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.     

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.     

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.     

Lipids and flaviviruses,present and future perspectives for the control of dengue,Zika, and West Nile viruses

Flaviviruses are emerging arthropod-borne pathogens that cause life-threatening diseases such as yellow fever, dengue, West Nile encephalitis, tick-borne encephalitis, Kyasanur Forest disease, tick-borne encephalitis, or Zika disease. This viral genus groups > 50 viral species of small enveloped plus strand RNA virus that are phylogenetically closely related to hepatitis C virus. Importantly, the flavivirus life cycle is intimately associated to host cell lipids. Along this line, flaviviruses rearrange intracellular membranes from the endoplasmic-reticulum of the infected cells to develop adequate platforms for viral replication and particle biogenesis. Moreover, flaviviruses dramatically orchestrate a profound reorganization of the host cell lipid metabolism to create a favorable environment for viral multiplication. Consistently, recent work has shown the importance of specific lipid classes in flavivirus infections. For instances, fatty acid synthesis is linked to viral replication, phosphatidylserine and phosphatidylethanolamine are involved on the entry of flaviviruses, sphingolipids (ceramide and sphingomyelin) play a key role on virus assembly and pathogenesis, and cholesterol is essential for innate immunity evasion in flavivirus-infected cells. Here, we revise the current knowledge on the interactions of the flaviviruses with the cellular lipid metabolism to identify potential targets for future antiviral development aimed to combat these relevant health-threatening pathogens.     

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.     

New Genetic Marker of Human Predisposition to Severe Forms of Tick-Borne Encephalitis

Molecular Biology - The causative agent of tick-borne encephalitis (a neurotropic RNA virus from the Flavivirus genus) can cause both severe paralytic forms of the disease (meningoencephalitis,...     

Viral genome integration with nuclear DNA in chronic viral infection]

A possibility of the integration of tick-borne encephalitis virus RNA in the host cell genome is studied under the condition of long-term chronic infection of HEp-2 cells. Molecular hybridization of viral RNA, labelled with 3H-uridine, and of DNA from intact and chronically infected cells was performed in 50% formamide with 0,4 M NaCl with subsequent investigation of the hybridization product by means of equilibrium centrifugation in CsSO4. 0,3 and 0,5 mg of DNA were used in hybridization experiments. It is shown that RNA obviously forms hybrids with DNA from chronically infected cells, whose density varied from 1,55 to 1,46 g/ml, depending on the proportion of RNA and DNA in the hybrids. When the amount of DNA increased, the number of DNA-RNA hybrids increased as well. It is concluded that in nuclear DNA of cells chronically infected with tick-borne encephalitis virus, there are DNA sites, homologous to viral RNA, which are absent in intact cells.     

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.     

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。     

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.     

Genetic Variability of Tick-Borne Encephalitis Virus Genome 5'-UTR from Northern Eurasia

Molecular Biology - This paper reports the analysis of the nucleotide sequences of the 5'-untranslated region (5'-UTR) of tick-borne encephalitis virus (TBEV) genomic RNA isolated from 39...     

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.     

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.     

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%).     

Role of sialic acids in the immune response formation in tick-borne encephalitis

The content of individual forms of sialic acids and total sialic acids in the lymphocytes of tick-borne encephalitis patients has been studied. The level of sialic acids has been found to depend on the clinical form of the disease and on the content of specific IgM and IgG to tick-borne encephalitis virus. Similar dependence has been established with respect to total sialic acids in lymphocytes.     

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.     

Mechanistic insight into the RNA-stimulated ATPase activity of tick-borne encephalitis virus helicase

The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase.