Synthetic fragments of the NS1 protein of the tick-borne encephalitis virus exhibiting a protective effect

Potentially immunoactive regions of the NS1 nonstructural protein of the tick-borne encephalitis virus that can stimulate the antibody formation in vivo and protect animals from this disease were chosen on the basis of theoretical calculations. Eleven 16- to 27-aa peptides containing the chosen regions were synthesized. The ability of the free peptides (without any high-molecular-mass carrier) to stimulate the production of antipeptide antibodies in mice of three lines and ensure the formation of protective immunity was studied. Most of these peptides were shown to exhibit the immunogenic activity in a free state. Five fragments that can protect mice from the infection by a lethal dose of tick-borne encephalitis virus were found.     

Synthesis,Immunogenicity, and Antigenicity of the Glycoprotein E Fragments of the Tick-Borne Encephalitis Virus

Six peptide fragments of the envelope protein E of the tick-borne encephalitis virus involving the predicted T-helper epitopes were synthesized. Their ability to induce antibodies without conjugation with any high-molecular-mass carrier was studied in mice of three lines. Five of six synthesized peptides exhibited immunogenic properties, which differed in dependence on the haplotype of immunized mice. The peptide binding to the antiviral antibodies was studied, and two peptides were revealed that demonstrated a high ability to recognize the viral antibodies in the horse and human sera. These peptides are promising for the development of diagnostic agents for the tick-borne encephalitis virus.     

Synthesis, immunogenicity and antigenic characteristics of the glycoprotein E fragments from the tick-borne encephalitis virus]

Six peptide fragments of the envelope protein E of the tick-borne encephalitis virus involving the predicted T-helper epitopes were synthesized. Their ability to induce antibodies without conjugation with any high-molecular-mass carrier was studied in mice of three lines. Five of six synthesized peptides exhibited immunogenic properties, which differed in dependence on the haplotype of immunized mice. The peptide binding to the antiviral antibodies was studied, and two peptides were revealed that demonstrated a high ability to recognize the viral antibodies in the horse and human sera. These peptides are promising for the development of diagnostic agents for the tick-borne encephalitis virus.     

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.     

Induction of antibodies to particular sites of the α7 subunit of the nicotinic acetylcholine receptor in mice of different lines

Five synthetic fragments of the N-terminal domain of the α7 subunit of the human nicotinic acetylcholine receptor (α7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2^d, H-2^b, and H-2^k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2^d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (α7 nAChRα. The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the α7 subunit: 1–23, 98–106, 159–168, and 173–188 (or 179–188).     

Chimeric T helper-B cell peptides induce protective response against Japanese encephalitis virus in mice

Virus neutralizing MAb binding and T helper cell stimulating peptide epitopes from structural and non-structural proteins of Japanese encephalitis virus were delineated. It was observed that priming by T helper peptides potentiated neutralizing antibody response against JE virus. Immunization with chimeric T helper - B cell peptides could thus protect mice from lethal challenge with JE virus.     

Study of the antigenic structure of tick-borne encephalitis virus using synthetic peptides]

A number of peptides, fragments of the envelope protein E of the tick-borne encephalitis virus (Sofjin strain), were synthesized. Their binding to the polyclonal antiserum to protein E was studied. Rats were immunized with both the free peptides and their KLH-conjugates, and the resulting antisera were tested for their reactivity toward protein E and for their neutralizing activity toward the virus in cell culture. The only peptide corresponding to the 98-113 sequence of protein E was shown to be bound by the protein E antiserum in EIA. Two-fold immunization of rats with KLH-conjugates of the peptides corresponding to the 98-113, 130-143, and 394-403 sequences of protein E resulted in antipeptide antibodies capable of binding the native protein E, and the antibodies to the 98-113 and 394-403 peptides were capable of neutralizing the virus.     

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.     

Expression of the NS1 gene of tick-borne encephalitis virus in gram-negative bacteria from the mouse nasopharynx

Bacteria were isolated from the nasopharynx of BALB/c mice and electroporated with pUR290(NS1)2 containing two copies of tick-borne encephalitis virus (TBEV) strain Sofjin NS1 under the control of the lac promoter. The plasmid persisted in transformants for at least ten passages. The NS1 gene expression was detected in Gram-negative enterobacteria via immunoblotting with monoclonal antibodies against TBEV nonstructural glycoprotein NS1. Recombinant NS1 was detected in bacterial cells and in the culture medium. Intranasal immunization with recombinant bacteria activated production of antibodies against NS1 in serum of BALB/c mice. The humoral immune response to NS1 failed to protect immunized mice from a TBEV challenge.     

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.     

Geographical variants in the immunological structure of the population in combined foci of tick-borne encephalitis and endemic rickettsiosis in the European part of the USSR

The zonal study of the immunological structure of the population revealed an increase in the number of infective agents with which the population comes into contact in the southern regions. In the medial boreal forest zone contacts of low intensity only with tick-borne encephalitis virus were detected (the immune stratum was found to be 5.5%, 2,178 persons were examined); in the southern boreal forest zone the foci of tick-borne encephalitis actively manifested (the immune stratum reached 38.9%, 2,854 persons were examined), and Q fever was constantly detected (1.6%). In the forest-steppe zone the population was found to have contacts with the causative agent of tick-borne encephalitis (the immune stratum was found to be 13.3%, 2,056 persons were examined), Q fever (1.2%) and tick-borne rickettsiosis (1.1%).     

