Study of Viral Coinfection of the Ixodes persulcatus Ticks Feeding on Humans in a Natural Focus of the South of the Far East.

The phenomenon of pathogen co-infection detected in a half-fed Ixodes persulcatus tick taken from a human in the south of the Far East was studied. Research was carried out on PEK, Vero, and Vero-E6 cell lines, outbred mice, and chicken embryos using ELISA, PCR, IMFA, plaque formation, and electron microscopy. The tick contained an antigen and a genetic marker of the tick-borne encephalitis virus (TBEV). The patient had post-vaccination antibodies in a titer of 1:200, as a result of which, obviously, an antibody-dependent elimination of TBEV occurred. The tick-borne co-isolate also contained an unknown pathogen (Kiparis-144 virus), which, in our opinion, was a trigger for the activation of chronic infection in suckling white mice. In the laboratory co-isolate, ectromelia virus was present, as evidenced by paw edema during the intradermal infection of mice, characteristic rashes on the chorioallantoic envelope of chicken embryos, and typical plaques on Vero-E6. The Kiparis-144 virus was not pathogenic for white mice and chicken embryos, but it successfully multiplied in the PEK, Vero, and Vero-E6 lines. Viral co-infection was confirmed by electron microscopy. Passaging on mice contributed to an increase in the virulence of the co-isolate, whose titer increased by 10,000 times by the fifth passage, which poses an epidemiological danger.

Antiviral and Antioxidant Properties of Echinochrome A.

The aim of this study was to examine the in vitro antioxidant and antiviral activities of echinochrome A and echinochrome-based antioxidant composition against tick-borne encephalitis virus (TBEV) and herpes simplex virus type 1 (HSV-1). The antioxidant composition, which is a mixture of echinochrome A, ascorbic acid, and α-tocopherol (5:5:1), showed higher antioxidant and antiviral effects than echinochrome A. We suppose that echinochrome A and its composition can both directly affect virus particles and indirectly enhance antioxidant defense mechanisms in the hosting cell. The obtained results allow considering the echinochrome A and the composition of antioxidants on its basis as the promising agents with the both antioxidant and antiviral activities.

Immunogenicity and Protective Activity of a Chimeric Protein Based on the Domain III of the Tick-Borne Encephalitis Virus E Protein and the OmpF Porin of Yersinia pseudotuberculosis Incorporated into the TI-Complex.

Tick-borne encephalitis (TBE) is a widespread, dangerous infection. Unfortunately, all attempts to create safe anti-TBE subunit vaccines are still unsuccessful due to their low immunogenicity. The goal of the present work was to investigate the immunogenicity of a recombinant chimeric protein created by the fusion of the EIII protein, comprising domain III and a stem region of the tick-borne encephalitis virus (TBEV) E protein, and the OmpF porin of Yersinia pseudotuberculosis (OmpF-EIII). Adjuvanted antigen delivery systems, the tubular immunostimulating complexes (TI-complexes) based on the monogalactosyldiacylglycerol from different marine macrophytes, were used to enhance the immunogenicity of OmpF-EIII. Also, the chimeric protein incorporated into the most effective TI-complex was used to study its protective activity. The content of anti-OmpF-EIII antibodies was estimated in mice blood serum by enzyme-linked immunosorbent assay (ELISA). To study protective activity, previously immunized mice were infected with TBEV strain Dal'negorsk (GenBank ID: FJ402886). The animal survival was monitored daily for 21 days. OmpF-EIII incorporated into the TI-complexes induced about a 30⁻60- and 5⁻10-fold increase in the production of anti-OmpF-EIII and anti-EIII antibodies, respectively, in comparison with the effect of an individual OmpF-EIII. The most effective vaccine construction provided 60% protection. Despite the dramatic effect on the specific antibody titer, the studied TI-complex did not provide a statistically significant increase in the protection of OmpF-EIII protein. However, our results provide the basis of the future search for approaches to design and optimize the anti-TBEV vaccine based on the OmpF-EIII protein.

The rearrangement of motif F in the flavivirus RNA-directed RNA polymerase.

