Search

Introduction. Ixodes ticks are vectors for pathogens of many infectious diseases. Recently, during the study of Rhipicephalus geigyi ticks collected from livestock in the Republic of Guinea, a new multicomponent flavi-like RNA virus, called Kindia tick virus (KITV), was discovered with an unusual mechanism for the implementation of genetic information. The aim of the work is to detect and study the genetic diversity of KITV in ixodes ticks collected in the territory of the Kindia province of the Republic of Guinea. Material and methods. In 2021, 324 specimens of ticks of the species Amblyomma variegatum, Rh. geigyi, Rh. annulatus, Rh. decoloratus, Rh. senegalensis were collected from cattle. The detection of viral RNA was carried out in individual samples of ticks by RT-PCR, followed by the determination of the nucleotide sequence and phylogenetic analysis. Results and discussion. KITV detection rates in ticks of the species Rh. geigyi was 12.2%, Rh. annulatus 4.4%, Rh. decoloratus 3.3%. However, the KITV genetic material has not been identified in Am. variegatum ticks, which are one of the dominant species in West Africa. For all virus isolates, a partial nucleotide sequences of each of the four viral segments (GenBank, OK345271OK345306) were determined. The phylogenetic analysis showed a high level of identity (98.599.8%) for each of the four segments of the viral genome with those previously found in the Republic of Guinea. The obtained KITV isolates are most genetically close to Mogiana tick virus, which was previously detected in South America in Rh. microplus ticks and significantly differed from other multicomponent viruses circulating in Europe and Asia, including the Russian Federation. Conclusion. KITV genetic material was found in three species of ixodid ticks collected from livestock in a number of prefectures of the Republic of Guinea. The infection rate in ticks was 3.312.2%. The continuation of research in this direction remains relevant.

In this article, we describe a newly discovered cypovirus strain that displays features ideally suited for the development of a modern biological insecticide: high potency, relatively broad host range, true regulating effect, flexible production (possibility to choose host species for production), interaction with enhancing adjuvants, and ecologically friendly. Based on an alignment of CPV genomes, we suggest that the enhanced host range of this new strain is the sequence of evolutionary events that occurred after coinfections involving different CPV species within the same host.

The circulation of seasonal influenza in 2020–2021 around the world was drastically reduced after the start of the COVID-19 pandemic and the implementation of mitigation strategies. The influenza virus circulation reemerged in 2021–2022 with the global spread of the new genetic clade 3C.2a1b.2a.2 of A(H3N2) viruses. The purpose of this study was to characterize influenza viruses in the 2021–2022 season in Russia and to analyze the receptor specificity properties of the 3C.2a1b.2a.2 A(H3N2) viruses. Clinical influenza samples were collected at the local Sanitary-and-Epidemiological Centers of Rospotrebnadzor. Whole genome sequencing was performed using NGS. The receptor specificity of hemagglutinin was evaluated using molecular modeling and bio-layer interferometry. Clinical samples from 854 cases of influenza A and B were studied; A(H3N2) viruses were in the majority of the samples. All genetically studied A(H3N2) viruses belonged to the new genetic clade 3C.2a1b.2a.2. Molecular modeling analysis suggested a higher affinity of hemagglutinin of 3C.2a1b.2a.2. A(H3N2) viruses to the α2,6 human receptor. In vitro analysis using a trisaccharide 6’-Sialyl-N-acetyllactosamine receptor analog did not resolve the differences in the receptor specificity of 3C.2a1b.2a.2 clade viruses from viruses belonging to the 3C.2a1b.2a.1 clade. Further investigation of the A(H3N2) viruses is required for the evaluation of their possible adaptive advantages. Constant monitoring and characterization of influenza are critical for epidemiological analysis.

Nowadays researchers provide more and more evidence that it is necessary to develop an ecologically friendly approach to pest control. This is reflected in a sharp increase in the value of the biological insecticide market in recent decades. In our study, we found a virus strain belonging to the genus Cypovirus (Reoviridae); the strain was isolated from Dendrolimus sibiricus: that possesses attractive features as a candidate for mass production of biological agents for lepidopteran-pest control. We describe morphological, molecular, and ecological features of the new Cypovirus strain. This strain was found to be highly virulent to D. sibiricus (half-lethal dose is 68 occlusion bodies per second-instar larva) and to have a relatively wide host range (infects representatives of five families of Lepidoptera: Erebidae, Sphingidae, Pieridae, Noctuidae, and Lasiocampidae). The virus strain showed a strong interaction with a nontoxic adjuvant (optical brightener), which decreased the lethal dose for both main and alternative hosts, decreased lethal time, and may expand the host range. Moreover, we demonstrated that the insecticidal features were preserved after passaging through the most economically suitable host. By providing strong arguments for possible usefulness of this strain in pest control, we call on virologists, pest control specialists and molecular biologists to give more attention to the Cypovirus genus, which may lead to new insights in the field of pest control research and may provide significant advantages to compare with baculoviruses and Bacillus thuringiensis products which are nowadays main source of bioinsecticides.Significance statementWithin this article we are describing unique set of features of newly discovered cypovirus strain which possess by significant premises for modern biological insecticides requirements: high potency, universality, true regulating effect, flexible production (possibility to choose host species for production), interaction with enhancing adjuvants, ecologically friendly. Basing on genome alignment we suggest that increasing of host range of new strain is the sequence of evolutionary event which was occurred after coinfection of different CPV species within same host. This finding open new perspective to consider CPVs as perspective agent of biocontrol products.

