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.

The Viromes of Mosquitoes from the Natural Landscapes of Western Siberia.

The metagenomic analysis of mosquitoes allows for the genetic characterization of mosquito-associated viruses in different regions of the world. This study applied a metagenomic approach to identify novel viral sequences in seven species of mosquitoes collected from the Novosibirsk region of western Siberia. Using NGS sequencing, we identified 15 coding-complete viral polyproteins (genomes) and 15 viral-like partial sequences in mosquitoes. The complete sequences for novel viruses or the partial sequences of capsid proteins, hypothetical viral proteins, and RdRps were used to identify their taxonomy. The novel viral sequences were classified within the orders Tymovirales and Picornavirales and the families Partitiviridae, Totiviridae, Tombusviridae, Iflaviridae, Nodaviridae, Permutotetraviridae, and Solemoviridae, with several attributed to four unclassified RNA viruses. Interestingly, the novel putative viruses and viral sequences were mainly associated with the mosquito Coquillettidia richardii. This study aimed to increase our understanding of the viral diversity in mosquitoes found in the natural habitats of Siberia, which is characterized by very long, snowy, and cold winters.

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.

Novel Flavi-like virus in ixodid ticks and patients in Russia.

Novel Haseki tick virus (HSTV) was detected in ixodid ticks and patients in the Asian part of Russia. Sequencing of the genome fragments corresponding whole polyprotein and viral RdRp demonstrated that HSTV is genetically close to unclassified Flavi-like viruses. Phylogenetic analysis of HSTV sequences showed that these viruses were close to Bole tick virus 4 (BLTV 4), which was detected early in Asia, Europe, Africa and the Caribbean region. The organization of the genome predicts that HSTV and BLTV 4 may also be classified as putative new genera within Flaviviridae with enlarged Flavi-like positive-sense ssRNA viral genomes. Cases of HSTV putative human incidents after Ixodes persulcatus attack were discovered in hospital patients with tick-borne infections in Vladivostok (Russia). The illness was associated with 3-5 days of fever, accompanied by acute respiratory lesions. Mixed human tick-borne infections (TBIs) were also detected for these patients as dual or triple coinfections for tick-borne encephalitis virus, Borrelia spp., Anaplasma spp., and HSTV. Thus, it is necessary to study HSTV antibody tests, virus isolation, and surveillance for HSTV sequences in different species of ticks, different geographical regions and patients after tick attacks.

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.

Detection of Babesia caballi, Theileria mutans and Th. velifera in ixodid ticks collected from cattle in Guinea in 2017-2018.

Intraerythrocytic protozoan parasites from the genera Babesia and Theileria may infect a wide range of animals and humans. The purpose of this study was to detect the 18S ribosomal RNA gene of Babesia spp. and Theileria spp. in ticks collected from household cows in the Republic of Guinea from 2017 to 2018 by PCR and then genotype the gene fragments by sequencing. A total of 907 ticks from 319 cows were collected in seven prefectures of Guinea (Boke, Faranah, Kankan, Kindia, Labe, Mamou and N'Zerekore). The following tick species on cattle were identified: Amblyomma variegatum (44.2%), Rhipicephalus decoloratus (34.7%), Rh. annulatus (10.3%), Rh. geigyi (7.3%) Hyalomma truncatum (2.4%), Rh. senegalensis (0.8%) and Haemaphysalis leachi (0.6%). Genetic markers for piroplasms were found in Am. variegatum, Rh. decoloratus, Rh. annulatus, and Rh. geigyi ticks, and the total infection rate for these ticks was 4.2%. The highest infection rate was found in Rh. annulatus ticks (10.9%). The piroplasms were genotyped as Babesia caballi, Theileria mutans and Theileria velifera by phylogenetic analysis of the 1150 bp 18S ribosomal RNA gene fragments. These pathogens were discovered in practically all studied prefectures in Guinea except for Mamou Prefecture. We propose that these ixodid ticks might play a major role in the transmission of piroplasm infections in domestic animals in Guinea.

Antiviral photodynamic therapy: Inactivation and inhibition of SARS-CoV-2 in vitro using methylene blue and Radachlorin.

INTRODUCTION: Recently, the COVID-19 pandemic has spread globally, necessitating the development of new methods for its prevention and treatment. The purpose of this study was to evaluate the antiviral activity of photodynamic therapy (PDT) against SARS-CoV-2 in vitro. METHODS: Vero E6 cells and SARS-CoV-2 isolated in Russia were used for PDT with methylene blue (MB) and Radachlorin. A continuous laser with wavelength λ = 662 nm in doses of 16 J/cm2 and 40 J/cm2 laser irradiation was used for PDT of a viral suspension and SARS-CoV-2-infected cells. The direct cytopathogenic effect of SARS-CoV-2 was evaluated via light microscopy to calculate the TCID50 in the samples and perform statistical analysis. RESULTS: Viral suspensions of SARS-CoV-2 that had a TCID50 greater than 103 were inactivated by PDT in the presence of MB and Radachlorin. Vero E6 cells were protected from 104 TCID50 of SARS-CoV-2 by PDT post infection. The range of protective concentrations was 1.0-10.0 µg/ml and 0.5-5.0 µg/ml for MB and Radachlorin, respectively. Additionally, it was found that MB and Radachlorin also possess significant antiviral activity even without PDT. The 50 % inhibitory concentration (IC50) against 102 TCID50 of SARS-CoV-2 was found to be 0.22 and 0.33 µg/mL with the addition of MB and Radachlorin, respectively, to cells concomitantly with virus, whereas in the case of applying the photosensitizers at 3.5 h post infection, the IC50 was 0.6 and 2.0 µg/mL for MB and Radachlorin, respectively. CONCLUSION: PDT shows high antiviral activity against SARS-CoV-2 when combined with MB and Radachlorin in vitro.

