Immune complex formation during the passive immunization of mice infected with the tick-borne encephalitis virus

The results of investigations, indicating that the development of the infectious process in experimental encephalitis is accompanied by the formation of immune complexes circulating in the blood and localized in the brain tissue, are presented. The intravenous injections of a homologous serum preparation into intact animals induces the appearance of a rather low level of circulating immune complexes, which precedes the elimination of antibodies. The intravenous injection of a specific serum preparation two days after the infection of the animals with tick-borne encephalitis virus is accompanied by the formation of immune complexes; the course of infection is not aggravated.     

Dry erythrocytic diagnostic agent for the determination of antiglobulins]

Dry erythrocytic diagnostic agents were obtained under experimental conditions for determination of antiglobulins forming in the organism of man and animals under the effect of serum preparations from the blood of horses and homologoum immunoglobulins. A study was made of the sera of 100 patients with tick-borne encephalitis treated with heterologous and homologous immunoglobulins of directed action; in response to the administration of horse gamma-globulin antiglobulins (in titres below 1 : 10000) appeared in the serum; they circulated in the blood for long periods and inhibited the accumulation of hormonal antibodies to the causative agent; in the majority of cases a high level of antiglobulins to the foreign protein correlated with the presence of remote side-reactions of the serum sickness type. In patients treated with immunoglobulin of human origin antiglobulins were determined in low titres, disappeared from the blood in 15--20 days and did not hinder the accumulation of antihemmagglutinins to the tick-borne encephalitis virus.     

Assay of interferon and viral antibodies in the cerebrospinal fluid in clinical neurology and psychiatry

Cerebrospinal fluids (CSF) of 245 neurological and 194 psychiatric patients were tested for viral antibodies and interferon. Complement dependent neutralizing antibodies to Herpesvirus hominis 1 were found in the CSF of patients with encephalitis (50.6%), meningitis (35.4%), lesions of peripheral nerves (36.9%), sclerosis multiplex (41.2%), schizophrenia (31.9%), senile dementia (51.4%), mental retardation (11.1%), ethylism (43.5%). Neutralizing antibodies to tick-borne encephalitis virus were found in the CSF of 38% patients with encephalitis, in 14% meningitis, 11% lesions of peripheral nerves and also in 5.6--11.8% of psychiatric patients. In encephalitis, meningitis and in lesions of peripheral nerves were found in the CSF frequently plaque neutralizing antobidies to the tick-borne orbivirus Lipovník, complement-fixing antibodies to lymphocytic choriomeningitis virus and hemagglutination inhibiting antibodies to measles virus. In multiple sclerosis were detected CSF antibodies to measles virus (44%), Herpesvirus hominis 1 (41.2%) and Lipovník virus (52.6%). In neurological patients were observed CSF antibodies simultaneously to two or three viruses in 16.7 to 40.6%, while in psychiatric patients in zero to 4.6%. CSF interferon was found in psychiatric patients with an equal or even higher incidence (33.7 to 57.1%) than in the neurological patients (29.6--38.6%, in multiple sclerosis only 16.7%). Non-interferon virus inhibitors were excluded. The evaluation of the ratio of serum and CSF titers of viral antibodies and of interferon indicated local synthesis of both in the central nervous system -- with the exception of antibodies to Herpesvirus hominis 1 in CSF of some patients with very high titres in serum and probable lesions of the blood brain barrier.     

Relationships between the content of circulating immune complexes, the concentration of serum immunoglobulins and the titers of specific antibodies in patients with tick-borne encephalitis

In the blood serum of tick-borne encephalitis (TBE) patients the detection rate and concentration of circulating immune complexes, as well as the content of serum IgA, IgM and IgG, were evaluated. The formation of immune complexes was found to depend on IgM and IgG specific antibodies to TBE virus, the period of the disease and the clinical form of virus infection.     