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.

In 2018, a Zika infection was detected in patients in the Republic of Guinea. We determined the complete genome sequence of the Faranah/18 strain of Zika virus (ZIKV) isolated using C6/36 cells. The genome sequences are significantly different from those of other African ZIKVs. This isolate has 13 aa substitutions in nonstructural polypeptides, an additional glycosylation site on protein E, and unusual localization of MSI1 binding sites in the 3’-UTR of the African lineage. Additionally, Faranah/18 formed a separate lineage into African genotypes with divergence times near 94 and 646-861 years from Senegal and Uganda ZIKVs, respectively.

Due to cessation of mass smallpox vaccination in 1980, the collective immunity of humans against orthopoxvirus infections has virtually been lost. Therefore, the risk of spreading zoonotic human orthopoxvirus infections caused by monkeypox and cowpox viruses has increased in the world. First-generation smallpox vaccines based on Vaccinia virus (VAC) are reactogenic and therefore not suitable for mass vaccination under current conditions. This necessitates the development of modern safe live vaccines based on VAC using genetic engineering. We created the VACΔ6 strain by transient dominant selection. In the VACΔ6 genome, f ive virulence genes were intentionally deleted, and one gene was inactivated by inserting a synthetic DNA fragment. The virus was passaged 71 times in CV-1 cells to obtain the VACΔ6 strain from the VAC LIVP clonal variant. Such a long passage history might have led to additional off-target mutations in VACΔ6 compared to the original LIVP variant. To prevent this, we performed a genome-wide sequencing of VAC LIVP, VACΔ6, and f ive intermediate viral strains to assess possible off-target mutations. A comparative analysis of complete viral genomes showed that, in addition to target mutations, only two nucleotide substitutions occurred spontaneously when obtaining VACΔ4 from the VACΔ3 strain; the mutations persisting in the VACΔ5 and VACΔ6 genomes. Both nucleotide substitutions are located in intergenic regions (positions 1431 and 189738 relative to LIVP), which indicates an extremely rare occurrence of off-target mutations when using transient dominant selection to obtain recombinant VAC variants with multiple insertions/deletions. To assess the genome stability of the resulting attenuated vaccine strain, 15 consecutive cycles of cultivation of the industrial VACΔ6 strain were performed in 4647 cells certif ied for vaccine production in accordance with the "Guidelines for Clinical Trials of Medicinal Products". PCR and sequencing analysis of six DNA fragments corresponding to the regions of disrupted genes in VACΔ6 showed that all viral DNA sequences remained unchanged after 15 passages in 4647 cells.В связи с прекращением после 1980 г. массовой противооспенной вакцинации в настоящее время практически полностью утрачен коллективный иммунитет человеческой популяции к ортопоксвирусным инфекциям. Вследствие этого увеличилась опасность распространения в мире зоонозных ортопоксвирусных инфекций человека, обусловленных вирусами оспы обезьян или оспы коров. Противооспенные вакцины первого поколения на основе вируса осповакцины (Vaccinia virus, VAC) являются реактогенными и поэтому в современных условиях не пригодны для массовой вакцинации. Это обусловливает необходимость разработки современных безопасных живых вакцин на основе VAC с применением методов генетической инженерии. С использованием метода временной доминантной селекции нами создан штамм VACΔ6, в геноме которого пять генов вирулентности направленно делетированы, а один ген инактивирован встройкой синтетического фрагмента ДНК. В процессе получения штамма VACΔ6 из клонового варианта VAC LIVP вирус прошел 71 пассаж в культуре клеток CV-1. Такая длинная пассажная история могла привести к дополнительным нецелевым изменениям в геноме штамма VACΔ6 относительно исходного LIVP. Поэтому для оценки возможных нецелевых изменений провели полногеномное секвенирование VAC LIVP, VACΔ6 и пяти промежуточных штаммов вируса. Сравнительный анализ полных вирусных геномов показал, что, помимо целевых нарушений, спонтанно произошли только две нуклеотидные замены при получении VACΔ4 из штамма VACΔ3 и сохранившиеся в геноме VACΔ5 и VACΔ6. При этом обе эти замены находятся в межгенных участках (позиции 1431 и 189738 относительно штамма LIVP), что указывает на крайне редкое возникновение нецелевых изменений при использовании методики временной доминантной селекции для получения рекомбинантных VAC со множественными встройками/делециями. Для выяснения стабильности генома полученного аттенуированного вакцинного штамма и в соответствии с «Руководством по проведению клинических исследований лекарственных средств…» выполнено 15 последовательных циклов культивирования производственного штамма вируса VACΔ6 в культуре клеток 4647, аттестованной для производства вакцины. ПЦР-анализ и секвенирование шести фрагментов ДНК, соответствующих районам нарушаемых генов VACΔ6, показали, что после 15 пассажей в культуре клеток 4647 все последовательности вирусной ДНК остались неизменными.