In the flavivirus genus, the non-structural protein NS5 plays a central role in RNA viral replication and constitutes a major target for drug discovery. One of the prime challenges in the study of NS5 protein is to investigate the interplay between the two protein domains, namely, the RNA-dependent RNA polymerase (RdRp) domain and the methyltransferase (MTase) domain. These investigations could clarify the multiple roles of NS5 protein in the virus life cycle. Here we present the results of sequence analyses and structural bioinformatics studies of NS5 protein, which suggest that the conserved motif F in the NS5 protein could act as a lock which controls the rearrangement of the domains and as a switch in the protein enzymatic activity.

Characteristics of far eastern strains of tick-borne encephalitis virus.

A comparative study of biological, molecular and genetic characteristics of a collection of ten strains of tick-borne encephalitis virus (TBEV) isolated in Primorsky Krai before 1960 and stored in a lyophilized state for a prolonged period (over 65 years) is presented. The collection includes the Sofjin strain isolated from the brain of a fatal case in Primorsky Krai in 1937 and transferred to the Scientific Research Institute of Epidemiology and Microbiology (Vladivostok) in 1953. All lyophilized viral strains demonstrated great preservation and high infectious activity in the model of 2-day-old non-inbred mice. Whole-genome sequencing showed that all strains belong to the Far East TBEV subtype, comprising three clusters of Sofjin-, Oshima- and Senzhang-like strains. We show that SofjinPYB, Sofjin (Vector) and Sofjin-HO strains form a separate branch of the phylogenetic tree and are closely related to Khabarovsk-Obor-4, but not to the original Sofjin strain. The Sofjin-1953, Sofijin-Chumakov, SofjinKSY and SofjinCDC strains are genetically close to each other and can be used as reference strains for comparative analysis of the tick-borne encephalitis virus population.

Molecular Mechanisms of Interaction Between Human Immune Cells and Far Eastern Tick-Borne Encephalitis Virus Strains.

Although studies have established that immune mechanisms are important in controlling tick-borne encephalitis virus (TBEV) infection, the interactions of different TBEV strains with cells of innate and adaptive immunity are not well understood. In this study, the ability of two Far Eastern subtype TBEV strains (Dal'negorsk and Primorye-183) with various degrees of pathogenicity for humans to modulate the expression of membrane molecules differently on human immune cells were investigated using a whole-blood flow cytometry-based assay. The whole-blood samples (from 10 healthy donors) were infected with TBEV strains and analyzed for the virus binding to the blood cells, as well as expression of adhesion (CD11b and ICAM-1) and activation (CD69, CD25, CD95) molecules on the surfaces of monocytes, granulocytes, natural killer (NK) cells, and T-lymphocytes (CD4+, CD8+) at selected times (3, 6, and 24 h post-infection). It was found that the highly pathogenic Dal'negorsk strain penetrated rapidly and was actively replicated in the blood cells, inducing downregulation of CD11b, ICAM-1, and CD69 on monocytes and a significant decrease of NK cells expressing CD69, CD25, CD95, and CD8 T-lymphocytes expressing CD69 compared with the mock-infected cells. The nonpathogenic Primorye-183 strain penetrated slowly and was replicated in the blood cells, but caused a significant increase in the adhesion and activation of molecule expression to trigger innate defense mechanisms and enable the rapid elimination of the virus from the organism. Thus, TBEV-induced activation or suppression of adhesion and activation receptors expression form an essential part of fundamental virus properties, that is, virulence and pathogenicity.

Louping ill virus (LIV) in the Far East.

This study focused on finding, culturing, and identifying the biological and genetic characteristics of three louping ill virus (LIV) strains in the south of the Russian Far East. The Primorye-155-77 and Primorye-20-79 virus strains were isolated from Ixodes persulcatus ticks, and the Primorye-185-91 strain was isolated from the blood of a person after a tick bite. According to the hemagglutination and neutralization tests, Primorye-155-77, Primorye-20-79 and Primorye-185-91 had weak reactivity with antibodies in an antiserum against tick-borne encephalitis virus. In Primorye-155-77 and Primorye-20-79, the sequences of the 5' ends of the 2456-nucleotide-long viral RNA including the 5' untranslated region (UTR) and genes of the capsid protein, prM protein and envelope E protein were determined. The complete genome sequence of Primorye-185-91 was determined. The E protein gene of the Negishi strain differed from those of three analyzed strains, as there were mutations resulting in the replacement of three amino acids: Ala163Thr, Asp193Asn and Ala313Thr. The homology of Primorye-185-91 to LIV 369/T2 was 97.57 %, and to the Penrith strain, it was 98.36 %. Phylogenetic analysis showed that Primorye-155-77, Primorye-20-79 and Primorye-185-91 are related to LI/A and LI/K strains isolated in England and Scotland and to the Negishi strain; these strains have a common progenitor. Negishi-like strains were represented by one subtype of louping ill virus, i.e. the British subtype (LIV-Brit). The possibility is discussed of a single introduction of the virus to the Far Eastern region (Japan and Primorsky Krai) from a single natural locus more than 50 years ago.