Biological properties of strains of tick-borne encephalitis virus isolated in the natural foci of the eastern part of the Russian plain

Strains of tick-borne encephalitis virus isolated in the natural foci of infection in the eastern part of the Russian Plain (the Kirov region) were examined for their biological properties. The strains examined were 69 strains isolated from ticks Ixodes persulcatus, 62 strains obtained from patients with the clinically manifest form of tick-borne encephalitis and 56 strains isolated from the blood of patients with the inapparent form of infection. Comparative studies on laboratory animals (albino mice, golden hamsters, suckling guinea pigs and other mammals) as well as comparative serologic studies provided evidence which suggested that all virus isolates from the Kirov region were antigenically identical with the strain "Sofin" isolated in the Far East and represent thus a single causative agent of the tick-borne encephalitis virus infection. This strain of virus is supposed to exist in two variants, in dependence on ecological conditions: one of these variants is the eastern variant (strain Sofin and strains from the Kirov region) and the other one is the western variant of tick-borne encephalitis virus.     

Use of protein A in the immunosorbent analysis of antibodies to the tick-borne encephalitis virus

The results of the studies made with a view to developing the method for the determination of specific antibodies to the antigen of tick-borne encephalitis virus in human blood serum and liquor are presented. The method is based on the capacity of Staphylococcus aureus protein A to bind with Fc-region of immunoglobulins, which makes it possible to use this protein as the "second" system of antibodies. The conditions for the sorption of the antigen on polystyrene test tubes and for binding 125I-or horse radish peroxidase-labeled protein A preparations with antibodies have been determined, and the method has been approved in tests made on sera and liquor obtained from donors and tick-borne encephalitis patients.     

Antibody-dependent harmful effect of non-immune spleen cells in acute experimental tick-borne encephalitis in mice

The injection of nonprotective dilutions of immune serum and nonimmune spleen cells into mice infected with tick-borne encephalitis virus induced a sharply pronounced immunopathological effect: the mean survival time of the recipients decreased by 3.6 days in comparison with the control animals. This effect was not linked with the increased replication of the virus in the brain. The antibody-dependent damaging action of spleen cells could be reproduced by using the cells of both syngeneic and allogeneic donors. This phenomenon developed only in those cases when antibodies to the infective agent under study were used. The combination of immune serum to Japanese encephalitis virus and nonimmune spleen cells produced no damaging effect. The hypothesis stating that the antibody-dependent damaging action of nonimmune spleen cells arises from the antibody-dependent cytotoxic action of immunocompetent cells on the infected cells of the central nervous system is discussed.     

Arboviruses in birds captured in Slovakia.

Virus neutralizing (VN) antibodies against Sindbis, tick-borne encephalitis (TBE), West Nile (WN), Tahyna and Calovo viruses were found in birds captured in Slovakia. In parallel, Sindbis, TBE and WN viruses were isolated from the blood, brain and liver of migrating birds.     

Insertion of microRNA targets into the flavivirus genome alters its highly neurovirulent phenotype

Flaviviruses such as West Nile, Japanese encephalitis, and tick-borne encephalitis (TBEV) viruses are important neurotropic human pathogens, causing a devastating and often fatal neuroinfection. Here, we demonstrate that incorporation into the viral genome of a target sequence for cellular microRNAs expressed in the central nervous system (CNS) enables alteration of the neurovirulence of the virus and control of the neuropathogenesis of flavivirus infection. As a model virus for this type of modification, we used a neurovirulent chimeric tick-borne encephalitis/dengue virus (TBEV/DEN4) that contained the structural protein genes of a highly pathogenic TBEV. The inclusion of just a single target copy for a brain tissue-expressed mir-9, mir-124a, mir-128a, mir-218, or let-7c microRNA into the TBEV/DEN4 genome was sufficient to prevent the development of otherwise lethal encephalitis in mice infected intracerebrally with a large dose of virus. Viruses bearing a complementary target for mir-9 or mir-124a were highly restricted in replication in primary neuronal cells, had limited access into the CNS of immunodeficient mice, and retained the ability to induce a strong humoral immune response in monkeys. This work suggests that microRNA targeting to control flavivirus tissue tropism and pathogenesis might represent a rational approach for virus attenuation and vaccine development.     