Induction of antibodies to particular sites of the alpha7 subunit of the nicotinic acetylcholine receptor in mice of different lines

Five synthetic fragments of the N-terminal domain of the alpha7 subunit of the human nicotinic acetylcholine receptor (alpha7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2d, H-2b, and H-2k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (alpha7 nAChRalpha). The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the alpha7 subunit: 1-23, 98-106, 159-168, and 173-188 (or 179-188).     

MicroRNA targeting of neurotropic flavivirus: effective control of virus escape and reversion to neurovirulent phenotype

Neurotropic flaviviruses can efficiently replicate in the developing and mature central nervous systems (CNS) of mice causing lethal encephalitis. Insertion of a single copy of a target for brain-expressed microRNAs (miRNAs) in the 3' noncoding region (3'NCR) of the flavivirus genome (chimeric tick-borne encephalitis virus/dengue virus) abolished virus neurovirulence in the mature mouse CNS. However, in the developing CNS of highly permissive suckling mice, the miRNA-targeted viruses can revert to a neurovirulent phenotype by accumulating deletions or mutations within the miRNA target sequence. Virus escape from miRNA-mediated suppression in the developing CNS was markedly diminished by increasing the number of miRNA target sites and by extending the distance between these sites in the virus genome. Insertion of multiple miRNA targets into the 3'NCR altered virus neuroinvasiveness, decreased neurovirulence and neuroinflammatory responses, and prevented neurodegeneration without loss of immunogenicity. Although the onset of encephalitis was delayed, a small number of suckling mice still succumbed to lethal intracerebral infection with the miRNA-targeted viruses. Sequence analysis of brain isolates from moribund mice revealed that the viruses escaped from miRNA-mediated suppression exclusively through the deletion of miRNA targets and viral genome sequence located between the two miRNA targets separated by the greatest distance. These findings offer a general strategy to control the reversion of virus to a virulent phenotype: a simultaneous miRNA targeting of the viral genome at many different functionally important regions could prevent virus escape from miRNA-based attenuation, since a deletion of the targeted genomic sequences located between the inserted miRNA binding sites would be lethal for the virus.     

Amino acid sequence of various tryptic peptides of the envelope protein of the tick-borne encephalitis virus

Protein E of the tick-borne encephalitis virus (TBEV) (strain Sofin) was treated with purified trypsin in 1% Triton X-100, and the peptides thus obtained were separated by micro-column reversed-phase chromatography. Four of the purified peptides were sequenced, their structures being in accordance with the nucleotide sequence of the viral protein E gene. Amino acid sequences of peptides deduced from the cDNA primary structure are: Ser-Val-Leu-Ile-Pro-Ser-His-Ala-Gln-Gly-Asp-Leu-Thr-Gly-Arg (N-terminal peptide of protein E); Thr-Glu-Gly-Ala-Gln-Asn-Trp-Asn-Ala-Glu-Arg: Trp-Leu-Glu-Gly-Asp-Ser-Leu-Arg; Leu-Val-Glu-Phe-Gly-Ala-Pro-His-Ala-Val-Lys.     

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.     

Expression of the Tick-borne Encephalitis Virus NS1 Gene in Gram-Negative Bacteria from the Mouse Nasopharynx

Bacteria were isolated from the nasopharynx of BALB/c mice and electroporated with pUR290(NS1)₂ containing two copies of tick-borne encephalitis virus (TBEV, strain Sofjin) NS1 under the control of the lac promoter. The plasmid persisted in transformants for at least ten passages. The NS1 gene expression was detected in Gram-negative enterobacteria by immunoblotting with monoclonal antibodies against the TBEV nonstructural glycoprotein NS1. Recombinant NS1 was detected in bacterial cells and in the culture medium. Intranasal immunization with recombinant bacteria caused antibodies against NS1 to appear in the serum of BALB/c mice. However, the humoral immune response to NS1 failed to protect mice from a TBEV challenge.     

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.     

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.     

Mapping of a conformational epitope shared between E1 and E2 on the serum-derived human hepatitis C virus envelope

Monoclonal antibody D32.10 produced by immunizing mice with a hepatitis C virus (HCV)-enriched pellet obtained from plasmapheresis of a chronically HCV1b-infected patient binds HCV particles derived from serum of different HCV1a- and HCV1b-infected patients. Moreover, this monoclonal has been shown to recognize both HCV envelope proteins E1 and E2. In an attempt to provide novel insight into the membrane topology of HCV envelope glycoproteins E1 and E2, we localized the epitope recognized by D32.10 on the E1 and/or E2 sequence using Ph.D.-12 phage display peptide library technology. Mimotopes selected from the phage display dodecapeptide library by D32.10 shared partial similarities with 297RHWTTQGCNC306 of the HCV E1 glycoprotein and with both 613YRLWHYPCT621 and 480PDQRPYCWHYPPKPC494 of the HCV E2 glycoprotein. Immunoreactivity of D32.10 with overlapping peptides corresponding to these three HCV regions confirmed these localizations and suggested that the three regions identified are likely closely juxtaposed on the surface of serum-derived particles as predicted by the secondary model structure of HCV E2 derived from the tick-borne encephalitis virus E protein. This assertion was supported by the detection of specific antibodies directed against these three E1E2 regions in sera from HCV-infected patients.     

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.