Human CD8+ T cell cross-reactivity across influenza A, B and C viruses.

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8+ T cells confer cross-protection against IAV strains, however the responses of CD8+ T cells to IBV and ICV are understudied. We investigated the breadth of CD8+ T cell cross-recognition and provide evidence of CD8+ T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8+ T cell epitopes from IBVs that were protective in mice and found memory CD8+ T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8+ T cells displayed tissue-resident memory phenotypes. Notably, CD38+Ki67+CD8+ effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8+ T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.

Unique Directional Motility of Influenza C Virus Controlled by Its Filamentous Morphology and Short-Range Motions.

Influenza virus motility is based on cooperation between two viral spike proteins, hemagglutinin (HA) and neuraminidase (NA), and is a major determinant of virus infectivity. To translocate a virus particle on the cell surface, HA molecules exchange viral receptors and NA molecules accelerate the receptor exchange of HA. This type of virus motility was recently identified in influenza A virus (IAV). To determine if other influenza virus types have a similar receptor exchange mechanism-driven motility, we investigated influenza C virus (ICV) motility on a receptor-fixed glass surface. This system excludes receptor mobility, which makes it more desirable than a cell surface for demonstrating virus motility by receptor exchange. Like IAV, ICV was observed to move across the receptor-fixed surface. However, in contrast to the random movement of IAV, a filamentous ICV strain, Ann Arbor/1/50 (AA), moved in a straight line, in a directed manner, and at a constant rate, whereas a spherical ICV strain, Taylor/1233/47 (Taylor), moved randomly, similar to IAV. The AA and Taylor viruses each moved with a combination of gradual (crawling) and rapid (gliding) motions, but the distances of crawling and gliding for the AA virus were shorter than those of the Taylor virus. Our findings indicate that like IAV, ICV also has a motility that is driven by the receptor exchange mechanism. However, compared with IAV movement, filamentous ICV movement is highly regulated in both direction and speed. Control of ICV movement is based on its specific motility employing short crawling and gliding motions as well as its own filamentous morphology.IMPORTANCE Influenza virus enters into a host cell for infection via cellular endocytosis. Human influenza virus infects epithelial cells of the respiratory tract, the surfaces of which are hidden by abundant cilia that are inactive in endocytosis. An open question is the manner by which the virus migrates to endocytosis-active domains. In analyzing individual virus behaviors through single-virus tracking, we identified a novel function of the hemagglutinin and esterase of influenza C virus (ICV) as the motility machinery. Hemagglutinin iteratively exchanges a viral receptor, causing virus movement. Esterase degrades the receptors along the trajectory traveled by the virus and prevents the virus from moving backward, causing directional movement. We propose that ICV has a unique motile machinery directionally controlled via hemagglutinin sensing the receptor density manipulated by esterase.

Influenza C and D Viruses Package Eight Organized Ribonucleoprotein Complexes.

Influenza A and B viruses have eight-segmented, single-stranded, negative-sense RNA genomes, whereas influenza C and D viruses have seven-segmented genomes. Each genomic RNA segment exists in the form of a ribonucleoprotein complex (RNP) in association with nucleoproteins and an RNA-dependent RNA polymerase in virions. Influenza D virus was recently isolated from swine and cattle, but its morphology is not fully studied. Here, we examined the morphological characteristics of D/bovine/Yamagata/10710/2016 (D/Yamagata) and C/Ann Arbor/50 (C/AA), focusing on RNPs packaged within the virions. By scanning transmission electron microscopic tomography, we found that more than 70% of D/Yamagata and C/AA virions packaged eight RNPs arranged in the "1+7" pattern as observed in influenza A and B viruses, even though type C and D virus genomes are segmented into only seven segments. These results imply that influenza viruses generally package eight RNPs arranged in the "1+7" pattern regardless of the number of RNA segments in their genome.IMPORTANCE The genomes of influenza A and B viruses are segmented into eight segments of negative-sense RNA, and those of influenza C and D viruses are segmented into seven segments. For progeny virions to be infectious, each virion needs to package all of their genomic segments. Several studies support the conclusion that influenza A and B viruses selectively package eight distinct genomic RNA segments; however, the packaging of influenza C and D viruses, which possess seven segmented genomes, is less understood. By using electron microscopy, we showed that influenza C and D viruses package eight RNA segments just as influenza A and B viruses do. These results suggest that influenza viruses prefer to package eight RNA segments within virions independent of the number of genome segments.

Effect of Phosphorylation of CM2 Protein on Influenza C Virus Replication.

