2A region. Within that group, several EV11 isolates from EU and MHD outbreaks formed a distinct cluster in VP1-2A and 5' NTR genome regions, designated EV11/B. All strains of this cluster possessed high virulence for monkeys compared with the prototype echoviruses. Subgrouping within this cluster correlated with year of virus isolation, not with the disease the viruses caused in infants (EU or MHD).

[Connection between severity of the course of tick-borne encephalitis with the concentration of interleukin-2 and interleukin-6 in blood]. / Izuchenie sviazi tiazhesti techeniia zabolevaniia s kontsentratsiei interleikina-2 i interleikina-6 v syvorotkakh krovi bol'nykh kleshchevym éntsefalitom.

AIM: To test IL-6 and IL-2 serum concentrations as indirect prognostic indicators in tick-borne encephalitis (TBE) course. MATERIAL AND METHODS: 13 TBE patients' sera were examined for IL-2 and IL-6. RESULTS: IL-6 was not detected in patients with TBE fever but was detected in meningoencephalytic TBE. CONCLUSION: TBE patients demonstrate a positive correlation between the disease severity and serum IL-6. This criterion is proposed as a prognostic factor of a TBE course.

Phylogenetic and serological characterization of echovirus 11 and echovirus 19 strains causing uveitis.

The strains of echovirus 19 (EV 19) and echovirus 11 (EV11), isolated from infants with similar clinical symptoms of acute enterovirus uveitis (EU) in Russia (Siberia) in 1980-1989, were investigated phylogenetically (nucleotide sequence of a 300 nt fragment in 5' NTR and VP4 junction) and serologically. The result confirmed that viruses belong to the Enterovirus genus, with 58-80% nt sequence homology with previously sequenced enteroviruses, and showed the genetical identity between the strains isolated during each of five outbreaks of the EU. The results also demonstrated that isolates from the last three outbreaks of EU belong to the same phylogenetic group despite the remarkable spatial and temporal distance between the outbreaks. The results confirm the role of these echoviruses in the etiology of the EU. Based on phylogenetic and serological comparisons the studied strains were divided into three distinct groups: group I, EV19/K (Krasnoyarsk, 1980-1981), group II, EV11/A (Krasnoyarsk, 1982), group III, EV11/B (Krasnoyarsk, 1986; Omsk, 1987-1988; Irkutsk, 1989). Minor details of the epidemiology of the outbreaks were also revealed.

[Virus-specific proteins of Crimean-Congo hemorrhagic fever]. / Virusspetsificheskie belki virusa Krymskoi-Kongo gemorragicheskoi likhoradki.

A variant of Asian strain Hodzha of Crimean-Congo hemorrhagic fever (Hodzha-A), adapted to Vero-E6 culture and yielding high harvest in it, was used for analysis of the synthesis of virus-specific proteins and protein composition of virions. Major virus-specific proteins with mol. weights 110, 83, 78, 55, 57, and 45 kD and minor low-molecular-weight proteins of 42, 36, 23, and 21 kD were detected by immunoprecipitation with hyperimmune ascitic fluid and polyacrylamide gel electrophoresis in infected cell lysates. N-type glycosylation of gp83 and gp78 was shown using tunicamicin. Viral proteins with mol. weights of 83, 78, 55, 57, and 45 kD were detected in infected cell culture fluid and in purified virion preparations after ultracentrifugation in sucrose density gradient. Two proteins with mol. weights 55-57 and about 180 kD were detected in purified nucleocapside preparations.

[Protective activity of a bacterial plasmid, bearing the gene for the tick-borne encephalitis virus NS1 nonstructural protein]. / Protektivnaia aktivnsot' bakterial'noi plazmidy, nesushchei gen nestrukturnogo belka NS1 virusa kleshchevogo éntsefalita.

Three intramuscular injections (50 micrograms each) with bacterial plasmid pMV45 carrying nonstructural gene of NS1 protein of tick-borne encephalitis (TBE) virus protected 88% Balb/c mice from lethal challenge with the virus. Antibodies to NS1 nonstructural protein were detected in the sera of vaccinated mice after the challenge. Absence of antibodies to E structural protein indicated absence of manifest infectious process in mice vaccinated with plasmid and challenged with a lethal dose of TBE virus.