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 regi...

An analysis of the incidence in the dynamics of years (1998-2018) showed that the leading place among infections transmitted by ixodid ticks in the Chelyabinsk region is occupied by tick-borne tick-borne borreliosis (IKB), tick-borne encephalitis (TBE) is less common. Long-term monitoring of the natural foci of TBE and IKB showed their high epizootological potential. In 2018, febrile forms prevailed in the structure of clinical forms of TBE - 61.9%, meningeal and focal forms were found in 28.5% and 9.4%, respectively. Focal forms were represented by meningoencephalitic and polioencephalomyelitis forms with the same frequency of 4.7%. In the structure of the nosological diagnosis of ICD in the South Urals, erythema forms of the disease prevail (69.5%). The detection frequency of Borrelia DNA of the pathogenic complex B.burgdorferi s.l. in the blood serum and leukocyte fraction of patients was 13%. In 4 patients, for the first time in the Chelyabinsk region, DNA of a new Borrelia miyamotoi Borrelia genotype was detected in whole blood samples and in the samples of the leukocyte fraction using PCR-RV. Phylogenetic analysis of B. miyamotoi was carried out on all sequences of glpQ and 23SrRNA gene fragments, deposited at NCBI and homologous sequences of Chelyabinsk-2018 isolates. The disease proceeded in non-erythema form, intoxication syndrome, myalgia, chills were observed in the clinical picture.

In studied blood serum samples from Russian patients hospitalized with suspected dengue fever, one or more markers of dengue fever have been detected, namely, viral RNA, NS1 antigen, and IgM or IgG antibodies. Moreover, only the combined use of various immunological and genetic methods provided a more effective diagnosis of dengue fever. The detection of IgG to dengue virus in patients with high titers of IgG to tick-borne encephalitis virus (TBEV) and, conversely, the detection of IgG to TBEV virus in patients with high titers of IgG to dengue has been documented. The results confirm the literature data on pronounced antigenic cross-sections between different representatives of flaviviruses, which may complicate the ELISA diagnosis of TBEV and dengue in patients. Genotyping of obtained dengue virus isolates showed that imported cases of dengue fever are caused by subtypes 1–4 of the dengue virus, with the prevalence of infection cases caused by dengue virus 1. Sequencing of these dengue virus isolates, including genome-wide, showed that viral isolates obtained on the territory of Russia are phylogenetically close to modern variants of the dengue virus circulating in the countries of Southeast Asia.

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.

Hepatitis A virus (HAV) continues to cause outbreaks of viral hepatitis, which remains a socially significant disease despite existing vaccines. During the production of vaccines, HAV strains that require prolonged incubation of infected cell cultures, up to 4 weeks, are used. Several HAV strains, which are fast-growing on cell cultures, up to 7–10 days, are known. This paper presents the results of virological characterization and analysis of the genome of the HAV strain MB-7/293 (HepA-293) derived from MB-7/4647, one of the fast-growing strains, which was obtained by adapting it to HEK293 cell culture (human embryonic kidney cells). It was shown that the MB-7/293 strain significantly exceeds the parent MB-7/4647 strain in productivity when cultured on HEK293 cell culture, i.e., in the yield of the infectious virus and the virus-specific antigen. Analysis of the nucleotide sequence of full-length genomic RNA of the MB-7/293 strain showed that it differs from the genome sequence of MB-7/4647 in three nucleotide substitutions in the 5'-untranslated region and 20 nucleotide substitutions in the coding region, which lead to substitutions of ten amino acid residues in the proteins VP3, VP1, 2B and 2C. Comparative analysis of the genomic sequences of the strains MB-7/293, MB-7/4647, and other HAV strains, including the previously described fast-growing strains, allowed to reveal mutations that affect the reproductive properties of the virus.

Kindia tick virus (KITV) is a novel multicomponent virus first detected by direct sequencing of Rhipicephalus geigyi ticks in Guinea in 2017. Here, we present a complete coding genome sequence for all four segments of KITV/2017/1. This virus appears to be evolutionarily related to unclassified flaviviruses, such as Alongshan virus.

