[Persistence of antibodies specific to West Nile virus in blood of reconvalescents of Volgograd region].

AIM: Determine the duration of persistence of IgM and IgG in reconvalescents of West Nile fever (WNF) 1 year after the disease in southern regions of Russia and evaluate effectiveness of PCR method for acute infection diagnostics. MATERIALS AND METHODS: Blood sera of 87 patients with WNF diagnosis was studied for the presence of West Nile virus (WNV) RNA and IgM and IgG by PCR and EIA. Samples of the first sera were collected in 2010 at days 2 - 30 after the onset of the disease, samples of the second sera--at days 5 - 23 and third--264 - 385 days later. RESULTS: During the first 2 weeks of the disease WNV RNA was detected in more than 50% of patients. In all the first sera IgM at titers of ≥ 1:800 were detected. Seroconversion of IgG titers of 4 and more times was observedin 83% (30/36) of patients. In 2011 IgG were detected in 91% of reconvalescents (79/87), IgM--in 57% (50/87), and in 25% (22/87) IgM titers were ≥ 1:800. CONCLUSION: The results obtained give evidence on the necessity of using several diagnostic criteria simultaneously for the confirmation of WNF clinical diagnosis.

[Tick-borne encephalitis with fulminant course and lethal outcome in patients after plural vaccination].

In the Kurgan region, the Siberian subtype of the tick-borne encephalitis virus (TBEV) is dominant. The vaccines prepared from Far-Eastern TBEV subtype are used in this area. Among TBE patients in 2007-2011, 23.79% were vaccinated according to complete or incomplete course. 76.9% of persons were vaccinated with Encevir vaccine, Tomsk. An unusual focal form of TBE with fulminant disease with lethal outcome was developed in a patient who was vaccinated 6 times with heterotype vaccines produced using the strains of the Far-Eastern TBE subtype. Inoculation of immunoglobulin in hospital produced aggravation of clinical symptoms, development of convulsions, brain oedema, and respiratory distress syndrome. The disease continues only 55 hours from first symptoms to fatal outcome. Siberian subtype of TBEV was isolated from patient spinal cord (Kurgan-118-2010 strain). Possible mechanisms of this disease are discussed.

[Polytypic strains in the genofund of tick-borne encephalitis virus].

Eighteen polytypic tick-borne encephalitis virus (TBEV) strains containing the fragments of E and NS1 protein genes of Siberian and Far Eastern, occasionally Siberian and European subtypes were isolated in the European and Asian parts of the tick-borne encephalitis (TBE) area. They were identified using real-time polymerase chain reaction, hybridization-fluorescence detection with genotype-specific probes, restriction fragment length polymorphism analysis, and E protein sequencing. The polytypic strains were isolated from individual Ixodes persulcatus ticks, their pools, from the blood of patients and the brain of dead patients. The isolation rates of the polytypic strains in the sympathry area of different TBEV subtypes ranged from 4.4% (the Irkutsk Region) to 15.1% (the Yaroslavl Region). In addition to 2 polytypic strains, a strain similar to the TBEV 886-84 strain was isolated. The TBEV subtypes entering into the composition of the polytypic strains show nongenetic interactions, such as neutral replication or competition. The polytypic strains are stable during passages in the cultured pig embryo kidney epithelial cells and on cloning. Mouse brain passage promotes dissociation of polytypic strains. The conditions for the formation of polytypic strains and their role in the etiology of TBE are discussed.

[Interaction of the Siberian and Far Eastern subtypes of tick-borne encephalitis virus in mammals with mixed infection. I. Factors influencing the type of interaction].

Polytypic strains containing the fragments of genes of Siberian and Far Eastern tick-borne encephalitis (TBE) virus subtypes were isolated from the brain of fatal TBE patients, the blood of TBE patients, and Ixodes persulcatus ticks in the foci of concomitant circulation of the two subtypes. The interaction of the Siberian and Far Eastern TBE virus subtypes was studied in the neural phase of the infection of albino mice and Syrian hamsters in order to understand conditions for formation of these strains and their role in the etiology of acute TBE. Their viral progeny was genotyped by reverse transcription-polymerase chain reaction and fluorescence hybridization assay with genotype-specific probes. Mixed infection showed an effect of synergism, independent reproduction of the two subtypes in the brain and spleen, competitive exclusion of one subtype from the viral population. The type of the Interaction depended on the species of animals, the properties of partner strains, and the target organ.

