Clinical and diagnostic manifestations of tickborne mixed infection in combination with COVID-19.

The coexistence of various pathogens inside the patient's body is one of the poorly studied and current issues. The aim of the study is to identify the relationship between the indicators of complex laboratory diagnostics and the clinical manifestations of a mixed disease during subsequent infection with the SARS-CoV-2 virus using the example of a case of chronic encephalitis-borreliosis infection. Seven blood serum samples were collected from the patient over the course of a year. For the etiological verification of the causative agents of TBE, Lyme disease and COVID-19, the methods of ELISA and PCR diagnostics were used. The patient was diagnosed with Lyme disease on the basis of the detection of IgG antibodies to Borrelia 5 months after the onset of the disease, since she denied the tick bite. In the clinical picture, there was an articular syndrome and erythema migrans. Later, IgG antibodies to the TBEV were found in the blood. Throughout the study, IgM antibodies to Borrelia were not detected. The exacerbation of Lyme disease could be judged by the clinical manifestations of this disease and by the growth of specific IgG antibodies. A feature of this case was that during an exacerbation of the Lyme disease, an infection with the SARS-CoV-2 virus occurred. Treatment (umifenovir, hydroxychloroquine, azithromycin, ceftriaxone) was prescribed, which improved the condition of the underlying disease, decreased joint pain, decreased IgG levels to borrelia. However, during this period, serological markers of TBEV appear: antigen, IgM antibodies, and the titer of IgG antibodies increases. Most likely, this was facilitated by the switching of the immune system to the SARS-CoV-2 virus, with the simultaneous suppression of borrelia with antibiotics and the appointment of hydroxychloroquine, which has an immunosuppressive effect. Despite the activation of the virus, clinical manifestations of TBE were not observed in the patient, which is most likely associated with infection with a weakly virulent TBEV strain. The further course of tick-borne infections revealed the dominant influence of B. burgdorferi in relation to TBEV. Laboratory studies have shown that suppression of the activity of the borreliosis process by etiotropic treatment subsequently led to the activation of the persistent TBEV.

Analysis of effectivity of the tickborne encephalitis virus detection methods in ixodid ticks.

Tick-borne encephalitis (TBE) is transmissible viral disease widely common in temperate zone of Eurasia. ELISA and PCR are used for express identification of the vector's infection, but the results of the two methods often do not agree. Aim of the work is comparative analysis for TBE virus of Ixodid ticks from nature using complex of methods, including ELISA, PCR, and isolation of the virus in laboratory mice. 18608 Ixodid ticks were collected during 2013-2019 in TBE natural foci of the Baikal Region. The ticks suspensions were examined individually, using ELISA (n=17610) and PCR (n=2999). Suckling mice were inoculated with the suspensions positive in the both tests. The TBEV antigen was found in 1.2 % of ticks in average. All ticks positive in ELISA were examined in PCR (Group 1). Randomly selected part of negative-ELISA samples were examined in PCR too (Group 2). The PCR results were positive in 68.9±3.13 % of the Group 1, with average Ct index 24.6±0.38. Positive results of PCR in Group 2 accounted for just 2.7±0.31 % with average Ct index 31.0±0.70. The average Ct margin of the Groups 1 and 2 is statistically significant (p < 0.001; df = 118). Isolation of strains was significantly more successful in Group 1 (21.7±2.77 %), than in Group 2 (8.2±5.26 %; p < 0.05; df = 50). ELISA is more useful for examining large amounts of ticks. To get a more complex picture about epidemically dangerous part of the vectors in TBE natural foci, the results of the two express-methods is better to sum. The isolation of the virus is useful to carry out of the samples positive in ELISA and PCR concurrently.

Indication Q fever pathogen in the south of Far east.

Q fever (coxiellosis) is a widespread natural focal disease in the world. The causative agent of coxiellosis is the gram-negative bacterium Coxiella burnetii, which is highly contagious and low virulence. The main carriers of C. burnetii are ixodid ticks, which feed on domestic and farm animals in anthropurgic foci. To address the possible circulation of the Q fever pathogen in the territory of the Primorsky Territory, 334 samples of various natural material collected in the spring-summer period of 2019 were studied. In the vicinity of the Vladivostok (on Reineke island), genetic markers of C. burnetii were detected in 19.7% of all tick species. In the Khankaisk region, coxiella DNA was detected more often (in 6.3%) in ticks of D. silvarum, in ticks of I. persulcatus and H. japonica, 1 case was detected. From 56 copies. ixodid ticks sucked to humans, C. burnetii DNA was detected in ticks of I. persulcatus in 38.8%, H. concinna - in 14.3%. In the serum of farm animals, the presence of coxiella in sheep in 3 samples was detected, in horses - in two. Sequencing of the obtained sequences showed the presence of the pathogen C. burnetii in the blood serum of animals. The ticks have stuck to people in 6 samples were identified C. burnetii and 6 samples - Coxiella-like endosymbiont. The presented results indicate the circulation of the causative agent of Q fever in the territory of the Primorsky Territory. To obtain a more complete description of the current epidemiological situation, it is necessary to conduct more extensive studies of natural material and blood of people with suspected Q fever.