The nature of replication of tick-borne encephalitis virus strains isolated from residents of the Russian Far East with inapparent and clinical forms of infection.

We describe the biological properties and molecular characteristics of complete genomes of 33 tick-borne encephalitis virus (TBEV) strains that induced different forms of infection, from inapparent to severe focal ones resulting in fatal outcome. Hemagglutinating activity of Oshima-like strains was higher at pH 5.8, while activity of Sofjin- and Senhzang-like strains were higher at pH 6.2 and 6.8, respectively. We determined susceptibility of porcine kidney (PK) cell cultures to these TBEV strains by cytopathic effect (CPE), plaque formation, and size of plaques. The clinical TBEV strains had higher virus titers both in tissue culture infectious dose 50(TCID50) and in plaque-forming unit (PFU) titers and larger plaques than the inapparent strains. A comparison of virus multiplication kinetics by PFU in culture fluid with kinetics of ELISA antigen and hemagglutinin accumulation suggested a different mechanism of interaction between these virus strains and PK cells at the initial stage of cell infection.

The relationship between the structure of the tick-borne encephalitis virus strains and their pathogenic properties.

Tick-borne encephalitis virus (TBEV) is transmitted to vertebrates by taiga or forest ticks through bites, inducing disease of variable severity. The reasons underlying these differences in the severity of the disease are unknown. In order to identify genetic factors affecting the pathogenicity of virus strains, we have sequenced and compared the complete genomes of 34 Far-Eastern subtype (FE) TBEV strains isolated from patients with different disease severity (Primorye, the Russian Far East). We analyzed the complete genomes of 11 human pathogenic strains isolated from the brains of dead patients with the encephalitic form of the disease (Efd), 4 strains from the blood of patients with the febrile form of TBE (Ffd), and 19 strains from patients with the subclinical form of TBE (Sfd). On the phylogenetic tree, pathogenic Efd strains formed two clusters containing the prototype strains, Senzhang and Sofjin, respectively. Sfd strains formed a third separate cluster, including the Oshima strain. The strains that caused the febrile form of the disease did not form a separate cluster. In the viral proteins, we found 198 positions with at least one amino acid residue substitution, of which only 17 amino acid residue substitutions were correlated with the variable pathogenicity of these strains in humans and they authentically differed between the groups. We considered the role of each amino acid substitution and assumed that the deletion of 111 amino acids in the capsid protein in combination with the amino acid substitutions R16K and S45F in the NS3 protease may affect the budding process of viral particles. These changes may be the major reason for the diminished pathogenicity of TBEV strains. We recommend Sfd strains for testing as attenuation vaccine candidates.

Comprehensive assessment of the genetics and virulence of tick-borne encephalitis virus strains isolated from patients with inapparent and clinical forms of the infection in the Russian Far East.

We analyzed the genetics and virulence of 35 strains of TBEV isolated from patients with different forms of the infection living in the southern Far East region of Russia. The results of moleculargenetics studies of the TBEV strains showed that most of the strains that cause inapparent infections form a single cluster (I) with the Oshima 5-10 strain from Japan on the phylogenetic tree. A comparison of the amino acid sequences of the viral polyproteins of the studied strains identified 17 amino acid residues distributed unevenly across the polyprotein that distinctly differed between the clusters of inapparent and virulent strains. We detected additional substitutions in the NS1 and NS5 proteins. These substitutions might influence the pathogenic potential of the strains. Using a model of inbred mice of different ages, we examined the virulence of these strains and showed the different pathogenic potentials of strains belonging to different clusters.

