The structure of inactivated mature tick-borne encephalitis virus at 3.0 Å resolution.

Tick-borne encephalitis virus (TBEV) causes a severe disease, tick-borne encephalitis (TBE), that has a substantial epidemiological importance for Northern Eurasia. Between 10,000 and 15,000 TBE cases are registered annually despite the availability of effective formaldehyde-inactivated full-virion vaccines due to insufficient vaccination coverage, as well as sporadic cases of vaccine breakthrough. The development of improved vaccines would benefit from the atomic resolution structure of the antigen. Here we report the refined single-particle cryo-electron microscopy (cryo-EM) structure of the inactivated mature TBEV vaccine strain Sofjin-Chumakov (Far-Eastern subtype) at a resolution of 3.0 Å. The increase of the resolution with respect to the previously published structures of TBEV strains Hypr and Kuutsalo-14 (European subtype) was reached due to improvement of the virus sample quality achieved by the optimized preparation methods. All the surface epitopes of TBEV were structurally conserved in the inactivated virions. ELISA studies with monoclonal antibodies supported the hypothesis of TBEV protein shell cross-linking upon inactivation with formaldehyde.

Human Adenovirus and Influenza A Virus Exacerbate SARS-CoV-2 Infection in Animal Models.

In this study, we investigated the features of the infectious process by simulating co-infection with SARS-CoV-2 and human adenovirus type 5 (HAdV-5) or influenza A virus (IAV) in vitro and in vivo. The determination of infectious activity of viruses and digital PCR demonstrated that during simultaneous and sequential HAdV-5 followed by SARS-CoV-2 infection in vitro and in vivo, the HAdV-5 infection does not interfere with replication of SARS-CoV-2. The hamsters co-infected and mono-infected with SARS-CoV-2 exhibited nearly identical viral titers and viral loads of SARS-CoV-2 in the lungs. The hamsters and ferrets co-infected by SARS-CoV-2- and IAV demonstrated more pronounced clinical manifestations than mono-infected animals. Additionally, the lung histological data illustrate that HAdV-5 or IAV and SARS-CoV-2 co-infection induces more severe pathological changes in the lungs than mono-infection. The expression of several genes specific to interferon and cytokine signaling pathways in the lungs of co-infected hamsters was more upregulated compared to single infected with SARS-CoV-2 animals. Thus, co-infection with HAdV-5 or IAV and SARS-CoV-2 leads to more severe pulmonary disease in animals.

Can Modern Molecular Modeling Methods Help Find the Area of Potential Vulnerability of Flaviviruses?

Flaviviruses are single-stranded RNA viruses that have emerged in recent decades and infect up to 400 million people annually, causing a variety of potentially severe pathophysiological processes including hepatitis, encephalitis, hemorrhagic fever, tissues and capillaries damage. The Flaviviridae family is represented by four genera comprising 89 known virus species. There are no effective therapies available against many pathogenic flaviviruses. One of the promising strategies for flavivirus infections prevention and therapy is the use of neutralizing antibodies (NAb) that can disable the virus particles from infecting the host cells. The envelope protein (E protein) of flaviviruses is a three-domain structure that mediates the fusion of viral and host membranes delivering the infectious material. We previously developed and characterized 10H10 mAb which interacts with the E protein of the tick-borne encephalitis virus (TBEV) and many other flaviviruses' E proteins. The aim of this work was to analyze the structure of E protein binding sites recognized by the 10H10 antibody, which is reactive with different flavivirus species. Here, we present experimental data and 3D modeling indicating that the 10H10 antibody recognizes the amino acid sequence between the two cysteines C92-C116 of the fusion loop (FL) region of flaviviruses' E proteins. Overall, our results indicate that the antibody-antigen complex can form a rigid or dynamic structure that provides antibody cross reactivity and efficient interaction with the fusion loop of E protein.

Detection of tick-borne pathogens in wild birds and their ticks in Western Siberia and high level of their mismatch.