Data obtained from monitoring cases of severe influenza, cases of vaccinated individuals, and unique cases were used to describe influenza viruses that circulated in Russia in the 2018–2019 epidemic season. A high proportion of the mutations D222G/N in A(H1N1)pdm09 HA was detected in fatal cases. Viruses of the B/Victoria lineage with deletions in HA were detected in Russia, and a reassortant seasonal influenza A(H1N2) virus was identified. A C-terminal truncation in the NS1 protein was detected in a substantial proportion of A(H3N2) viruses.

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.

The occurrence of Mediterranean fever with periods of increase and decrease has been recorded in the Crimean peninsula. The city of Sevastopol and its vicinity are known endemic areas for this disease. Some of the most active agents in the spread of this rickettsiosis are feral and abandoned dogs. The aim of this study was to test ticks parasitizing dogs in Sevastopol for the presence of Rickettsia using molecular methods. The testing of ticks was carried out using real-time PCR and the ‘Real Best DNA Rickettsia species’ kit (AO ‘Vector-Best’) followed by sequence identification of the rickettsial DNA detected. The DNA marker for Rickettsia species (a conservative area of citrate synthase gene, gltA) was detected in 16 of 84 (19.1%) samples of Rhipicephalus sanguineus ticks tested. Larger fragments of gltA, ompA and sca4 were amplified and sequenced for 10 of 16 PCR-positive samples. Rickettsia DNA amplified from eight of the samples matched the sequence of Rickettsia conorii conorii Malish, the causative agent of Mediterranean fever. The sequences of Rickettsia DNA from two other ticks had the closest match to homologous fragments of Rickettsia massiliae, a pathogenic spotted fever rickettsia that was identified in the Crimean Peninsula for the first time as part of this study. The detection of two pathogenic species of Rickettsia in the studied ticks suggests the potential for two rickettsial diseases in the region and warrants further epidemiological and clinical studies.

This study aimed to assess the antiviral susceptibility of influenza A(H5N8) viruses isolated in Russia in 2014–2018. Genetic analysis of 57 Russian isolates with full genome sequences did not find any markers of reduced susceptibility to baloxavir. Only one strain bore an amino acid substitution associated with adamantane resistance (M2-S31N). The neuraminidase of 1 strain had an NA-N293/294S (N8/N2 numbering) substitution associated with reduced inhibition by oseltamivir and normal inhibition by zanamivir, which was confirmed phenotypically. There were no other strains with reduced inhibition by oseltamivir and zanamivir in the phenotypic analysis. In order to estimate the worldwide prevalence of influenza A(H5N8) viruses bearing genetic markers of antiviral resistance, genome sequences deposited in the GISAID database were analyzed (database access: October 2020). The M2 protein of A(H5N8) viruses from the 2.3.4.4c clade had an M2-S31N substitution associated with reduced susceptibility to adamantanes. On the contrary, the majority (94%) of viruses from the 2.3.4.4b clade had the M2-S31 genotype. Fewer than 1% of analyzed viruses had amino acid substitutions associated with reduced susceptibility to baloxavir (PA-E199G, PA-E199E/G) or reduced or highly reduced inhibition by neuraminidase inhibitors (NA-R150/152K, NA-I221/222M, NA-I221/222I/M, NA-I221/222V, NA-I115/117V, NA-G145/147R, NA-R291/292R/K). An NA-N293/294S substitution was not present in sequences from the GISAID database. To the best of our knowledge, influenza A(H5N8) viruses with reduced inhibition by oseltamivir bearing an NA-N293/294S substitution have not been previously reported in epidemiological surveillance studies.

Outbreaks of influenza, which is a contagious respiratory disease, occur throughout the world annually, affecting millions of people with many fatal cases. The D222G/N mutations in the hemagglutinin (HA) gene of A(H1N1)pdm09 are associated with severe and fatal human influenza cases. These mutations lead to increased virus replication in the lower respiratory tract (LRT) and may result in life-threatening pneumonia. Targeted NGS analysis revealed the presence of mutations in major and minor variants in 57% of fatal cases, with the proportion of viral variants with mutations varying from 1% to 98% in each individual sample in the epidemic season 2018–2019 in Russia. Co-occurrence of the mutations D222G and D222N was detected in a substantial number of the studied fatal cases (41%). The D222G/N mutations were detected at a low frequency (less than 1%) in the rest of the studied samples from fatal and nonfatal cases of influenza. The presence of HA D222Y/V/A mutations was detected in a few fatal cases. The high rate of occurrence of HA D222G/N mutations in A(H1N1)pdm09 viruses, their increased ability to replicate in the LRT and their association with fatal outcomes points to the importance of monitoring the mutations in circulating A(H1N1)pdm09 viruses for the evaluation of their epidemiological significance and for the consideration of disease prevention and treatment options.