Use of the specific rosette formation test in tick-borne encephalitis for the purpose of laboratory diagnosis

In the study of blood samples collected from patients with tick-borne encephalitis, the modified antigen-specific rosette formation test with erythrocytes, loaded with tick-borne encephalitis virus antigen via the specific immunoglobulin, has been used. The number of rosette-forming cells has been the highest during the acute period of the disease. The use of this test has confirmed the clinical diagnosis of the disease, together with hemagglutination inhibition serving as the main diagnostic test, in 35% of cases. The results of this study make it possible to recommend the antigen-specific rosette formation test for the early diagnosis of tick-borne encephalitis.     

Immunoenzyme analysis with visual demonstration for detecting antibodies to the tick-borne encephalitis virus

Good prospects for the use of enzyme immunoassay (EIA) with the simple visual indication of results have been shown with the detection of specific antibodies to tick-borne encephalitis virus in blood serum used as an example. When compared with such highly sensitive method as radioimmunoassay, visual EIA is inferior in both sensitivity and selectivity, but its special advantage is that it requires no instrument for evaluating the result.     

蜱传脑炎病毒非结构蛋白NS2B-NS3与NS5的研究进展

蜱传脑炎病毒是引起严重的中枢神经系统疾病蜱传脑炎的病原体,每年在欧洲、俄罗斯远东地区、日本和中国北部报道的蜱传脑炎病例数约为10000-12000例,且在我国和多个欧洲国家的发病率逐渐增高,正成为人类健康的潜在危害。主动免疫是预防蜱传脑炎的有效措施,包括我国在内的多个国家已研制出安全性较高的疫苗,但在我国流行省份的疫苗接种较为有限,特异性抗病毒药物的研发或许是治疗蜱传脑炎病毒感染的研究方向之一。蜱传脑炎病毒非结构蛋白NS2B-NS3与NS5因为在病毒基因组复制、加帽和宿主免疫调节中的重要作用,成为关键的抗病毒药物研发靶点。本文综述了蜱传脑炎病毒非结构蛋白NS2B-NS3与NS5的三维结构和抑制剂研发工作,为深入探究该病毒感染的分子机制和抗病毒药物研发提供参考。     

SiRNA Inhibits Replication of Langat Virus, a Member of the Tick-Borne Encephalitis Virus Complex in Organotypic Rat Brain Slices

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000–3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3′ untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.     

Results of using the ELISA method in studying tick-borne encephalitis and rabies

The results of using the indirect variant of ELISA for the study of serum samples from humans and white mice for the presence of antibodies to tick-borne encephalitis virus and rabies virus are presented. The high sensitivity and specificity of this method have been confirmed.     

Pre- and postexposure protection by passive immunoglobulin but no enhancement of infection with a flavivirus in a mouse model.

Antibody-dependent enhancement of flavivirus infection, which except for dengue virus is without clear proof in vivo, is still under debate. Recently, postexposure immunoglobulin prophylaxis against tick-borne encephalitis virus, a flavivirus, was claimed to possibly have worsened the outcome of infection due to antibody-dependent enhancement. In the present study, antibody-dependent enhancement and pre- or postexposure protection by passive administration of tick-borne encephalitis virus immunoglobulin were evaluated in a mouse model. Preexposure treatment with homologous murine or heterologous human immunoglobulin provided complete protection against lethal challenge with tick-borne encephalitis virus. For postexposure treatment with antibody, the degree of protection correlated with the amount of immunoglobulin administered and was inversely related to the time interval between infection and treatment. Indications of enhancement of infection would have been increased lethality or reduced mean survival time, but neither was observed under the conditions used in our experiments despite the broad range of immunoglobulin and virus challenge doses applied. In contrast to these in vivo results, antibody-dependent enhancement of tick-borne encephalitis virus infection of murine peritoneal macrophages was readily demonstrable in vitro. Thus, antibody-dependent enhancement of viral infection in vitro does not necessarily predict enhancement in vivo.     