[Interaction of the Siberian and Far Eastern subtypes of tick-borne encephalitis virus in mammals with mixed infection. Competition of the subtypes in acute and inapparent infection].

Long-term monitoring of natural tick-borne encephalitis virus (TBEV) populations could reveal the change of TBEV subtypes, the displacement of the Far Eastern (FE) subtype, and its substitution for the Siberian (Sib) subtype. Acute and inapparent mixed infections were studied in Syrian hamsters to understand this phenomenon. The animals were inoculated with the Sib subtype and then with the FE one of TBEV (JQ845440-YaroslavI-Aver-08 and Fj214132-Kemerovo-Phateev-1954 strains). The inapparent form developed more frequently in mixed infection. Viral progeny was genotyped by reverse transcription polymerase chain reaction and hybridization fluorescence detection using genotype-specific probes. Independent reproduction of strains in the brain gave way to competition. The FE subtype dominated in hamster youngsters with acute infection. The Sib subtype had selective benefits in asymptomatic infection (adult hamsters infected intracerebrally and subcutaneously and youngsters infected subcutaneously). The competition of the subtypes was imperfect.

[Evolution of tick-borne encephalitis and a problem of evolution of its causative agent].

The evolution of tick-borne encephalitis (TBE) is marked by the expanded nosological area, the transformation of landscapes, the formation of anthropurgic foci, the change of environmental systems, the increase of mortality rate mainly among urban dwellers, as well as pathomorphism. The evolution of natural TBE virus (TBEV) populations was studied in Eastern and Western Siberia, Middle Urals, and the European part of the nosological area. The paper first describes the types of evolutionary transformations of viral populations under the conditions of a varying environmental and epidemiological situation. These include: 1) the change of TBEV subtypes over 50-60 years; substitution of the Far-Eastern subtype for its Siberian subtype (the Sverdlovsk and Kemerovo regions); 2) the steady-state circulation of one Siberian subtype with mutanttypes being accumulated (the Vologda region); 3) co-existence of the Far-Eastern and Siberian subtypes with the common vector Ixodes persulcatus (the Yaroslavl and Irkutsk regions, etc.); 4) original mixed TBEV strains including the gene sites of proteins E and NSI of two subtypes. There is new evidence that the Siberian subtype is able to induce focal TBE forms, leading to death.

Variabilities in the antigenic structure of persisting tick-borne encephalitis virus strains.

Antigenic variants in the E protein from persisting tick-borne encephalitis (TBE) virus strains were analyzed using monoclonal antibodies (MAbs) to an analogous protein of the reference Sofyin strain. MAbs to sequential epitopes demonstrated their ability to differentiate persisting TBE virus strains from Sofyin and from each other. Two MAbs (2H3 and 13D6) showed a higher neutralizing activity in the interaction with persisting TBE virus variants as compared to the Sofyin strain. Based on the obtained data, a comparison was made of topologically identical epitopes from the E protein of reference and persisting virus strains. The possibility of increasing the neutralizing activity of MAbs through alterations in the primary structure of sequential antigenic sites is discussed.

Persistence of tick-borne encephalitis virus IV. Virus localization after intracerebral inoculation.

Tick-borne encephalitis (TBE) virus was isolated from the brains and spinal cords, blood, livers, lymph nodes and kidneys from Macaca rhesus monkeys showing acute and subacute fatal encephalitis. In subacute encephalitis, virus titres in the CNS were lower than in acute disease (3.0--6.2 against 3.8--8.3 log LD50/ml). TBE virus localization in chronic encephalitis was largely the same as in acute and subacute disease. In monkeys with a chronic course and stable paralysis of the upper extremity, infectious TBE virus was isolated on day 383 from subcortical ganglia and spinal cord. In lymph nodes and spleen, it could be detected only by a combination of methods (co-cultivation in association with fluorescent antibody technique and complement-fixation test, explantation of organ fragments) more sensitive than is the inoculation of mice with organ homogenates. TBE virus was detected by the same methods on day 90 in the CNS and internal organs of a monkey with chronic encephalitis in the stage of remission.

Persistence of tick-borne encephalitis virus in monkeys. V. Virus localization after subcutaneous inoculation.