Human carcinoma A431 cells were subcutaneously injected into nude mice at points remote from each other. One of the two xenografts developed afterwards was used for the treatment with a recombinant vaccinia virus, while the other xenograft served as an artificial metastasis. We used the attenuated recombinant vaccinia virus (VACV) VVdGF-GFP2 of the L-IVP strain (GenBank accession number KP233807) with the deletion of two virulence genes, i.e., the genes of the virus growth factor and thymidine kinase, with the gene for the green fluorescent protein (GFP2) inserted in the place of the latter. The treatment was performed by a single intratumoral injection of recombinant VACV at a dose of 107 PFU/mouse. VACV was detected in cells of the artificial metastasis as early as two days after infection. After eight days, the virus concentration was comparable with that in the infected tumor (~109 PFU/mL). Electron microscopy revealed selective replication of the recombinant virus in the tumor cells. The targeted accumulation of GFP2 in both tumor and metastasis was shown in the UV images of the mice obtained using the In-vivo Multispectral Imaging System (Bruker, Germany). The complete destruction of the tumor was detected after 12 days, and that of metastasis, after 20 days after the injection of VVdGF-GFP2. The destruction process was accompanied by pronounced edema and leukocyte infiltration of the tumor tissue. The recombinant virus induced a significant reduction in the sizes of the tumor and metastasis. By the end of the experiment (35 days), the xenografts in the control mice were 10 times larger than those in the treated mice (5000 vs 500 mm3). Our study has shown that the attenuated VACV administered by the peripheral route can not only destroy the primary tumor but also has a distinct antimetastatic effect.

The occurrence of Mediterranean fever with periods of increase and decrease has been recorded in the Crimean peninsula. The city of Sevastopol and its vicinity are known endemic areas for this disease. Some of the most active agents in the spread of this rickettsiosis are feral and abandoned dogs. The aim of this study was to test ticks parasitizing dogs in Sevastopol for the presence of Rickettsia using molecular methods. The testing of ticks was carried out using real-time PCR and the ‘Real Best DNA Rickettsia species’ kit (AO ‘Vector-Best’) followed by sequence identification of the rickettsial DNA detected. The DNA marker for Rickettsia species (a conservative area of citrate synthase gene, gltA) was detected in 16 of 84 (19.1%) samples of Rhipicephalus sanguineus ticks tested. Larger fragments of gltA, ompA and sca4 were amplified and sequenced for 10 of 16 PCR-positive samples. Rickettsia DNA amplified from eight of the samples matched the sequence of Rickettsia conorii conorii Malish, the causative agent of Mediterranean fever. The sequences of Rickettsia DNA from two other ticks had the closest match to homologous fragments of Rickettsia massiliae, a pathogenic spotted fever rickettsia that was identified in the Crimean Peninsula for the first time as part of this study. The detection of two pathogenic species of Rickettsia in the studied ticks suggests the potential for two rickettsial diseases in the region and warrants further epidemiological and clinical studies.

The 2017-2018 influenza epidemic season in Russia was characterized by a relatively low morbidity and mortality. We evaluated herd immunity prior to the 2017-2018 influenza season in hemagglutination inhibition assay, and performed characterization of influenza viruses isolated from severe or fatal influenza cases and from influenza cases in people vaccinated in the fall of 2017. During the 2017-2018 epidemic season, 87 influenza A and B viruses were isolated and viruses of the 75 influenza cases, including selected viral isolates and viruses analyzed directly from the original clinical material, were genetically characterized. The analyzed A(H1N1)pdm09 viruses belonged to clade 6B.1, B/Yamagata-like viruses belonged to clade 3, and B/Victoria-like viruses belonged to clade 1A and they were antigenically similar to the corresponding vaccine strains. A(H3N2) viruses belonged to clade 3C.2a and were difficult to characterize antigenically and the analysis indicated antigenic differences from the corresponding egg-grown vaccine strain. The next generation sequencing revealed the presence of D222/G/N polymorphism in the hemagglutinin gene in 32% of the analyzed A(H1N1)pdm09 lethal cases. This study demonstrated the importance of monitoring D222G/N polymorphism, including detection of minor viral variants with the mutations, in the hemagglutinin gene of A(H1N1)pdm09 for epidemiological surveillance. One strain of influenza virus A(H1N1)pdm09 was resistant to oseltamivir and had the H275Y amino acid substitution in the NA protein. All other isolates were susceptible to NA inhibitors. Prior to the 2017-2018 epidemic season, 67.4 million people were vaccinated, which accounted for 46.6% of the country's population. Just before the epidemic season 33-47% and 24-30% of blood sera samples collected within the territory of Russia showed the presence of protective antibody titers against vaccine strains of influenza A and influenza B/Victoria-like, respectively. Mass vaccination of the population had evidently reduced the severity of the flu epidemic during the 2017-2018 influenza epidemic season in Russia.

[This corrects the article DOI: 10.1371/journal.pone.0220401.].