Molecular and Genetic Bases of Inhibition of Tick-Borne Encephalitis Virus Replication by Eprosartan and Ribavirin.

The antiviral activity of eprosartan (compound selected in silico) towards highly and low-virulent strains of tick-borne encephalitis virus was compared in vitro with activity of ribavirin. Study of the cytopathogenic activity of the virus on SPEV cells by ELISA, IFAT, and PCR showed similar results: both substances (eprosartan and ribavirin) promoted elimination of tick-borne encephalitis virus. Ribavirin exhibited intracellular inhibition towards both strains: the selectivity index for highly virulent Dal'negorsk strain was 160, for low-virulent Primorye-437 strain - 113. Eprosartan inhibited intracellular replication of Dal'negorsk strain (13.7) and less so that of Primorye-437 strain (2.9). The efficiency of virtual screening of the ligand (eprosartan) was demonstrated for highly virulent, but not low virulent tick-borne encephalitis strain.

Verification of mono- and mixed contaminations transmitting agents tick-borne infections.

In the territory of the Primorsky region, the nosoareas of causative agents of tick-borne infections often coincide and lead to mixed infection of ticks, which causes the mixed pathology in humans . We investigated ixodid ticks taken from people during the epidemic season of 2019 for the spectrum of pathogens of transmissible infections to determine the degree of their mono- and mixed infection. 651 specimens of ixodid ticks investigated. RNA/DNA pathogens was determined by the method of polymerase chain reaction (PCR), TBEV antigen by ELISA. The presence in ticks of pathogens of the following diseases was detected: Lyme borreliosis (35.3%), tick-borne replasing fever (11.3%), human granulocytic anaplasmosis (2.6%), human monocytic ehrlichiosis (2.4%), tick-borne encephalitis (1.8%), tick-borne ricketsiosis (1.0%). As agents of mono-infections, the virus of TBE, ehrlichia, anaplasma and rickettsia in ticks occurred less frequently than in combination with other pathogens. A high frequency of occurrence B. burgdorferi s.l. in mono-infection detected (58.7%). B. miyamotoi has met evenly at ticks with mono- and mixed infections. Total mixed infection of ticks was 23.1%. Borrelia have been identified in all mixed cases. Variants of double combinations (95.5 % of cases from all mixed infections) were as follows: B. burgdorferi s. l. + A. phagocytophilum, B. burgdorferi s.l. + E. chaffeensis / E. muris, B. burgdorferi s.l. + TBEV, B. burgdorferi s.l. + R. heilongjiangensis, B. burgdorferi s.l. + B. miyamotoi. The most frequent combination was B. burgdorferi s.l. + B. miyamotoi (37.8% of all mixed infections). In triple infection of B. burgdorferi s.l. + A. phagocytophilum + TBEV and B. burgdorferi s.l. + A. phagocytophilum + B. miyamotoi, the frequency of occurrence for each combination was 2.2%. Given the above, a complex laboratory diagnosis of tick-borne infections in individuals who have been bitten by a tick is necessary, including the detection of all pathogens common in focal areas.

Mechanisms of Protective Actions of Specific Antibodies against the Tick-Borne Encephalitis Virus.

The protective mechanisms of specific antibodies against tick-borne encephalitis virus were demonstrated on in vitro model. The effect of specific IgG on tick-borne encephalitis virus was comprehensively assessed in virucidal, preventive, direct antiviral, and intracellular actions by ELISA and virus titration results. The IC50 values were obtained for virucidal (3.8±0.7 U/ml), preventive (42.8±9.9 U/ml), direct antiviral (7.2±0.9 U/ml), and intracellular action (1.7±0.4 U/ml). During titration of the samples, complete elimination of the virus was observed at IgG concentration of 16 U/ml (virucidal), 320 U/ml (preventive), 32 U/ml (direct antiviral), and 8 U/ml (intracellular action). It was demonstrated that specific IgG produces a complex inhibitory effect on tick-borne encephalitis virus: it possesses both direct neutralizing activity on the virus and reduces its adsorption and intracellular replication.

[Verification of dengue fever cases imported to the territory of the south Far East.]