The Tomsk region located in the south of Western Siberia is one of the most high-risk areas for tick-borne diseases due to elevated incidence of tick-borne encephalitis and Lyme disease in humans. Wild birds may be considered as one of the reservoirs for tick-borne pathogens and hosts for infected ticks. A high mobility of wild birds leads to unpredictable possibilities for the dissemination of tick-borne pathogens into new geographical regions. The primary goal of this study was to evaluate the prevalence of tick-borne pathogens in wild birds and ticks that feed on them as well as to determine the role of different species of birds in maintaining the tick-borne infectious foci. We analysed the samples of 443 wild birds (60 species) and 378 ticks belonging to the genus Ixodes Latraille, 1795 collected from the wild birds, for detecting occurrence of eight tick-borne pathogens, the namely tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and species of Borrelia, Rickettsia, Ehrlichia, Anaplasma, Bartonella and Babesia Starcovici, 1893, using RT-PCR/or PCR and enzyme immunoassay. One or more tick-borne infection markers were detected in 43 species of birds. All markers were detected in samples collected from fieldfare Turdus pilaris Linnaeus, Blyth's reed warbler Acrocephalus dumetorum Blyth, common redstart Phoenicurus phoenicurus (Linnaeus), and common chaffinch Fringilla coelebs Linnaeus. Although all pathogens have been identified in birds and ticks, we found that in the majority of cases (75.5 %), there were mismatches of pathogens in birds and ticks collected from them. Wild birds and their ticks may play an extremely important role in the dissemination of tick-borne pathogens into different geographical regions.

Variability in the 3' untranslated regions of the genomes of the different tick-borne encephalitis virus subtypes.

Tick-borne encephalitis viruses (TBEVs) are usually divided into three major subtypes: European (TBEV-Eu), Siberian (TBEV-Sib) and Far Eastern (TBEV-FE). The TBEV-Eu strains have the longest genomes, and TBEV-FE strains have the smallest genomes. Changes in the variable region of the untranslated region (V3' UTR) play a major role in determining the viral genome length. Analyses of the 3' UTRs of the different subtypes of TBEV have revealed significant changes in the secondary structures of the V3' UTR of TBEV. More complex secondary structures of the V3' UTR regions are typical for TBEV-Eu. The Siberian strain Tomsk-PT122 was isolated from birds and has an unusual 3' UTR. Several short fragment (24-26 nucleotides) insertions derived from the viral E (2) and NS4a (1) genes have been found in the V3' UTR of Tomsk-PT122. Additionally, the length of the V3' UTR increases from 21 to 37 nucleotides during passages of the C11-13 strain of TBEV-Sib into PEK, 293 and Neuro-2a cells. The elongation of the V3' UTRs of Tomsk-PT122 and C11-13 is the first direct evidence of an intragenomic 3' UTR modification (insertion) for TBEV. Thus, the obtained results suggest that changing the length of the V3' UTR in the genome is typical for different TBEV subtypes and can play an essential role in effective TBEV replication in different host cells.

Adaptation of tick-borne encephalitis virus from human brain to different cell cultures induces multiple genomic substitutions.

The C11-13 strain from the Siberian subtype of tick-borne encephalitis virus (TBEV) was isolated from human brain using pig embryo kidney (PEK), 293, and Neuro-2a cells. Analysis of the complete viral genome of the C11-13 variants during six passages in these cells revealed that the cell-adapted C11-13 variants had multiple amino acid substitutions as compared to TBEV from human brain. Seven out of eight amino acids substitutions in the high-replicating C11-13(PEK) variant mapped to non-structural proteins; 13 out of 14 substitutions in the well-replicating C11-13(293) variant, and all four substitutions in the low-replicating C11-13(Neuro-2a) variant were also localized in non-structural proteins, predominantly in the NS2a (2), NS3 (6) and NS5 (3) proteins. The substitutions NS2a1067 (Asn → Asp), NS2a1168(Leu → Val) in the N-terminus of NS2a and NS31745(His → Gln) in the helicase domain of NS3 were found in all selected variants. We postulate that multiple substitutions in the NS2a, NS3 and NS5 genes play a key role in adaptation of TBEV to different cells.

Detection of Rickettsia helvetica and Candidatus R. tarasevichiae DNA in Ixodes persulcatus ticks collected in Northeastern European Russia (Komi Republic).

