The influenza virus RNA polymerase as an innate immune agonist and antagonist.

Influenza A viruses cause a mild-to-severe respiratory disease that affects millions of people each year. One of the many determinants of disease outcome is the innate immune response to the viral infection. While antiviral responses are essential for viral clearance, excessive innate immune activation promotes lung damage and disease. The influenza A virus RNA polymerase is one of viral proteins that affect innate immune activation during infection, but the mechanisms behind this activity are not well understood. In this review, we discuss how the viral RNA polymerase can both activate and suppress innate immune responses by either producing immunostimulatory RNA species or directly targeting the components of the innate immune signalling pathway, respectively. Furthermore, we provide a comprehensive overview of the polymerase residues, and their mutations, associated with changes in innate immune activation, and discuss their putative effects on polymerase function based on recent advances in our understanding of the influenza A virus RNA polymerase structure.Engineering and Physical Sciences Research Council scholarship EP/ S515322/1 National Institutes of Health grant R21AI14717

Single-Cell Virus Sequencing of Influenza Infections That Trigger Innate Immunity.

Influenza virus-infected cells vary widely in their expression of viral genes and only occasionally activate innate immunity. Here, we develop a new method to assess how the genetic variation in viral populations contributes to this heterogeneity. We do this by determining the transcriptome and full-length sequences of all viral genes in single cells infected with a nominally "pure" stock of influenza virus. Most cells are infected by virions with defects, some of which increase the frequency of innate-immune activation. These immunostimulatory defects are diverse and include mutations that perturb the function of the viral polymerase protein PB1, large internal deletions in viral genes, and failure to express the virus's interferon antagonist NS1. However, immune activation remains stochastic in cells infected by virions with these defects and occasionally is triggered even by virions that express unmutated copies of all genes. Our work shows that the diverse spectrum of defects in influenza virus populations contributes to-but does not completely explain-the heterogeneity in viral gene expression and immune activation in single infected cells.IMPORTANCE Because influenza virus has a high mutation rate, many cells are infected by mutated virions. But so far, it has been impossible to fully characterize the sequence of the virion infecting any given cell, since conventional techniques such as flow cytometry and single-cell transcriptome sequencing (scRNA-seq) only detect if a protein or transcript is present, not its sequence. Here we develop a new approach that uses long-read PacBio sequencing to determine the sequences of virions infecting single cells. We show that viral genetic variation explains some but not all of the cell-to-cell variability in viral gene expression and innate immune induction. Overall, our study provides the first complete picture of how viral mutations affect the course of infection in single cells

Inhibition of antigen-specific and non-specific stimulation of bovine T and B cells by lymphostatin from attaching and effacing Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are enteric bacterial pathogens of worldwide importance. Most EPEC and non-O157 EHEC strains express lymphostatin (also known as LifA), a chromosomally encoded 365-kDa protein. We previously demonstrated that lymphostatin is a putative glycosyltransferase that is important in intestinal colonization of cattle by EHEC serogroup O5, O111, and O26 strains. However, the nature and consequences of the interaction between lymphostatin and immune cells from the bovine host are ill defined. Using purified recombinant protein, we demonstrated that lymphostatin inhibits mitogen-activated proliferation of bovine T cells and, to a lesser extent, proliferation of cytokine-stimulated B cells, but not NK cells. It broadly affected the T cell compartment, inhibiting all cell subsets (CD4, CD8, WC-1, and γδ T cell receptor [γδ-TCR]) and cytokines examined (interleukin 2 [IL-2], IL-4, IL-10, IL-17A, and gamma interferon [IFN-γ]) and rendered T cells refractory to mitogen for a least 18 h after transient exposure. Lymphostatin was also able to inhibit proliferation of T cells stimulated by IL-2 and by antigen presentation using a Theileria-transformed cell line and autologous T cells from Theileria-infected cattle. We conclude that lymphostatin is likely to act early in T cell activation, as stimulation of T cells with concanavalin A, but not phorbol 12-myristate 13-acetate combined with ionomycin, was inhibited. Finally, a homologue of lymphostatin from E. coli O157:H7 (ToxB; L7095) was also found to possess comparable inhibitory activity against T cells, indicating a potentially conserved strategy for interference in adaptive responses by attaching and effacing E. coli