There are various diagnostic and research methods for detecting cases of Dengue fever, the effectiveness of which is given in this work. MATERIALS AND METHODS: On biomaterial from 70 people, verification of imported cases of Dengue fever into the south of the Far East from 2012 to 2019 is shown. Serological and virological methods were used, as well as PCR. RESULTS: Using the immunochromatographic rapid test, the Dengue virus (DENV) NS1 antigen and antibodies to DENV (IgM and IgG) were detected in human blood. We examined 12 patients from the infectious diseases department with unknown fever and the blood of 58 people who applied to clinics in Vladivostok after returning from tourist trips. Dengue fever was diagnosed in 23 patients (32.8%), of which antigen was detected in 56%, IgM antibodies in 91.3% and IgG in 52.1%. In 2 cases (8.7%), only antigen was detected in patients. Three strains of the pathogen were isolated by virological methods from 18 blood samples, two of which turned out to be the DENV of the 1st genotype and one - of the DENV of the 2nd genotype. Using RT-PCR, 38 blood samples were tested positive in the immunochromatographic rapid test, of which in 16 cases (42.1%) a DENV marker was detected, in 11 cases it was genotype 1, in three cases genotype 2, and one each - genotypes 3 and 4. CONCLUSIONS: 1. The most reliable method of rapid verification (in 100%) the primary infection DENV was the comprehensive determination of antigen and antibodies of the IgM class; 2. With antigenemia, blood should be used to isolate the virus, as well as to diagnose the disease by PCR and to establish the genotype of the DENV; 3. When using only PCR to indicate Dengue virus, a significant proportion of the disease cases will not be diagnosed.

Inhibitory Activity of Scutellaria baicalensis Flavonoids against Tick-Borne Encephalitis Virus.

We studied virus-inhibiting activity of Baikal skullcap (Scutellaria baicalensis) flavonoids against tick-borne encephalitis virus using various model schemes. The half-maximum cytotoxic concentration (CC50) for the plant extract was found (363.9±58.6 µg/ml). Based on the CC50 and IC50, selective index (SI) was calculated for viricidal (53.4), preventive (50.5), and direct antiviral actions (39.1) and for-intracellular replication of the virus (40.4). Suppression of virus reproduction ≥2.0 lg TCID50 was observed at extract concentration ≥5 µg/ml (viricidal effect), ≥11.2 µg/ml (preventive and direct antiviral effects), and ≥9 µg/ml (intracellular replication). Flavonoids of Baikal skullcap extract produced an in vitro inhibitory effect on tick-borne encephalitis virus due to their direct viricidal activity and direct inhibition of adsorption and intracellular replication of tick-borne encephalitis virus, which determines their value as highly effective antiviral drugs.

[Comparative analysis of methods efficiency of virification of the tick-borne encephalite virus.]

Based on experimental studies with 10-fold dilutions of two the strains TBEV of the Far Eastern subtype, different in moleculargenetic characteristic complex data of simultaneously taking into account three indicators of their verification (virus titer, ELISA and PCR-RT) were obtained. The efficiency of detecting a genetic marker in PCR compared with ELISA for a weakly pathogenic strain with defects in the genetic structure was higher by a factor of 10, and for a highly pathogenic strain by a factor of 5,000. At the same time, positive results in both reactions with respect to two strains were detected with a virus titer of not less than 1-1.5 log TCID50, i.e. this level of virus in the sample is defined as epidemically significant. An algorithm for conducting research on the verification of TBEV is proposed: 1) Ticks collected from vegetation can be examined by ELISA or by PCR. All positive results can be summarized and considered viral ticks; 2) All samples with positive results only in PCR or ELISA must be investigated in two reactions in order to obtain confirmation of the possible infectivity of the pathogen; 3) To obtain a fast complex result of infection of the removed ticks from patients or blood after a tick bite, studies should be carried out simultaneously in two reactions simultaneously in ELISA and PCR; 4) Isolation virus should be carried out in biological samples (ticks collected from vegetation, ticks removed from patients, the blood of patients with suspected TBE, mammals) only with the same results in PCR and ELISA. Thus, such an approach to verifying TBEV in a tick or in the blood of patients will improve the reliability of laboratory diagnostics, identifying not only markers of TBEV, but also determining the infectivity of the pathogen, which may be the basis for the appointment of early intensive antiviral therapy.

Effect of Glycosaminoglycans on Pathogenic Properties Far-Eastern Tick-Borne Encephalitis Virus.