The chicken anemia virus gene that encodes apoptin, a selective killer of cancer cells, was synthe-sized and inserted into the vaccinia virus (strain L-IVP) genome. The insertion replaces a major part of the viral C11R gene that encodes viral growth factor, which is important for virulence. The recombinant virus VVdGF-ApoS24/2 was obtained by transient dominant selection using the gene of puromycin resistance as a selective marker. The expression of the apoptin gene from a synthetic early-late promoter of vaccinia virus ensured the efficient accumulation of the target protein in VVdGF-ApoS24/2-infected cells. Although recombinant apoptin carried the signal peptide of the virus growth factor at the N-end, the protein was not secreted into the culture medium. The recombinant virus VVdGF-ApoS24/2 exhibited significantly higher selective lytic activity in human cancer cell lines (A549, A431, U87MG, RD, and MCF7) than the parent strain L-IVP and its VVdGF2/6 variant with C11R deletion. These results suggest that the use of apoptin can be an efficient means of enhancing the natural anticancer activity of vaccinia virus.

Most of the live vaccine doses of vaccinia virus donated to the Intensified Smallpox Eradication Programme after 1971 were prepared using the L-IVP strain. A mixture of three clones of the L-IVP strain was sequenced using MySEQ. Consensus sequence similarity with the vaccinia virus Lister strain is 99.5%.

The cDNA fragments 466–966 and 878–1088 coding for the precursor M (prM) protein and polypeptide M31–75-E1–30 of the Russian strain LEIV-Vlg99-27889-human of the West Nile virus (WNV) were synthesized and cloned. The corresponding prM and M31–75-E1–30 recombinant polypeptides were purified by affinity chromatography. The prM polypeptide interacted with a polyclonal serum against WNV in ELISA and immunoblotting, demonstrating the immunochemical similarity of the recombinant polypeptide and the native WNV prM protein. Six species-specific monoclonal antibodies (mAbs) against the prM recombinant polypeptide recognized at least four epitopes on the recombinant polypeptides. In addition, mAb 7D11 displayed a virus-neutralizing activity. The patterns of mAb interactions with the prM, M31–75-E1–30, E1–180, and E260–466 recombinant polypeptides revealed cross-reacting epitopes in regions 260–466 of the E protein and 31–75 of the M31–75-E1–30 polypeptide and the WNV prM protein. A spatial model revealed structural similarity of the C-terminal regions of the E and M proteins of WNV, supporting the results of immunochemical experiments. Based on virus neutralization by mAb 7D11, which recognized an epitope mapping to region 31–75 of the WNV M protein, an important function in virus penetration into the cell was assumed for the C-terminal region of the M protein.

The use of a scanning flow cytometer (SFC) to study the evolution of monomers, dimers and higher multimers of latex particles at the initial stage of the immunoagglutination is described. The SFC can measure the light-scattering pattern (indicatrix) of an individual particle over an angular range of 10-60 deg. A comparison of the experimentally measured and theoretically calculated indicatrices allows one to discriminate different types of latex particles (i.e. monomers, dimers, etc.) and, therefore, to study the evolution of immunoagglutination process. Validity of the approach was verified by simultaneous measurements of light-scattering patterns and fluorescence from individual polymer particles. Immunoagglutination was initiated by mixing bovine serum albumin (BSA)-covered latex particles (of 1.8 um in diameter) with anti-BSA IgG. The analysis of experimental data was performed on the basis of a mathematical model of diffusion-limited immunoagglutination aggregation with a steric factor. The steric factor was determined by the size and the number of binding sites on the surface of a latex particle. The obtained data are in good agreement with the proposed mathematical modeling., 12 pages, 6 figures

A statistical approach is presented to model the kinetics of cell distribution in the process of ligand–receptor binding on cell surfaces. The approach takes into account the variation of the amount of receptors on cells assuming the homogeneity of monovalent binding sites and ligand molecules. The analytical expressions for the kinetics of cell distribution have been derived in the reaction-limited approximation. In order to demonstrate the applicability of the mathematical model, the kinetics of binding the rabbit, anti-mouse IgG with Ig-receptors of the murine hybridoma cells has been measured. Anti-mouse IgG was labeled with fluorescein isothiocyanate (FITC). The kinetics of cell distribution on ligand–receptor complexes was observed during the reaction process by real-time measuring of the fluorescence and light-scattering traces of individual cells with the scanning flow cytometer. The experimental data were fitted by the mathematical model in order to obtain the binding rate constant and the initial cell distribution on the amount of receptors.