In 28 Macaca rhesus monkeys inoculated subcutaneously (s.c.) with different strains and mutants of tick-borne encephalitis (TBE) virus and developing asymptomatic infection, TBE virus and the virus-specific antigen were found at different intervals up to 302 days post inoculation (p.i.) in the CNS and internal organs (liver, spleen, lymph nodes, kidneys). When cyclophosphane (CP) was used as an immunosuppressor, no significant changes in virus distribution were observed with the exception of more frequent isolations at early intervals from kidneys and at late intervals from spleen. With or without CP administration virus was found in the spinal cord at 3--6 days, in the cerebellum and subcortical ganglia at 11--14 days, in the cerebral cortex at 19 days p.i. Early after inoculation the virus was more frequently isolated from the CNS than from the internal organs; later (93--302 days p.i.) the persisting virus or virus-specific antigen were predominantly found in internal organs. The set of sensitive methods used facilitated to detect the persistence of TBE virus in noninfectious form in monkeys with asymptomatic infection.

Persistence of tick-borne encephalitis virus in monkeys. I. Features of experimental infection.

Sixty-seven Macaca rhesus monkeys were inoculated with 2 mutants and 3 virulent strains of tick-borne encephalitis (TBE) virus including strains isolated from patients with a chronic form of TBE. A model of the clinical course of acute, subacute, and chronic encephalitis was produced by intracerebral inoculation and that of asymptomatic infection was produced by subcutaneous inoculation [with or without administration of cyclophosphane (CP)]. Virus persistence developed after inoculation with all the strains, after non-fatal encephalitis and inapparent infection irrespective of CP administration. In monkeys recovering from encephalitis the virus persisted for at least 783 days. After asymptomatic infection, it persisted for 302 days; neither virus nor virus-specific antigen was detected at 356, 367, and 620 days.

Persistence of tick-borne encephalitis virus in monkeys. II. Effectiveness of methods used for virus detection.

Fifty monkeys (Macaca rhesus) inoculated with tick-borne encephalitis (TBE)virus intracerebrally or subcutaneously were examined. The efficiencies of different virus detection methods at 90 to 783 days after inoculation were: explantation of organs - 41.2%; co-cultivation of trypsinized organ cells and indicator cells in the presence of 5-bromo-2-iododeoxyuridine with detection of the virus-specific antigen by immunofluorescence - 43.7%, detection of infectious virus and complement-fixing antigen - 13.6%; immunofluorescence examination of organ impression smears - 29.3%; and examination of organ homogenates with detection of virus pathogenic for mice - 1.7%. the latter method was the least sensitive for the detection of persisting TBE virus, in spite of that in the first 3 weeks of infection its efficacy was 88.1% in examinations of clinically ill monkeys and 23.8% in the inapparent form of the infection.

Persistence of tic-borne encephalitis virus in monkeys. III. Phenotypes of the persisting virus.

The properties of tick-borne encephalitis (TBE) virus persisting for 90-383 days after intracerebral and subcutaneous inoculation of Macaca rhesus monkeys were studied, namely (1) the type of infection produced directly in the tissues of the experimental monkeys; (2) the activating effect of co-cultivation and explantation procedures; and (3) the phenotype of the isolates by a set of markers. The virus was detected and analysed in 52 instances. Directly in monkey tissues the virus induced a productive infection rarely (5.8%) but more frequently (71.2%) an abortive infection detectable by immunofluorescence (presence of virus-specific antigen). In 23% of instances a nonproductive infection was observed in monkey tissues. Like abortive infection it could be activated by the co-cultivation of cells and explantation procedures. The latter exerted a more marked activating effect than co-cultivation. The strains isolated from monkey tissues in productive infection or activated by explanation or co-cultivation were heterogeneous in their properties. The following virus phenotypes were found: virus highly virulent for mice, cytocidal and antigenically complete; a cytocidal virus of low virulence, possessing haemagglutinin; and a cytocidal virus apathogenic for mice, devoid of haemagglutinin but synthesizing complement-fixing antigen and an antigen detectable by immunofluorescence.

[The search for antibiotics not exerting activating action in the persistence of the tick-borne encephalitis virus]. / Poisk antibiotikov, ne okazyvaiushchikh aktiviruiushchego deistviia pri persistentsii virusa kleshchevogo éntsefalita.

Chronic tick-borne encephalitis (TBE) frequently develops in the presence of concomitant diseases requiring antibiotic therapy. Because some antibiotics, e.g. streptomycin, strongly activate persisting TBE virus, a study was carried out in search of antibiotics without the activating effect. The experiments were carried out in Syrian hamsters inoculated subcutaneously with the Vasil'chenko strain of TBE virus which at 60-348 days of the persistent infection were given florimycin, levomycetin and kanamycin. The antibiotics were administered for 3 weeks. Levomycetin showed no activating properties, while kanamycin and florimycin exerted weak activating effect on the persisting TBE virus (isolated from 5% of the specimens) without marked immunosuppressive effect or manifestation of the infection. The TBE virus strains isolated on the 205th day from the brain and 348th day from the spleen of the hamsters given kanamycin and florimycin had higher virulence than the original strain.

