[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).

Characterization of a siberian virus isolated from a patient with progressive chronic tick-borne encephalitis.

A strain of Tick-borne encephalitis virus designated Zausaev (Za) was isolated in Siberia from a patient who died of a progressive (2-year) form of tick-borne encephalitis 10 years after being bitten by a tick. The complete genomic sequence of this virus was determined, and an attempt was made to correlate the sequence with the biological characteristics of the virus. Phylogenetic analysis demonstrated that this virus belongs to the Siberian subtype of Tick-borne encephalitis virus. Comparison of Za virus with two related viruses, a Far Eastern isolate, Sofjin, and a Siberian isolate, Vasilchenko, revealed differences among the three viruses in pathogenicity for Syrian hamsters, cytopathogenicity for PS cells, plaque morphology, and the electrophoretic profiles of virus-specific nonstructural proteins. Comparative amino acid alignments revealed 10 individual amino acid substitutions in the Za virus polyprotein sequence that were different from those of other tick-borne flaviviruses. Notably, the dimeric form of the Za virus NS1 protein migrated in polyacrylamide gels as a heterogeneous group of molecules with a significantly higher electrophoretic mobility than those of the Sofjin and Vasilchenko viruses. Two amino acid substitutions, T(277)-->V and E(279)-->G, within the NS1 dimerization domain are probably responsible for the altered oligomerization of Za virus NS1. These studies suggest that the patient from whom Za virus was isolated died due to increased pathogenicity of the latent virus following spontaneous mutagenesis.

[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.

[Some mechanisms of the action of oligonucleotides: stimulation of the immune system and decreased infection of the brain in tickborne encephalitis]. / Nekotorye mekhanizmy deistviia oligonukleotidov: stimuliatsiia immunnoi sistemy i umen'shenie porazhenii golovnogo mozga pri kleshchevom éntsefalite.

Morphological changes in the thymus, spleen, and brain are analyzed in white mice injected 16-component oligonucleotide (ON) pE16 complementary to the NS3 protein gene sequences of tickborne encephalitis (TBE) virus in doses of 1 to 0.001 nM. ON stimulated thymic and splenic cells. Besides the stimulating effect, injection of ON to mice infected with TBE enhanced the destruction of lymphocytes and boosted the macrophagal activity, which was paralleled by a decrease in the intensity of virus-specific injuries in the brain. Thus, the antiviral activity of ON may be due to not only the fact that it is complementary to the TBE virus genome, but to stimulation of the immune system, specifically, the thymus and T-related elements, as well.

[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.

[The clinical picture and pathogenesis of polyradiculoneuropathy in tick-borne encephalitis]. / Klinika i patogenez poliradikulonevropatii pri kleshchevom éntsefalite.

Based on examination of tick-borne encephalitis patients with ascending polyradiculoneuropathy the authors describe the character of the disease, its clinical picture and the results of laboratory studies, etc. Differential diagnosis is made between the indicated syndrome associated with tick-borne encephalitis and sporadic polyradiculoneuropathies. A detailed description is given for the first time of the clinical and pathomorphological picture of that gravest form of tick-borne encephalitis. As regards the character of the clinical and pathomorphological alterations, the ascending polyradiculoneuropathy associated with tick-borne encephalitis is meningoencephalomyelitis with the radicular syndrome which often determines the disease gravity and prognosis.

[The antiviral action of medicinal plant extracts in experimental tick-borne encephalitis]. / Protivovirusnoe deistvie ékstraktov lekarstvennykh rastenii pri éksperimental'nom kleshchevom éntsefalite.

Some mechanisms of inducing resistance to experimental infection with tick-borne encephalitis virus were studied in experimental mice treated with aqueous extracts of berries of Vaccinium vitis-idaea, black currant, Vaccinium myrtillus, and of greater celandine grass. The condition of the immune system organs (spleen and thymus) after treatment with the extracts under study was analysed. A correlation was found between the degree of developing resistance to infection, virus accumulation in the brain, blood, spleen and thymus and changes in some parameters (spleen and thymus indices) of these immunocompetent organs. Possible mechanisms of induction of resistance to virus by herb extracts are discussed.

[The genetic characteristics of the Vergina serotype of the tick-borne encephalitis virus and its pathogenetic traits]. / Genetichskaia kharakteristika serotipa Vergina virusa kleshchevogo éntsefalita i osobennosti ego patogennosti.