Introduction. ARI occupying the first place in the structure of total human morbidity. The aim of the study was to investigate the species diversity of the viruses causing ARI among residents of the Novosibirsk region during epidemic season (October to April). Materials and methods. 164 nasopharyngeal swabs were collected and analyzed. Viral RNA/DNA, cDNA synthesis and PCR were carried out employing “RIBO-prep”, “Reverta-L”, “AmpliSens Influenza virus A/B-FL” and “AmpliSens ARI-screen-FL” kits (CRI of Epidemiology). Results. Etiological agent of the disease was found in 69 (43%) samples. Monoinfection was found in 58 (35%).In 14 (9%) samples were detected serogroup I coronaviruses, in 13 (8%) rhinoviruses, in 7 (4%) respiratory syncytial virus, in 6 (4%) parainfluenza virus type 1, in 5 (3%) parainfluenza virus type 3. Adenoviruses and bocavirus were identified in 3 (2%) samples. Parainfluenza virus type 2 and 4, metapneumovirus, serogroup II coronaviruses (HKU1 and OC43) were presented in 2 (1%) samples. In 11 (7%) samples was found mixed infection. Conclusion. The majority of common colds were caused by serogroup I coronaviruses (NL63 and 229E), rhinoviruses and mixed infections. The peak of species variability of viruses caused acute respiratory infections was determined in age group of children 2–4 years old. In older age groups the species variability of analyzed viruses was decreased, rhinovirus infection becomes prevalent.

Two chimeric antibodies (ch) 13D6 and 10C2 against the glycoprotein E of tick-borne encephalitis virus (TBEV) were constructed by fusing variable regions of murine monoclonal antibodies (Mabs) 13D6 and 10C2 to human constant regions. Monovalent analogues of these antibodies in format of single-chain antibodies (scFv or sc) were developed, as well. The ch13D6, ch10C2, sc13D6 and sc10C2 exhibited binding characteristics similar to parental Mabs. Only the ch13D6 and sc13D6 were able to neutralize TBEV infectivity in vitro. The in vitro neutralization provided by ch13D6 suggests that this antibody can be further developed into a potent prophylaxis and therapy for tick-borne encephalitis (TBE) infection.

A recombinant pSC13D6 plasmid DNA was constructed based on cDNA fragments of genes encoding variable domains of heavy and light chains of the MKA 13D6 monoclonal antibody against glycoprotein of the tick-borne encephalitis (TBE) virus. This plasmid provided expression in Escherichia coli cells of the sc13D6 single-chain antibody against the TBE virus. The produced antibodies could bind to the TBE virus, strain 205, and the TBE virus recombinant E protein. The affinity constant of purified sc13D6 was (3.0 +/- 0.2) x 10(7) M(-1) for the equilibrium state and (2.8 +/- 0.3) x 10(7) M(-1) in the case of antigen-antibody formation on the surface. The obtained single-chain antibody could inhibit the infection potency of the TBE virus on a monolayer of eukaryotic cells. The calculated IC50 value for sc13D6 was 16.7 microg/ml.

We have studied the light-scattering properties of human erythrocytes both experimentally and theoretically. In the experimental study measurements were performed with a Scanning Flow Cytometer (SFC). The SFC can measure the light-scattering pattern (indicatrix) of an individual particle over an angular range of 10-60 degrees. We have observed polymorphism in the measured set of indicatrices. To understand the reason for this polymorphism, we have made a theoretical study of the scattering properties of erythrocytes. The Wentzel-Kramer-Brillouin approximation has been employed to calculate indicatrices of individual erythrocytes in different orientations relative to the incident light beam. The indicatrices were calculated over an angular range of 15-35 degrees. A comparison of the experimentally measured and theoretically calculated indicatrices shows that the polymorphism is due mainly to the different orientation of the erythrocytes in the flow. The effect caused by the Poiseuille profile of the flow on an individual erythrocyte within the SFC cuvette capillary was studied theoretically by use of the Stokes approximation. Rotation of an erythrocyte was predicted by this theoretical analysis, and this prediction was further verified by comparison with experimental results.

Complementary DNA fragments (nucleotides 466-966 and 878-1088) encoding prM protein and polypeptide M31-75-E1-30 of West Nile virus (WNV), strain LEIV-Vlg99-27889-human, were obtained and cloned. Recombinant polypeptides prM and M3175-E1-30 having amino acid sequences corresponding to the cloned cDNA fragments were purified by affinity chromatography. According to ELISA and Western blotting prM protein interacted with polyclonal antibodies against WNV. This is indicative the immunochemical similarity of WNV recombinant and native protein prM. 6 types of species-specific monoclonal antibodies (MAbs) raised against recombinant polypeptide prM recognized at least four epitopes within recombinant polypeptides prM and M31-75-E1-30. MAbs 7D11 were active in the virus - neutralization assay. Analysis of interaction of the MAbs with recombinant polypeptides prM, M31-75-EI-30, E1-180, E260-466 revealed cross-reactive epitopes within 260-466 amino acid residues (aa) of WNV protein E, 31-75 aa of polypeptide M31-75-E1-30 and protein prM. Proposed spatial model of proteins E and M C-end fragments shown similarity of their three-dimensional structures confirming results of immunochemical assay. Neutralization of viral infectivity by MAbs 7D11 raised against epitope within 31-75 aa t of protein M is evidence of important function of C-end region in the process of flaviviral penetration into host cell.