БЕЗОПАСНОСТЬ И ИММУНОЛОГИЧЕСКАЯ ЭФФЕКТИВНОСТЬ СОЧЕТАННОЙ ИММУНИЗАЦИИ ДЕТЕЙ В ВОЗРАСТЕ 6—7 ЛЕТ ВАКЦИНАМИ НАЦИОНАЛЬНОГО КАЛЕНДАРЯ ПРОФИЛАКТИЧЕСКИХ ПРИВИВОК

We estimated the safety of the vaccination for prevention of influenza with Grippol® plus vaccine alongside with vaccination with combined preparations for the prevention of diphtheria and tetanus (Td) and measles, rubella, mumps in children aged 6—7 years. We determined that combined immunization with the indicated vaccines proves good tolerability and low reactogenicity. Vaccine Grippol® Plus shows low reactogenicity , high immunologenicity and does not cause cross-suppression of antibodies in co-administration with other vaccines on vaccination calendar. Also concomitant vaccination with Grippol® plus and other vaccines does not inhibit the development of a specific immune response against influenza.Оценивали безопасность сочетанного введения вакцины для профилактики гриппа Гриппол® плюс с комбинированными препаратами для профилактики дифтерии и столбняка (АДС-М) и кори, краснухи, эпидемического паротита у детей в возрасте 6—7 лет. Показана хорошая переносимость и низкая реактогенность сочетанной иммунизации указанными вакцинами. Вакцина Гриппол® плюс является низкореактогенной, высокоиммуногенной, и не вызывает перекрестной супрессии антител при одновременном введении с другими вакцинами календаря профилактических прививок. В свою очередь, сочетанная вакцинация Гриппол® плюс с другими вакцинами не подавляет развития специфического иммунного ответа против гриппа

Durability of Concrete Reinforced by Specific Rebar

Design of reinforced concrete is based on assuming that applied rebar has no corrosion damages at its surface. However, conventional construction practice allows the rebar embedded into concrete to have surface rust. Being transported in open railway cars, by river or marine transports, being in warehouses of intermediate traders and then affected during this time by an atmospheric precipitation and sometimes even aggressive agents of the environment (for example, being in direct contact with remains of chlorides which are earlier transported in the same cars), rebar arrives to construction site with corrosion defects of different types and various extents. Corrosion damages are caused not only by the conditions of its transportation and storage, but also by poor quality of the steel itself. This may lead to dangerous consequences such as sudden collapses of building constructions without preliminary visible deflections and deformations. One of the reasons of sudden structure collapses related to steel quality is strong structure heterogeneity stressed out by the slag immerge at the surface from the bar’s core. The emergence of slag to a surface of the rebar is especially noticeable in rebar produced by a multilane casting method. The solution of durability problems of reinforced concrete structures is based on competent design, identification and application of necessary methods of corrosion resistance for steel rebar, quality control in producer facilities as well as on the construction site of the consumer, increase in durability of bonding of rebar with concrete, and sufficient choice of primary and secondary anticorrosive protection for rebar and concrete

Investigating the mechanisms behind the immunostimulatory activity of influenza A virus RNA polymerase mutations