NS2B/NS3 protease: allosteric effect of mutations associated with the pathogenicity of tick-borne encephalitis virus.

The sequences of the protease domain of the tick-borne encephalitis (TBE) virus NS3 protein have two amino acid substitutions, 16 R→K and 45 S→F, in the highly pathogenic and poorly pathogenic strains of the virus, respectively. Two models of the NS2B-NS3 protease complex for the highly pathogenic and poorly pathogenic strains of the virus were constructed by homology modeling using the crystal structure of West Nile virus NS2B-NS3 protease as a template; 20 ns molecular dynamic simulations were performed for both models, the trajectories of the dynamic simulations were compared, and the averaged distance between the two models was calculated for each residue. Conformational differences between two models were revealed in the identified pocket. The different conformations of the pocket resulted in different orientations of the NS2B segment located near the catalytic triad. In the model of the highly pathogenic TBE virus the identified pocket had a more open conformation compared to the poorly pathogenic model. We propose that conformational changes in the active protease center, caused by two amino acid substitutions, can influence enzyme functioning and the virulence of the virus.

Seroprevalence of West Nile virus in wild birds in far eastern Russia using a focus reduction neutralization test.

West Nile (WN) virus has been spreading geographically to non-endemic areas in various parts of the world. However, little is known about the extent of WN virus infection in Russia. Japanese encephalitis (JE) virus, which is closely related to WN virus, is prevalent throughout East Asia. We evaluated the effectiveness of a focus reduction neutralization test in young chicks inoculated with JE and WN viruses, and conducted a survey of WN infection among wild birds in Far Eastern Russia. Following single virus infection, only neutralizing antibodies specific to the homologous virus were detected in chicks. The neutralization test was then applied to serum samples from 145 wild birds for WN and JE virus. Twenty-one samples were positive for neutralizing antibodies to WN. These results suggest that WN virus is prevalent among wild birds in the Far Eastern region of Russia.

Characterization of Powassan viruses from Far Eastern Russia.

We report the isolation and detailed characterization of the novel strain, Partizansk/2006, of Powassan virus (POWV) from a human case of infection, which occurred in Primorsky krai, Russia, in 2006. Comparative complete genome sequence analysis of the Far Eastern strains Spassk-9 (1975), Nadezdinsk-1991 and Partizansk/2006 of POWV revealed that these strains are 99.8% similar to the LB strain, which was isolated in Canada in 1958. Phylogenetic analysis of 5' UTR sequences of five other strains of POWV isolated from 1972 to 1986 in Primorsky krai produced similar results. Presumably, Far Eastern POWV has common putative ancestor with LB strain POWV from North America, and the time of divergence of these POWVs is relatively short. We conclude that POWV has become endemic in Far Eastern Russia.

Characterization of neutralizing antibodies to Far Eastern of tick-borne encephalitis virus subtype and the antibody avidity for four tick-borne encephalitis vaccines in human.

We assessed the humoral immunity of 290 vaccinated persons against tick-borne encephalitis (TBE). During the first year and 2 years after the primary three vaccinations the antibodies to the Far Eastern subtype tick-borne encephalitis virus strain P-73 were detected by neutralization test after immunization with FSME-Immune Inject vaccine (Baxter Vaccine AG, Austria) in 88.2% and 78.1% vaccinated persons, respectively; with Encepur((R)) Adult vaccine (Novartis Vaccines, Germany), in 100% and 100%; with the vaccine of the Chumakov Institute of Poliomyelitis and Viral Encephalitides RAMSci (Russia), in 100% and 94.1%; with EnceVir vaccine (Russia), in 88.2% and 83.9%; and after combined vaccination, in 100% and 92.7%. The dynamics of the decrease in IgG avidity index correlated with the changes of antibody titers determined by neutralization test. After the primary vaccination course, the titers of virus-neutralizing antibodies were high (6.3-7.4 log(2)) when the avidity index of IgG antibodies were 31% and more; thus, this level can be considered immunologically significant. Two years after the primary vaccination course, the IgG avidity indexes of 60% and more can be regarded as significant on the background of the GMT decrease of virus-neutralizing antibodies. These results allow us to recommend all four vaccines for mass vaccination and an assay of IgG avidity, along with neutralization test, for a more adequate assessment of the level of postvaccination immune response.