The number of tick-borne infections in the northern European regions of Russia has increased considerably in the last years. In the present study, 676 unfed adult Ixodes persulcatus ticks were collected in the Komi Republic from 2011 to 2013 to study tick-borne rickettsioses. Rickettsia spp. DNA was detected by PCR in 51 (7.6%) ticks. The nucleotide sequence analysis of gltA fragments (765bp) from 51 ticks indicated that 60.8% and 39.2% of the ticks were infected with Rickettsia helvetica and Candidatus R. tarasevichiae, respectively. The gltA fragments showed 100% identity with those of Candidatus R. tarasevichiae previously discovered in Siberia and China, whereas R. helvetica showed 99.9% sequence identity with European isolates. The ompB had 8 nucleotide substitutions, 6 of which resulted in amino acid substitutions. In the sca9 gene, 3 nucleotide substitutions were detected, and only one resulted in amino acid substitution. The smpA, ompW, and ß-lactamase genes of R. helvetica also showed a high level of sequence identity.

Complete mitogenome of the ixodid tick Ixodes pavlovskyi (Acari: Ixodida).

Here, we present complete mitochondrial DNA sequence of Ixodes pavlovskyi Pom., 1946 for the first time. The mitogenome is 14,575 bp in length and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The overall base composition is 40.1% T, 13.8% C, 37.9% A and 8.1% G. Four protein-coding genes are initiated by ATT codon, three genes--by ATA codon and ATG start codon is found for six genes. Only tRNA-Lys, tRNA-Ile, tRNA-Arg are folded into the cloverleaf secondary structure, other tRNA have atypical structure with reduced T- or D-arms.

Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVß3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process.

Surveillance of tick-borne encephalitis virus in wild birds and ticks in Tomsk city and its suburbs (Western Siberia).

To study the role of wild birds in the transmission of tick borne encephalitis virus (TBEV), we investigated randomly captured wild birds bearing ixodid ticks in a very highly endemic TBE region located in Tomsk city and its suburbs in the south of Western Siberia, Russia. The 779 wild birds representing 60 species were captured carrying a total of 841 ticks, Ixodes pavlovskyi Pom., 1946 (n=531), Ixodes persulcatus P. Sch., 1930 (n=244), and Ixodes plumbeus Leach. 1815 (n=66). The highest average number of ticks per bird in a particular species was found for the fieldfare (Turdus pilaris Linnaeus, 1758) (5.60 ticks/bird) and the tree pipit (Anthus trivialis Linnaeus, 1758) (13.25 ticks/bird). Samples from wild birds and ticks collected in highly endemic periods from 2006 to 2011 were tested for the TBEV markers using monoclonal modified enzyme immunoassay (EIA) and RT-PCR. TBEV RNA and antigen were found in 9.7% and 22.8% samples collected from wild birds, respectively. TBEV markers were also detected in 14.1% I. persulcatus ticks, 5.2% I. pavlovskyi, and 4.2% I. plumbeus ticks collected from wild birds. Two TBEV strains were also isolated on PKE (pig kidney embryo) cells from fieldfare and Blyth's reed warbler (Acrocephalus dumetorum Blyth, 1849). Sequencing of 5'-NCR of TBEV revealed that all TBEV isolates belong to Far Eastern (dominate) and Siberian genotypes. Several phylogenetic subgroups included TBEV sequences novel for the Tomsk region. Our data suggest that wild birds are potential disseminators of TBEV, TBEV-infected ixodid ticks, and possibly other tick-borne infections.

Variability of the tick-borne encephalitis virus genome in the 5' noncoding region derived from ticks Ixodes persulcatus and Ixodes pavlovskyi in Western Siberia.