Activation of innate immune signalling during influenza A virus (IAV) infection is essential for the development of protective antiviral responses. However, uncontrolled release of proinflammatory cytokines is also associated with severe and fatal disease caused by highly pathogenic avian and pandemic IAV strains. IAV infections are primarily detected by the retinoic acid-inducible gene I (RIG-I), which recognises the partially double-stranded, 5′-triphosphorylated RNA structure that is formed by the protein-free 3′ and 5′ termini (viral promoter) of viral genomic segments as well as aberrant replication products. However, it remains unknown how RIG-I gains access to the viral promoter when it is bound by the viral polymerase, and if other viral RNA species could play a role in RIG-I activation. The viral RNA polymerase itself was shown to act as both an innate immune agonist by producing immunostimulatory RNA species, and as an antagonist by directly interacting with the components of the RIG-I signalling pathway. However, the mechanisms underlying these roles of the viral polymerase in the IAV infection cycle are not completely understood. In this thesis, I investigated the mechanisms behind the immunostimulatory activity of two mutations (D27N and T677A) in the polymerase basic 1 (PB1) subunit that were identified in a single-cell sequencing screen. Viruses containing the PB1 D27N and T677A mutations activated interferon (IFN) responses via the RIG-I signalling pathway. The immunostimulatory activity of the T677A mutation was also recapitulated in an *in vitro* ribonucleoprotein reconstitution assay. Surprisingly, IFN induction by the T677A polymerase was to a large extent driven by its transcriptional activity. Co-immunoprecipitation assays showed that RIG-I bound capped positive-sense RNA produced by the T677A polymerase. This finding suggests the existence of a novel mechanism by which defects in viral RNA synthesis activate innate immune signalling during viral infection. The D27N and T677A viruses were also severely attenuated in cell culture. Reduced fitness of the D27N virus was partly associated with the defects in segment 6 packaging, which led to low levels of neuraminidase expression and impaired haemagglutinin maturation. Adaptation of the T677A and D27N viruses during serial passaging suggested that changes in other viral proteins compensated for the fitness defects created by the PB1 mutations. These changes could be further explored to understand the interplay between the RNA polymerase and other viral proteins at different stages of the viral life cycle

Single-Cell Virus Sequencing of Influenza Infections That Trigger Innate Immunity.

Influenza virus-infected cells vary widely in their expression of viral genes and only occasionally activate innate immunity. Here, we develop a new method to assess how the genetic variation in viral populations contributes to this heterogeneity. We do this by determining the transcriptome and full-length sequences of all viral genes in single cells infected with a nominally "pure" stock of influenza virus. Most cells are infected by virions with defects, some of which increase the frequency of innate-immune activation. These immunostimulatory defects are diverse and include mutations that perturb the function of the viral polymerase protein PB1, large internal deletions in viral genes, and failure to express the virus's interferon antagonist NS1. However, immune activation remains stochastic in cells infected by virions with these defects and occasionally is triggered even by virions that express unmutated copies of all genes. Our work shows that the diverse spectrum of defects in influenza virus populations contributes to-but does not completely explain-the heterogeneity in viral gene expression and immune activation in single infected cells.IMPORTANCE Because influenza virus has a high mutation rate, many cells are infected by mutated virions. But so far, it has been impossible to fully characterize the sequence of the virion infecting any given cell, since conventional techniques such as flow cytometry and single-cell transcriptome sequencing (scRNA-seq) only detect if a protein or transcript is present, not its sequence. Here we develop a new approach that uses long-read PacBio sequencing to determine the sequences of virions infecting single cells. We show that viral genetic variation explains some but not all of the cell-to-cell variability in viral gene expression and innate immune induction. Overall, our study provides the first complete picture of how viral mutations affect the course of infection in single cells

SAFETY AND IMMUNOLOGIC EFFICACY OF COMBINED IMMUNIZATION IN CHILDREN AGED 6—7 YEARS WITH VACCINES FROM THE NATIONAL CALENDAR OF PROPHYLACTICS VACCINES

We estimated the safety of the vaccination for prevention of influenza with Grippol® plus vaccine alongside with vaccination with combined preparations for the prevention of diphtheria and tetanus (Td) and measles, rubella, mumps in children aged 6—7 years. We determined that combined immunization with the indicated vaccines proves good tolerability and low reactogenicity. Vaccine Grippol® Plus shows low reactogenicity , high immunologenicity and does not cause cross-suppression of antibodies in co-administration with other vaccines on vaccination calendar. Also concomitant vaccination with Grippol® plus and other vaccines does not inhibit the development of a specific immune response against influenza