Evaluation of vaccine Encepur Adult for induction of human neutralizing antibodies against recent Far Eastern subtype strains of tick-borne encephalitis virus.

We studied humoral immune response of 44 volunteers from Primorsky krai (Russia) immunized with the vaccine Encepur Adult. Induction of the humoral response towards the recently isolated tick-borne encephalitis virus (TBEV) strains P-69, P-202, and P-73 was evaluated by neutralization test and enzyme immunoassay. These strains belong to Far Eastern TBEV subtype based on their genotype and antigenic structure but maintain significant genetic and antigenic variability. The average geometric titers of neutralizing antibodies to P-69, P-202, and P-73 strains were 1:28, 1:34, and 1:128, respectively. The percentage of volunteers with neutralizing antibodies to these strains after complete course immunization was 63.9, 97.6, and 95.5%, respectively. We concluded that Encepur Adult vaccine induced pronounced humoral immune response towards genetically and antigenically heterogeneous strains of the Far Eastern TBEV subtype.

Novel variant of tickborne encephalitis virus, Russia.

We isolated a novel strain of tickborne encephalitis virus (TBEV), Glubinnoe/2004, from a patient with a fatal case in Russia. We sequenced the strain, whose landmark features included 57 amino acid substitutions and 5 modified cleavage sites. Phylogenetically, Glubinnoe/2004 is a novel variant that belongs to the Eastern type of TBEV.

Epizootiologic survey for Babesia microti among small wild mammals in northeastern Eurasia and a geographic diversity in the beta-tubulin gene sequences.

We previously reported that small wild rodents in Japan harbor two types of novel Babesia microti-like parasites (designated as Hobetsu and Kobe types), but not the type commonly found in the northeastern United States (U.S. type) where human babesiosis is endemic. To determine whether these new types of parasites are distributed in places surrounding Japan, an epizootiologic survey was undertaken in three geographically distant areas in northeastern Eurasia; South Korea, Vladivostok in Russia, and Xinjiang in China. Blood samples were collected from a total of 387 animals comprising 24 species. DNAs extracted from the samples were tested by nested PCR targeting babesial nuclear small-subunit rRNA gene (rDNA), which revealed that small rodents harboring B. microti exist in all three survey areas. Sequence analysis showed that all PCR-positive samples had rDNA sequences virtually identical to that of U.S.-type B. microti. However, when beta-tubulin gene sequences were compared, evident geographic variations were seen. By use of primers specific for each of the beta-tubulin genes of Kobe-, Hobetsu-, and U.S.-type parasites, a type-specific PCR was developed. Parasite with Hobetsu- or Kobe-type sequence was not detected from any of the three survey areas. These findings suggest that U.S.-type B. microti is widely distributed among small wild mammals in temperate zones of not only North America, but also Eurasia, whereas that Hobetsu- and Kobe-type parasites may be uniquely distributed in Japan.

Distribution and characterization of tick-borne encephalitis viruses from Siberia and far-eastern Asia.

In this study, tick-borne encephalitis (TBE) viruses from Siberia and far-eastern Asia were characterized in order to determine virus subtype distribution. TBE viruses were isolated from ticks (Ixodes persulcatus) collected in the far-eastern (Khabarovsk and Vladivostok) and Siberian (Irkutsk) regions of Russia in 1999. Phylogenetic analysis showed that isolates formed distinct clusters of far-eastern and Siberian subtypes. There was also a minor difference in antigenicity between the Irkutsk isolates and other TBE virus strains, as demonstrated by the reactivity of monoclonal antibodies. Amino acid alignments of the E gene showed that the Irkutsk isolates had a single amino acid change at position 234 (Q or H); this amino acid position is considered to be a 'signature' of Siberian subtype TBE viruses. Strains isolated in Irkutsk also exhibited equivalent or somewhat higher virulence in mice compared with far-eastern TBE virus isolates. All viruses isolated in this study (i.e. far-east Asian and Siberian isolates) have 3' non-coding regions (NCRs) of almost the same length, which contrasts with the various sizes of 3'NCRs of other TBE viruses strains reported previously. The data presented in this study show that the 3'NCR is uniform among TBE viruses isolated from Siberia and far-eastern Asia and that the 3'NCR is essential for TBE virus growth in tick and/or rodent host cells.