We studied the effect of sulfated glycosaminoglycan on the infection properties of high-virulence Dal'negorsk strain and low-virulence Primorye-437 of tick-borne encephalitis virus. Differences in reproductive activity of these strains and their tropism to the target cells were revealed. Glycosaminoglycan reduced pathogenetic activity of high-virulence strain in vitro, but had no effect on low-virulence strain. The interaction of imperfect virus particles of non-pathogen strain with the glycosaminoglycan led to their accumulation in cell, but in the culture medium of SPEV cells infected with experimental and control samples, accumulation of virus particles did not differ. The results on activity of glycosaminoglycan binding with strains differing by their biological and molecular genetic characteristics can be used to assess their pathogenic potential.

[Molecular-genetic monitoring as the basis of modern epidemiological surveillance of tick-borne infections.]

Molecular genetic monitoring of natural focal of tick-borne infections in the epidemic season of 2018 revealed infectiousness of ixodid ticks causative agents of tick-borne encephalitis (0.58% of cases), Lyme disease (31% of cases), human monocytic ehrlichiosis (1.6% of cases) and granulocytic anaplasmosis (3.9% of cases) is registered and also co-infections of ticks by these infections (2.9% of cases) is revealed in natural and anthropourgic foci (B. burgdorferi s.l.+A. phagocytophilum, B. burgdorferi s.l.+E. chaffeensis/E. muris and B. burgdorferi s.l.+tick-borne encephalitis virus). The major epidemiological importance of ticks of the species I. persulcatus is found, their share being 87,6%. The majority of patients being bitten by a tick were from the southern and southeast areas of Primorye. Contamination of ticks with Borrelia was revealed not only in I. persulcatus, but also in ticks of the Haemaphysalis and Dermacentor. The infectiousness of ticks of B. burgdorferi s.l. (42,3%), tick-borne encephalitis virus (7,7%) and A. phagocytophilum (15,4%) was highest on Russky Island.

[Phylogenetic analysis and distribution of far eastern tick-borne encephalitis virus subtype (Flaviridae, Flavirus, TBEV-FE) from Asia.]

To date, a lot of data on molecular genetic characteristics of different tick-borne encephalitis virus strains has appeared. Only on the basis of the E protein genome, sequences of about 1,500 TBEV strains were registered in GenBank. PURPOSE: The purpose of the work - revision and comparative analysis of data on complete genomes sequences of the Far Eastern subtype of TBE virus strains distributed in the Asian part of Eurasian continent. MATERIAL AND METHODS: The data on the complete genomes of 84 strains of TBEV isolated in Asia were used; phylogenetic analysis was performed. RESULTS AND DISCUSSION: it was shown that variants of the TBEV of the Far Eastern subtype are circulating here and form three separate clusters (Sofjin, Senzhang- и Shkotovo-like strains). Sofjin strain (Sofjin-1953, Sofjin-Chumakov, Sofjin-KSY) was considered to be the reference for Far Eastern TBE virus subtype strains and a cluster of Sofjin-like strains. Sofjin-like strains were not found in China and Japan, but widely distributed throughout the area of Primorsky and Khabarovsk krai. The group of Senzhang-like strains was distributed in China, Eastern Siberia, Khabarovsk krai and northern Primorsky krai, but was not found in Japan (Hokkaido). According to molecular genetic characteristics the youngest and more genetically homogeneous group was the Shkotovo-like strains, isolated in the southern part of Primorsky krai, however not found on Hokkaido Island (Japan). CONCLUSION: revision of the complete genome characteristics of TBEV strains revealed the features of micro-evolutionary process of viral populations in the Asian part of Eurasia, show the individual affection of strains to certain territories, as well as detect random finds of such strains in the territories of other natural foci.

Mechanisms of Antiviral Activity of the Polyphenol Complex from Seagrass of the Zosteraceae Family against Tick-Borne Encephalitis Virus.

Antiviral activity of the polyphenol complex from seagrass of the Zosteraceae family against highly pathogenic strain of the tick-borne encephalitis virus was studied on passaged culture of porcine embryo kidney cells. The antiviral effect of the test compound manifested in a decrease in the infectious titer of the virus and depended on the concentration and application schemes. Polyphenol complex in a concentration of 100 µg/ml suppressed accumulation of the pathogen in the cell culture: pretreatment of the virus reduced its titer by 4 log, pretreatment of cells by 1.4 log, and application of the compound 1 h after cell infection by 2.8 log. Antiviral action of the test compound is determined by direct inactivation of the virus and inhibition of virus replication at the early stage, which attests to potential of this compound in the treatment of tick-borne encephalitis.

THE STRUCTURAL CHANGES OF MACROPHAGES INFECTED WITH TICK-BORNE ENCEPHALITIS VIRUS.