[Streptomycin--an activator of persisting tick-borne encephalitis virus]. / Streptomitsin--aktivator persistiruiushchego virusa kleshchevogo éntsefalita.

The effect of streptomycin (C) on persistence of tick-borne encephalitis (TBE) virus in Syrian hamsters infected with 3 strains of the virus (41/65, Aina/1448, Vasilchenko ) intracerebrally or subcutaneously was studied. In the animals not given C the infectious virus could be detected in the brain for 8-14 days but not later although their organs (mostly brains and spleens) contained the hemagglutinating antigen and viral antigen detectable by immunofluorescence. Intramuscularly C was given twice daily for 13-35 days in a daily dose of 200 mg/kg. The C-treated hamsters yielded 7 virulent TBE virus strains: 3 from the brain, 3 from the spleen, and one from the blood. No virus could be isolated from the liver, kidneys, or lungs despite the use of various methods for isolation including tissue explantation. The activating effect of C was observed against the background of 4-fold decrease in the titre of complement-fixing and antihemagglutinating antibodies. C exerted its activating effect both at early (70 days) and late (9 months) stages of TBE virus persistence. The activating effect of C appears to be due to its immunosuppressive properties and neurotoxic action on the CNS.

[Mechanisms of the stimulating action of the Rauscher leukemia virus on togavirus reproduction in the bodies of BALB/c strain mice]. / Izuchenie mekhanizmov stimuliruiushchego deistviia virusa leikoza Raushera na reproduktsiiu togavirusov v organizme myshei linii BALB/c.

Reciprocal stimulation or inhibition are observed in double infection of mice with Rauscher leukemia virus (RLV) and togaviruses Sindbis or West Nile (WN). Stimulation of togavirus infections is manifested by the enhancement of the visceral phase of pathogenesis without subsequent activation of togarivus reproduction in the CNS. This effect is accompanied by enhanced togavirus replication in splenocytes, a decrease in the number of antibody-producing cells in the spleen, a decrease of antihemagglutinins titer in the blood without any significant change in the virus-neutralizing antibody and interferon titers. RLV-induced immunosuppression is termporary and of varying intensity with regard to individual parameters of immune response and to different variants of WN virus (highly virulent, poorly virulent). It is assumed that the differentiated and temporary nature of the immunosupressive effect of RLV is conducive to selective stimulation of the visceral phase of togavirus reproduction followed by inhibition of the neural phase under the influence of restored immune mechanisms and interferon. Because of the defects of humoral and cellular immunity, however, no complete elimination of togavirus occurs and conditions for its long-term persistence are created.

[Strategy for choosing antibiotics for treating bacterial infections associated with chronic tick-borne encephalitis]. / Strategiia vybora antibiotikov dlia terapii bakterial'nykh infektsii, assotsiiruiushchikhsia s khronicheskim kleshchevym éntsefalitom.

The capacity of wide-spectrum antibiotics kefzol and ristomycin to activate the persisting tick-borne encephalitis (TBE) virus and cause an exacerbation of chronic process was investigated in Syrian hamsters in whom a prolonged (77 to 270 days) persistent TBE infection was induced by three TBE strains: Vasilchenko, V-383, and 205. The degree of antibiotic-induced activation was assessed using the criteria characterizing the reproduction and peculiarities of persisting TBE virus, immunodepression, and morphologic changes in the central nervous system. Effects of kefzol and ristomycin were compared with those of 8 antibiotics studied previously. Ristomycin, levomycetin (chloramphycin), penicillin, ampicillin (ampital), and levoridan were referred to drugs devoid of evident provoking effect. Kefzol (cefamezin), florimycin (viomycin), and kanamycin (kanamytrex) were characterized as weak activators and streptomycin and tetracycline as potent activators of the persisting TBE virus. These data may be used when selecting alternative agents for therapy of secondary bacterial infections concomitant with TBE.

[Effect of gentamycin on persistence of tick-borne encephalitis]. / Effekt gentamitsina pri persistentsii virusa kleshchevogo éntsefalita.