The Greek Vergina strain of tick-borne encephalitis (TBE) virus was studied in comparison with 7 other strains by molecular hybridization of nucleic acids and by clinicomorphological markers of pathogenicity for monkeys and Syrian hamsters. By the genetical features the Vergina strain differed from the eastern and western TBE subtypes but was found to be similar to the strains of other subtypes of the Urals-Siberian, east-Siberian (Aina-1448) and Central Asian antigenic variant. This group of strains hybridized with cDNA at 65 degrees C only in the absence of 50% formamide, reacted with probe 1115 complementary to protein E gene, with 1-3 probes complementary to the conservative region of the genome but did not react with the probes corresponding to the variable regions of the genome. The Vergina strain is close to TBE genotype III. The Vergina strain was found to be virulent inducing subacute meningoencephalomyelitis which developed slowly and was accompanied by less marked morphological lesions in the cerebral cortex than those induced by the eastern subtype. The Vergina strain was demonstrated to persist in the brain, liver, spleen, and lymph node tissues.

Antigenic comparison of Yuli and Vnukovo-32 virus strains in monkeys: specific protection by commercial antirabies preparations.

A combined vaccination schedule using commercial antirabies immunoglobulin G and experimental vaccine from strains Vnukovo-32 or Yuli beginning 2 hr before intracerebral (i.c.) challenge with a high dose of Yuli virus conferred no protection to Cercopithecus aethiops monkeys. In monkeys inoculated into lip with a middle dose of Yuli virus, administration of large amounts of antirabic IgG (up to 5000 national units, NU/kg) had a clearcut effect. The disease in Yuli virus-infected monkeys showed typical signs of acute encephalitis with lethal outcome, although one animal which developed typical encephalitis recovered as evidenced by increased virus-neutralizing antibodies in its serum. Inflammatory and degenerative lesions developed in the CNS of animals with signs of acute encephalomyelitis; their intensity was less prominent in those monkeys which underwent the combined treatment. In the cytoplasm of brain neurons of monkeys infected with Yuli virus relatively small Babes-Negri bodies with more or less apparent internal structure were detected.

Molecular hybridization with cloned fragments of tick-borne encephalitis (TBE) virus cDNA in acute and chronic TBE infection.

Recombinant plasmid DNA was used as a probe to detect tick-borne encephalitis (TBE) virus RNA during incubation period, acute disease and persistent infection of syrian hamsters. Within the first three weeks post-infection the results of direct virus isolation and RNA detection in the brain agreed by a rate of 100%, the virus titre ranging between 10(1.9) to 10(10.5) LD50/ml and viral RNA concentration at 1-1000 pg. At the same time TBE virus RNA was detected in the spleen when the virus titre was greater than or equal to 10(6.5) LD50/ml. By 8 months post infection (p.i.) viral RNA was found in the brain, liver, and spleen in the absence of infectious TBE virus. No viral RNA was present in the thymus. In addition, electron microscopic findings in hamster brain confirmed the hypothesis that TBE virus persistence was accompanied by formation of virus-specific structures but impaired virion maturation.

[The effect of myelopeptides on the persistence of tick-borne encephalitis virus in monkeys]. / Vliianie mielopeptidov na persistentsiiu virusa kleshchevogo éntsefalita v organizme obez'ian.

Myelopeptides (MP), bioregulatory molecules of bone marrow origin, exert a protective effect in persistence of tick-borne encephalitis virus in cynomolgus monkeys (Macaca fascicularis). The experiments involved 32 monkeys. The effect of MP was observed after one or two subcutaneous injections in a dose of 1 mg within 1.5-2 months after virus infection. The effect consists in 25-fold reduction of the frequency of virus persistence, marked limitation of the zone of spread of the persisting virus, including the central nervous system (CNS), decrease in virulence of the persisting virus, and lack of morphological signs of progress of the pathological process in the CNS. The protective effect was also observed when the infected monkeys were treated with MP and inactivated concentrated TBE vaccine. At the same time, the vaccine alone exerted a much less marked effect on the persisting TBE virus producing only a 2-fold reduction in the frequency of persistence without limitation of the zones of virus spread. In acute TBE in BALB/c mice, the effect of MP is observed irregularly. The marked protective effect of MP in TBE virus persistence in monkeys is not associated with stimulation of humoral immunity but is mediated by other immunological mechanisms requiring further study.

[Pathogenesis of persistent and chronic forms of tick-borne encephalitis (experimental study)]. / Patogenez persistentnoi i khronicheskoi form kleshchevogo éntsefalita (éksperimental'noe issledovanie).

A long-term experiment was conducted to study various aspects of the pathogenesis of persistent and chronic tick-borne encephalitis (TBE). Virological, serological, pathomorphological, electron microscopic and immunofluorescent techniques have been utilized in this study. Persistent TBE infection of Syrian hamsters examined over the period from 40 days to 2 years was characterized by the presence of virus-specific antigens in the organs and of specific antibodies in the blood serum. The persisting TBE virus was found to be predominantly localized in the central nervous system and spleen. Nerve cells underwent ultrastructural changes which were characteristic of flavivirus infection and related to the morphogenesis of viral particles. The authors have developed an experimental model of a primary progressive form of TBE with early and late manifestations of clinical symptoms of the disease.