Correlation of TBE Incidence with Red Deer and Roe Deer Abundance in Slovenia

Tick-borne encephalitis (TBE) is a virus infection which sometimes causes human disease. The TBE virus is found in ticks and certain vertebrate tick hosts in restricted endemic localities termed TBE foci. The formation of natural foci is a combination of several factors: the vectors, a suitable and numerous enough number of hosts and in a habitat with suitable vegetation and climate. The present study investigated the influence of deer on the incidence of tick-borne encephalitis. We were able to obtain data from deer culls. Using this data, the abundance of deer was estimated and temporal and spatial analysis was performed. The abundance of deer has increased in the past decades, as well as the incidence of tick-borne encephalitis. Temporal analysis confirmed a correlation between red deer abundance and tick-borne encephalitis occurrence. Additionally, spatial analysis established, that in areas with high incidence of tick-borne encephalitis red deer density is higher, compared to areas with no or few human cases of tick-borne encephalitis. However, such correlation could not be confirmed between roe deer density and the incidence of tick-borne encephalitis. This is presumably due to roe deer density being above a certain threshold so that availability of tick reproduction hosts has no apparent effect on ticks'' host finding and consequently may not be possible to correlate with incidence of human TBE.     

Newly recognized mosquito-associated viruses in mainland China, in the last two decades

There are four principal arboviruses in mainland China. Two kinds of them are mosquito-borne viruses, namely Japanese encephalitis virus and dengue virus, which lead to Japanese encephalitis, and dengue fever/dengue hemorrhagic fever respectively; the other two are tick-borne viruses, namely tick-borne encephalitis virus and Crimean-Congo hemorrhagic fever virus (also known as Xinjiang hemorrhagic fever virus), which contribute to tick-borne encephalitis and Xinjiang hemorrhagic fever respectively. With exception of these four main arboviruses, many other mosquito-associated viruses have been isolated and identified in recent years. These newly isolated and identified mosquito-associated viruses are probably responsible for human and animal infections and diseases. The purpose of this review is to describe the newly isolated mosquito-associated viruses in mainland China which belong to five viral families, including their virological properties, phylogenetic relationships, serological evidence, as well as to appeal the public health concentration worldwide.     

Coding nucleotide sequences of tick-borne encephalitis virus strains isolated from human blood without clinical symptoms of infection

Genomes of four tick-borne encephalitis virus strains, isolated from the blood of the individuals after tick bites and causing no clinical symptoms of infection, were characterized. Analysis of translated polypeptides revealed 21 amino acid positions typical of this group of strains and distinguishing them from the other tick-borne encephalitis virus strains of Far Eastern subtype examined earlier. Only three mutations led to substantial amino acid changes, which probably could affect the infection process severity. It is suggested that two associated mutations, deletion of amino acid 111 in the capsid protein C and substitution (Ser1534 → Phe) in the NS3 protein influence strictly coordinated polyprotein processing, disturbing correct arrangement of viral particles. This process can result in the development of defect viral particles, containing no RNA. Mutation (Ser917 → Gly) in nonstructural protein NS1 results in the substitution of hydrophilic amino acid, specific to highly virulent strains, by the hydrophobic one. This could influence the effectiveness of viral replication complex, thereby affecting the infectivity of tick-borne encephalitis virus strains.     

Sexual transmission of the tick-borne encephalitis virus in ixodid ticks (Ixodidae)

Sexual transmission of tick-borne encephalitis virus from infected ixodid males to noninfected females is shown: in Ixodes persulcatus in 50% (6 of 12) and in Hyalomma anatolicum in 6.2% (1 of 16). The sexual transmission of tick-borne encephalitis virus is shown to provide a transmissible transfer of the virus into eggs in ixodid ticks. Electron microscope studies of the sexual system of ixodid males infected with the virus have revealed numerous morphologically mature virus particles in lumens of endoplasmic reticulum, in vacuoles of Golgi complex of spermatocytes and in association with tubular elements of spermatids.