A study of temporal and quantitative characteristics of inhibition of replication of Venezuelan equine encephalomyelitis (VEE) virus, strain TC-83, in Vero and CPE on PK cells showed purified polyclonal rabbit antibodies to human recombinant laminin-binding protein (LBP) to be able to block completely the development of cytopathic effect (CPE) in such cells, when infected with 10(7) CPE60. The extent of VEE infection inhibition in Vero was in direct proportion to a concentration of specific antibodies within a range of 0.44-3 microg/100 microl. When antibodies were added to Vero cells after they were infected, there was a gradual attenuation of the inhibition effect, which stopped almost completely 9 hours after the antibodies were placed. Inhibition was effective at 4 degrees C and 37 degrees C. A lack of synthesis of viral glycoprotein E2 in Vero cells infected in the presence of antibodies to LBP is an extra argument proving that the VEE replication is inhibited at early infection stages. The data obtained demonstrated the general LBP significance for the penetration of VEE into mammalian cells and the related importance of designing new antiviral drugs against alpha-viral infection, which are based on blocking the mechanism of receptor penetration of the virus into the cell.

Flow cytometry is a powerful tool for the analysis of individual particles in a flow. Differential light scattering (an indicatrix) was used for many years to obtain morphologic information about microorganisms. The indicatrices play the same role for individual particle recognition as a spectrum for substance characterization. We combined two techniques to analyze the indicatrix of the cells for the purpose of developing a database of light-scattering functions of cells.The scanning flow cytometer (SFC) allows the measurement of the entire indicatrix of individual particles at polar angles ranging from 5 degrees to 100 degrees. In this work, light-scattering properties of Escherichia coli have been studied both experimentally and theoretically with the SFC and the T-matrix method, respectively. The T-matrix method was used because of the nonspherical shape of E. coli cells, which were modeled by a prolate spheroid.The indicatrices of E. coli cells were stimulated with T-matrix method at polar angles ranging from 10 degrees to 60 degrees. The absolute cross-section of light scattering of E. coli has been determined comparing the cross section of polystyrene particles modeled by a homogeneous sphere. The E. coli indicatrices were compared for logarithmic and stationary phases of cell growth.The indicatrices of E. coli were reproducible and could be used for identification of these cells in biologic suspensions. The angular location of the indicatrix minimum can be used in separation of cells in logarithmic and stationary phases. To use effectively the indicatrices for that purpose, the light-scattering properties of other microorganisms have to be studied.

The differential light-scattering pattern, an indicatrix, provides the most complete characterization of the optical properties of a particle. Particle classification can be performed on the basis of particle parameters retrieved from the indicatrices. This classification extends the ability of flow cytometry in particle recognition.The scanning flow cytometer (SFC) permits an acquisition of traces of light scattering signals, i.e., native SFC traces, from single particles. The acquired native SFC traces are transformed into indicatrices. The performance of the SFC in measurements of indicatrices has been demonstrated for the following particles: lymphocytes, erythrocytes, polystyrene particles, and milk-fat particles.The structure and profile of the indicatrix for each particle type have been found to be unique. Classification of polystyrene particles has been performed on the basis of the map formed by particle refractive index and size. The polystyrene particles were classified using this map into different size categories ranging from 1.4-7 microm, with a size deviation of 0.07 microm.The method based on analysis of native SFC traces shows better performance in particle classification than the method based on the particle refractive index and size map. The classification performance of the SFC will be useful, for example, for particle sorting and particle identification, and with additional fluorescent measurements may have applications in multiparameter particle-based immunoassay.

We have studied the kinetics of binding of dissolved antigens to antibody-receptors on the surface of the intact living cells. We have combined the Scanning Flow Cytometry with fluorescent immunoassay. Measurements of the kinetics were carried out for rabbit anti-mouse immunoglobulins that bind to receptors of the mouse hybridomas. Anti-mouse immunoglobulins were labeled with fluorescein isothiocyanate molecules (FITC). The individual cells were identified from the native Scanning Flow Cytometer records in our experiment. The kinetics of the formation of antigen-antibody complexes was measured from the fluorescence from the individual cells. A kinetic scheme for the heterogeneous reactions between ligands and receptors has been suggested. This scheme utilizes the concept of a distribution function of an amount of active receptors on the cell surface. The analytical solution for the system of the kinetic equations has been found in the reaction-limited approximation. The experimental kinetics were fitted by this mathematical model to determine the rate constant for binding reaction of the used antigen-antibody pair.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Light scattering is a powerful and useful phenomenon for identification of microscopic particles. The angular dependency of light-scattering intensity, an indicatrix, contains complete information about particle size, internal structure, shape, refractive index.1