Effect of gentamicin, an aminoglycoside antibiotic, on the persistence of tick-borne encephalitis (TBE) virus in Syrian hamsters and Macaca iris in remote periods (70-434 days) after inoculation is studied. Attempts at virus isolation from animals treated with gentamicin failed. Unlike other aminoglycosides, e.g. streptomycin, gentamicin exerted no immunodepressive effect; moreover, immunocorrection was observed in some experiments on monkeys and hamsters. None of the 10 previously tested antibiotics elicited such an effect or inhibited the persisting TBE virus. Morphological study of the central nervous system in hamsters and monkeys showed that injection of gentamicin did not cause an exacerbation of chronic encephalitis. The mechanism of immunocorrecting effect of gentamicin is to be further investigated.

[Characteristics of the persistence of the tick-borne encephalitis virus against a background of experimentally induced allergic encephalomyelitis in M. rhesus monkeys]. / Osobennosti persistentsiii virusa kleshchevogo éntsefalita na fone éksperimental'no indutsirovannogo allergicheskogo éntsefalomielita u obez'ian M. rhesus.

Asymptomatic persistence of tick-borne encephalitis (TBE) virus was modeled by subcutaneous inoculation; the virus and the antigen were detected up to 307 days by the cell co-cultivation, tissue explants, and immunofluorescence methods. At early and late periods of the persistent infection (1 1/2, 8 1/2, or 10 1/2 months) for the purpose of its stimulation, an autoimmune disease, experimental allergic encephalomyelitis (EAE), running a course of the type of delayed hyperergic reaction was induced in the animals by inoculation of an encephalitogenic mixture (EM). After EM inoculation, a chronic demyelinating process developed in the CNS without the involvement of neurons; proliferation of immunocompetent cell elements was observed in the spleen and lymph nodes; a short-time or stable stimulation of TBE-specific humoral immunity was observed. Against the background of the development of chronic EAE and a short-time stimulation of humoral immunity, persistence of TBE virus was prolonged for over 2 years, its location being unchanged (different parts of the brain, spleen). Despite long-term persistence of TBE virus in the CNS, no clinical and morphological symptoms of chronic TBE were found. Possible mechanisms of the effect of this stimulating factor favouring the activation of the persisting TBE virus on the one hand and limiting this process on the other are discussed.

[Change in the nature of the persistence of alpha- and flaviviruses in the body of BALB/c strain mice with a mixed infection with leukovirus]. / Izmenenie kharaktera persistentsii al'fa- i flavivirusov v organizme myshei linii BALB/c pri smeshannoi infektsii s leikovirusom.

The features of pathogenesis of infection caused in adult Balb/c mice intraperitoneally infected with Sindbis virus, virulent or attenuated strains of West Nile (WN) virus, individually or in combination with Rauscher leukemia virus (RLV) were studied. The influence of the latter on the course of togavirus infections was characterized by 3 features: (a) different effects on the visceral and neural phases of the pathogenesis (increased period of viremia and virus reproduction in the viscera did not lead to stimulation of virus reproduction in the CNS); (b) changes in the time of togavirus persistence in the infectious form; (c) the dependence of the observed effect on the togavirus properties.

[Comparative analysis of virulence of the Siberian and Far-East subtypes of the tick-born encephalitis virus]. / Sravnitel'nyi analiz virulentnosti sibirskogo i dal'nevostochnogo podtipov virusa kleshchevogo entsefalita.

The Siberian subtype of the tick-borne encephalitis virus (TEV) is different from the Far-East subtype by a moderate virulence observed in Siberian hamsters and by a low infection development rate (100 strains were compared). No differences were found in neuro-invasiveness. Clinical findings and experiments with monkeys denote the ability of the Siberian subtype to provoke severe forms of tick-borne encephalitis (TBE). The inflammation-and-degenerative changes were localized in the brain cortex, subcortical ganglions, nuclei of medulla oblongata, in the cortex and nuclei of the cerebellum as well as in the anterior horns of the spinal cord. 18 disease cases triggered by the Siberian TEV subtypes in residents of the Western and Eastern Siberia and of Central Russia (Yaroslavl Region), including 7 acute TBE cases (5 lethal outcomes), as well as 11 chronic TBE cases are analyzed. The viral RNA was found in the cortex, medulla oblongata, horn and in the cervical part of the spinal cord of those diseased of acute TBE. Sequences of genotyped strains were presented to Gen Bank, NCBI (AY363846-AY363865).