Neurovirulence of the intertypic poliovirus recombinant v3/a1-25: characterization of strains isolated from the spinal cord of diseased monkeys and evaluation of the contribution of the 3' half of the genome.

A tsRNA- intertypic recombinant, v3/a1-25, which has the 5' and 3' halves of the genome derived from the neurovirulent type 3 poliovirus strain 452/62 3D and the attenuated type 1 poliovirus strain LSc-gr3, respectively, was previously shown to cause severe paralytic poliomyelitis after intracerebral inoculation of monkeys. To ascertain whether the illness was caused by the recombinant itself or by temperature-resistant trRNA+ mutants that might have arisen in the inoculated monkeys, five independent virus strains have been isolated from the spinal cord of the diseased animals. While two of these isolates exhibited RNA+ and RNA +/- phenotypes, respectively, the other three strains retained the parental RNA- character. Except for the RNA+ strain, the RNase T1 oligonucleotide maps of the genomes of all the isolates revealed only a minimal deviation from the parental pattern. These results were interpreted to mean that v3/a1-25 is intrinsically neurovirulent despite the presence of a tsRNA- mutation(s) in the 3' half of its genome. Nevertheless, this mutation, or other peculiarities of the 3' half of the recombinant genome, may somewhat alleviate the pathogenicity of the virus. This notion was inferred from the fact that, when used in a relatively small dose (about 10(3) p.f.u.), v3/a1-25 appeared to exhibit a lower level of neurovirulence compared to either the wild-type parent 452/62 3D, or a closely related intertypic recombinant having the genome 3' half derived from a neurovirulent trRNA +/- type 1 poliovirus strain. The problem of genetic determination of poliovirus neurovirulence and attenuation is briefly discussed.

Experimental encephalitis in monkeys caused by the Powassan virus.

We have carried out a comparative study of the experimental infection of monkeys with the P-40 strain of the Powassen virus, isolated in the Primor'e Territory of the USSR, and with the Canadian prototype LB strain. The Powassan virus was found to be pathogenic for Macaca rhesus. The clinical and pathomorphological picture of the experimental encephalitis was studied, and the full identity of the infection produced in the monkeys by the P-40 strain and the Canadian LB strain of the Powassan virus was demonstrated. On electron microscopic examination of the central nervous system the virus was detected in the neurons, glial cells, and intercellular spaces. The virions of the strains studied have identical morphological parameters, being 37-45 nm in diameter and of spherical shape. The data obtained indicated a marked neurotropism of the virus. They will contribute to the elucidation of the role of the virus in the infection pathology of humans, i.e., in the differentiation of encephalitis cases not associated etiologically with the virus of the spring-summer tickborne encephalitis.

[Variants of the host immune response to the tick-borne encephalitis virus]. / Varianty immunnogo otveta khoziaina na virus kleshchevogo éntsefalita.

Syrian hamsters intracerebrally infected with tick-borne encephalitis (TBE) virus were used to study the correlation between virulence of the strains, their serotype, form and course of the disease, average survival time (AST) of hamsters, features of CNS pathomorphology, and the changes of values of cell-mediated and humoral immunity. Thirty one strains of TBE virus isolated in Siberia and Far East were studied. Virulence of the strains ranged from 100% to 5%, AST from 5 to 237 days. Hamsters developed acute lethal encephalitis, subacute encephalitis, or asymptomatic infection. Most virulent strains produced early and extensive lesions in the brain stem. In encephalitis with subacute course pathomorphological changes in the CNS developed slower and the brain stem was less involved. Each variant of TBE course was associated with a certain pattern of host immune response formed as early as the first day of infection. Highly virulent strains inducing acute encephalitis and 100% lethality within 5-9 days produced immune response characterized by high antigen reactivity of lymphocytes by the level of rosette-forming cells, marked specific sensitization in splenocyte migration-inhibition test, moderate antibody-producing cell reaction, increased thymus weight index, low values of nonspecific resistance with low levels of antihemagglutinins and virus-neutralizing antibody by the end of the first week. Different immunologic condition was associated with asymptomatic infection and increase of AST to 100-237 days.

Persistence of tick-borne encephalitis virus in monkeys. VI. Pathomorphology of chronic infection in central nervous system.

Histological changes were studied in the central nervous system (CNS) of 58 Macaca rhesus monkeys, infected intracerebrally (i.c.) and subcutaneously (s.c.) with tick-borne encephalitis (TBE) virus. Subacute degenerative process in the CNS occurred in 7 monkeys on days 15-24 post-infection (p.i.), while chronic degenerative changes were observed in 5 monkeys examined on days 45, 90, 279, 383, 789 p.i. Pathological changes in the CNS of 11 monkeys correlated with the clinical picture of subacute and chronic encephalitis. Chronic lesions were observed in the CNS of 1 monkey with asymptotic infection. The pathomorphology of the CNS lesions was characterized by progressive development of focal changes of various duration.