We report the prevalence of Siberian and Far Eastern subtypes of tick-borne encephalitis virus (TBEV) in Ixodes persulcatus and Ix. pavlovskyi ticks collected in Tomsk and its suburbs during 2006-2008. The TBEV was detected in 5.7% ticks collected in the city, where Ix. pavlovskyi ticks were dominated and 7.5% ticks from suburban foci with prevalence Ix. persulcatus ticks. Genotyping of the virus showed that Siberian subtype (89.5%) is predominant in individual ticks of Tomsk suburbs; however, the proportion of Far Eastern subtype in two urban sites reached 47%. Phylogenetic analysis demonstrated that Siberian subtype variants from individual ticks were quite divergent and original. Only one subclade was found to be similar to Zausaev strain of TBEV, which is the etiological agent of lethal chronic form of tick-borne encephalitis infection. The average level of homology of 5' noncoding region (5'-NCR) of TBEV in the individual ticks was 95% for Far Eastern subtype and 89% for Siberian subtype of TBEV. Multiple substitutions in 5'-NCR were found in viral RNA derived from individual ticks. The A2 and C1 elements of Y-shaped structure and putative site for viral RNA polymerase were most variable regions for TBEV 5'-NCR. The B1 and B2 elements and the start codon were practically conserved. The viral RNA from three TBEV-infected pig kidney embryo cells after three passages (out of 21 polymerase chain reaction-positive ticks) were found to multiple substitutions in 5'-NCR in comparison with viral RNA from individual parent tick. However, these three variants did not replicate efficiently in pig kidney embryo cells that may be connected with a considerable modification of Y-shaped structure of 5'-NCR. The efficiently replicating isolate Kolarovo had only seven substitutions in the 5'-NCR and typical Y-shaped structure for Siberian subtype of TBEV. Our data support the idea that hypervariability of the 5'-NCR reflects viral strategy to select the fittest RNA molecule for productive viral infection in mammalian and tick cells.

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.

Immunochemical and single molecule force spectroscopy studies of specific interaction between the laminin binding protein and the West Nile virus surface glycoprotein E domain II.

ELISA and Western blot immunochemical data attest an effective and highly specific interaction of the surface glycoprotein E domain II (DII) of the tick born encephalitis and Dengue viruses with the laminin binding protein (LBP). Based on a highly conservative structure of the DII in different flaviviruses we propose a similarly effective interaction between the LBP and the DII of the surface glycoprotein E of the West Nile virus. We report the results of studies of this interaction by immunochemical and single molecule force spectroscopy methods. The specific binding between these species is confirmed by both methods.

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.

Neutralizing monoclonal antibodies against Russian strain of the West Nile virus.

We have developed a panel of 16 hybridomas secreting neutralizing monoclonal antibodies (Nt- MAbs) to Russian isolate (LEIV-Vlg99-27889-human) of the West Nile virus (WNV). Most of the Nt-Mabs were either IgG1 or IgG3 subtypes. Nine of the 16 neutralizing MAbs detected WNV protein E in Western blot. According to their Nt-activities, Western blot results and cross-reactivity, the MAbs were divided into four groups. Monoclonal antibodies from group I were able to neutralize WNV strains Vlg99-27889, Vlg00-27924, Hp-94, A-1640, A-72, Tur-2914, and Eg101. The Nt-activity of MAbs from groups II-IV towards these WNV strains was variable. Recombinant fragments E(1-180), E(1-321), and E(260-466) of protein E were used for preliminary mapping of domains recognized by Nt-MAbs. Only five Nt-MAbs were able to react with the recombinant polypeptides. The MAbs 9E2, 7G9, 11G3, and 7E6 from group Ia recognized Nt-epitope(s) between amino acids 321 and 466 of protein E and Nt-MAb 4F11 (group III) reacted with residues 1-180. This demonstrates that two discrete regions of protein E are involved in neutralization of WNV. Our data on immunochemical, biological activities of Nt-MAbs and mapping of Nt-epitopes using recombinant polypeptides suggest at least 13 different Nt-epitopes for WNV.

Induction of alternatively spliced spermidine/spermine N1-acetyltransferase mRNA in the human kidney cells infected by venezuelan equine encephalitis and tick-borne encephalitis viruses.

293 and RH cells derived from human embryo kidney were infected by Venezuelan equine encephalitis and tick-borne encephalitis viruses and cDNA libraries representing cellular mRNAs induced or suppressed due to the infection were prepared using suppressive subtractive hybridization. Among the up-regulated clones the RT-PCR and Northern analyses revealed an unusual transcript of the spermidine/spermine N1-acetyltransferase (SSAT) gene that was shown to be an alternatively spliced form containing an additional 110-bp exon. The alternatively spliced transcript is polyadenylated and can be expected to yield only a truncated 71 amino acid polypeptide. This first evidence of the host gene alternatively spliced mRNA induction by RNA viruses raises the questions of its biological role, regulation mechanisms of alternative splicing, and significance for the virus life cycle.