Complete mitogenome of the ixodid tick Dermacentor reticulatus (Acari: Ixodida).

Here, we present the complete mitochondrial DNA sequence of Dermacentor reticulatus. The mitogenome is 14,806 bp and contains 13 protein-coding, 2 rRNA, and 22 tRNA genes, along with 2 control regions. Dermacentor reticulatus mitogenome has the common mitochondrial gene order of Metastriata ticks. It is phylogenetically close to the mitogenomes of Dermacentor ticks, of which D. everestanus mitogenome is the closest with 85.7% similarity. These data provide insights into the phylogenetic relations among Dermacentor ticks.

Rotavirus Seasonality: An Application of Singular Spectrum Analysis and Polyharmonic Modeling.

The dynamics of many viral infections, including rotaviral infections (RIs), are known to have a complex non-linear, non-stationary structure with strong seasonality indicative of virus and host sensitivity to environmental conditions. However, analytical tools suitable for the identification of seasonal peaks are limited. We introduced a two-step procedure to determine seasonal patterns in RI and examined the relationship between daily rates of rotaviral infection and ambient temperature in cold climates in three Russian cities: Chelyabinsk, Yekaterinburg, and Barnaul from 2005 to 2011. We described the structure of temporal variations using a new class of singular spectral analysis (SSA) models based on the "Caterpillar" algorithm. We then fitted Poisson polyharmonic regression (PPHR) models and examined the relationship between daily RI rates and ambient temperature. In SSA models, RI rates reached their seasonal peaks around 24 February, 5 March, and 12 March (i.e., the 55.17 ± 3.21, 64.17 ± 5.12, and 71.11 ± 7.48 day of the year) in Chelyabinsk, Yekaterinburg, and Barnaul, respectively. Yet, in all three cities, the minimum temperature was observed, on average, to be on 15 January, which translates to a lag between the peak in disease incidence and time of temperature minimum of 38-40 days for Chelyabinsk, 45-49 days in Yekaterinburg, and 56-59 days in Barnaul. The proposed approach takes advantage of an accurate description of the time series data offered by the SSA-model coupled with a straightforward interpretation of the PPHR model. By better tailoring analytical methodology to estimate seasonal features and understand the relationships between infection and environmental conditions, regional and global disease forecasting can be further improved.

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.

Bioselection of coxsackievirus B6 strain variants with altered tropism to human cancer cell lines.

Cancer cells develop increased sensitivity to members of many virus families and, in particular, can be efficiently infected and lysed by many low-pathogenic human enteroviruses. However, because of their great genetic heterogeneity, cancer cells display different levels of sensitivity to particular enterovirus strains, which may substantially limit the chances of a positive clinical response. We show that a non-pathogenic strain of coxsackievirus B6 (LEV15) can efficiently replicate to high titers in the malignant human cell lines C33A, DU145, AsPC-1 and SK-Mel28, although it displays much lower replication efficiency in A431 and A549 cells and very limited replication ability in RD and MCF7 cells, as well as in the normal lung fibroblast cell line MRC-5 and the immortalized mammary epithelial cell line MCF10A. By serial passaging in RD, MCF7 and A431 cells, we obtained LEV15 strain variants that had acquired high replication capacity in the appropriate carcinoma cell lines without losing their high replication capability in the original set of cancer cell lines and had limited replication capability in untransformed cells. The strains demonstrated improved oncolytic properties in nude-mouse xenografts. We identified nucleotide changes responsible for the phenotypes and suggest a bioselection approach for a generation of oncolytic virus strains with a wider spectrum of affected tumors.

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.

A region in the C-terminal domain of ribosomal protein SA required for binding of SA to the human 40S ribosomal subunit.

The human ribosomal protein SA, known also as a precursor of the cell-surface laminin receptor, LAMR, is a protein of the 40S ribosomal subunit. It is homologous to eubacterial ribosomal protein S2p, but has a eukaryote-specific C-terminal domain (CTD) that is responsible in LAMR for the binding of laminin as well as prions and several viruses. Using serial deletions in the SA CTD, we showed that region between amino acids 236-262 is required for binding of the protein to 40S ribosomal subunits. All SA mutants containing this region protected nucleotides in hairpin 40 (which is not bound to any protein in the eubacterial 30S ribosomal subunit) of the 18S rRNA from hydroxyl radical attack. Comparison of our data with the cryo-EM models of the mammalian 40S ribosomal subunit allowed us to locate the SA CTD in the spatial structure of the 40S subunit.

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.