Macrophages belong to the innate immune cells and play a key role in the pathogenesis of viral infections. The results of ultrastructural study of macrophages infected with tick-borne encephalitis virus (TBEV), the Flavivirus family, pathogens of human infections, affecting the nervous system, were presented. With the assistance of virological methods was found that the TBEV are absorbed by macrophages and replication in them. An ultrastructural study has shown that the virus enters into the cytoplasm by local destruction of plasmalemma and newly synthesized virus particles exited from the cell by same. Simultaneously there is a seal of perinuclear cytoplasm space, where found in a large number of ribosomes, microfilaments, ribonucleoprotein fibers and viral special structure: nucleocapsids, tubular formations and viral layers (fabrics). On the surface of last structures the newly synthesized virus particles were visualized. Thus, the evidence shows that macrophages play a role in the spread of TBEV, being for their the target cell. As active antigen presenting cells the macrophages can modulate the protective response of the body and influence on the pathogenesis of tick-borne encephalitis.

Antioxidants as Potential Antiviral Agents for Flavivirus Infections.

The review considers potential opportunities of certain antioxidants as antiviral agents for flavivirus infections. Currently flavivirus infections represent a significant public health problem for various regions of the world, since they result in high morbidity and mortality. Oxidative stress induced by viruses, including the flaviviruses, not only interferes with the body's important metabolic processes, but also regulates replication of the virus. A broad spectrum of antiviral activity of the natural phenolic antioxidants, i.e. rosmarinic acid and luteolin as components of the polyphenol complex isolated from the seagrass family Zsteraceae is decribed. The antiviral activity of rosmarinic acid and luteolin is shown to be due to their high antioxidant, antiinflammatory and neuroprotective potential. Particular attention is paid to the analysis of the activity of the polyphenol complex and its components against the tickborne encephalitis virus. The antiviral properties of the compounds with a broad spectrum of the biological action provide an oppor- tunity to consider them as promising candidates for combined therapy of tick-borne encephalitis.

[Antiviral activity of various drugs with different mechanisms of action in patients with experimental tick-borne encephalitis].

A possible approach to effective, pathogenetically valid treatment of patients with the tick-borne encephalitis (TBE) is a complex therapy with the immunotropic preparations isolated from natural objects. This work is devoted to the comparative study of the antiviral activity of the tinrostim (immunoactive peptide from the optical ganglia of the squid Berritiuthis magister) and some officinal drugs used for prevention and treatment of the TBE (ribavirin, reaferon-EC, cycloferon, 4-jodantipyrin, immunoglobulin human against encephalitis ixodicum) in the experimental models of the TBE. All tested drugs significantly inhibited the proliferation of the highly virulent strain of the TBEV in the sensitive PK cell cultures: ribavirin and immunoglobulin against TBE completely inhibited viral replication (by 100%); cycloferon - by 75%; tinrostim, reaferon-EC, and jodantipyrin - by 50-60%. Therapeutic efficacy of the compounds was evaluated on a model of acute lethal TBE in mice: treatment with cycloferon and immunoglobulin against TBE prevented the mortality in 35-45% of infected animals; tinrostim - in 25%; ribavirin, reaferon-EC, and jodantipyrin - in 5-10%. The combination of the immunoactive peptide, tinrostim, with officinal drugs (ribavirin, cycloferon) was more effective than the treatment with a single drug, thereby indicating the prospects of the use of this therapy for treating TBE.

[Biochemical markers of virus cytopathogenicity in macrophages].

The results of macrophage metabolism studies at their infection by viruses differing in the level of virulence are presented. With the purpose of optimizing the estimation of viral cytopathogenic effects on macrophages, an index of cell reactions, which allows one to reveal the degree of virus influence in standard units, is offered. Generally, the application of high-sensitivity methods for functional activity determination and identification of the correlative communication between its changes and morphological features of cells can be prescribed to objective identification methods of not only viral reproduction, but also differentiation of types and the degree of their cytopathogenic effects.

Neutrophil apoptosis induction by tick-borne encephalitis virus.

Tick-borne encephalitis virus infects neutrophils and induces their apoptosis, judging from moderate increase of succinate dehydrogenase activity and a trend to anaerobic energy production in neutrophils infected with the virus (shown by an increase of lactate dehydrogenase activity).

[Investigation of luromarin efficacy on mice with experimental tick-borne encephalitis].

Oral use of Luromarin, extracted from sea algae Zostera asiatica, was efficient in protection of mice from lethal infection induced by highly pathogenic strain of TBE virus, by extending their average lifespan. Luromarin demonstrated potentiating action in combination with ribavirin and cycloferon.