CM2 is the second membrane protein of the influenza C virus and has been demonstrated to play a role in the uncoating and genome packaging processes in influenza C virus replication. Although the effects of N-linked glycosylation, disulfide-linked oligomerization, and palmitoylation of CM2 on virus replication have been analyzed, the effect of the phosphorylation of CM2 on virus replication remains to be determined. In this study, a phosphorylation site(s) at residue 78 and/or 103 of CM2 was replaced with an alanine residue(s), and the effects of the loss of phosphorylation on influenza C virus replication were analyzed. No significant differences were observed in the packaging of the reporter gene between influenza C virus-like particles (VLPs) produced from 293T cells expressing wild-type CM2 and those from the cells expressing the CM2 mutants lacking the phosphorylation site(s). Reporter gene expression in HMV-II cells infected with VLPs containing the CM2 mutants was inhibited in comparison with that in cells infected with wild-type VLPs. The virus production of the recombinant influenza C virus possessing CM2 mutants containing a serine-to-alanine change at residue 78 was significantly lower than that of wild-type recombinant influenza C virus. Furthermore, the virus growth of the recombinant viruses possessing CM2 with a serine-to-aspartic acid change at position 78, to mimic constitutive phosphorylation, was virtually identical to that of the wild-type virus. These results suggest that phosphorylation of CM2 plays a role in efficient virus replication, probably through the addition of a negative charge to the Ser78 phosphorylation site.IMPORTANCE It is well-known that many host and viral proteins are posttranslationally modified by phosphorylation, which plays a role in the functions of these proteins. In influenza A and B viruses, phosphorylation of viral proteins NP, M1, NS1, and the nuclear export protein (NEP), which are not integrated into the membranes, affects the functions of these proteins, thereby affecting virus replication. However, it was reported that phosphorylation of the influenza A virus M2 ion channel protein, which is integrated into the membrane, has no effect on virus replication in vitro or in vivo We previously demonstrated that the influenza C virus CM2 ion channel protein is modified by N-glycosylation, oligomerization, palmitoylation, and phosphorylation and have analyzed the effects of these modifications, except phosphorylation, on virus replication. This is the first report demonstrating that phosphorylation of the influenza C virus CM2 ion channel protein, unlike that of the influenza A virus M2 protein, plays a role in virus replication.

Intrinsic temperature sensitivity of influenza C virus hemagglutinin-esterase-fusion protein.

Influenza C virus replicates more efficiently at 33°C than at 37°C. To determine whether hemagglutinin-esterase-fusion protein (HEF), a surface glycoprotein of influenza C virus, is a restricting factor for this temperature sensitivity, we analyzed the biological and biochemical properties of HEF at 33°C and 37°C. We found that HEF exhibits intrinsic temperature sensitivities for surface expression and fusion activity.

The influenza C virus CM2 protein can alter intracellular pH, and its transmembrane domain can substitute for that of the influenza A virus M2 protein and support infectious virus production.

The influenza C virus CM2 protein and a chimeric influenza A virus M2 protein (MCM) containing the CM2 transmembrane domain were assessed for their ability to functionally replace the M2 protein. While all three proteins could alter cytosolic pH to various degrees when expressed from cDNA, only M2 and MCM could at least partially restore infectious virus production to M2-deficient influenza A viruses. The data suggest that while the CM2 ion channel activity is similar to that of M2, sequences in the extracellular and/or cytoplasmic domains play important roles in infectious virus production.

Role of the CM2 protein in the influenza C virus replication cycle.

CM2 is the second membrane protein of influenza C virus. Although its biochemical characteristics, coding strategy, and properties as an ion channel have been extensively studied, the role(s) of CM2 in the virus replication cycle remains to be clarified. In order to elucidate this role, in the present study we generated CM2-deficient influenza C virus-like particles (VLPs) and examined the VLP-producing 293T cells, VLPs, and VLP-infected HMV-II cells. Quantification of viral RNA (vRNA) in the VLPs by real-time PCR revealed that the CM2-deficient VLPs contain approximately one-third of the vRNA found in wild-type VLPs although no significant differences were detected in the expression levels of viral components in VLP-producing cells or in the number and morphology of the generated VLPs. This finding suggests that CM2 is involved in the genome packaging process into VLPs. Furthermore, HMV-II cells infected with CM2-deficient VLPs exhibited significantly reduced reporter gene expression. Although CM2-deficient VLPs could be internalized into HMV-II cells as efficiently as wild-type VLPs, a smaller amount of vRNA was detected in the nuclear fraction of CM2-deficient VLP-infected cells than in that of wild-type VLP-infected cells, suggesting that the uncoating process of the CM2-deficient VLPs in the infected cells did not proceed in an appropriate manner. Taken together, the data obtained in the present study indicate that CM2 has a potential role in the genome packaging and uncoating processes of the virus replication cycle.

[Influenza virus]. / Gripo virusas.

Every year, especially during the cold season, many people catch an acute respiratory disease, namely flu. It is easy to catch this disease; therefore, it spreads very rapidly and often becomes an epidemic or a global pandemic. Airway inflammation and other body ailments, which form in a very short period, torment the patient several weeks. After that, the symptoms of the disease usually disappear as quickly as they emerged. The great epidemics of flu have rather unique characteristics; therefore, it is possible to identify descriptions of such epidemics in historic sources. Already in the 4th century bc, Hippocrates himself wrote about one of them. It is known now that flu epidemics emerge rather frequently, but there are no regular intervals between those events. The epidemics can differ in their consequences, but usually they cause an increased mortality of elderly people. The great flu epidemics of the last century took millions of human lives. In 1918-19, during "The Spanish" pandemic of flu, there were around 40-50 millions of deaths all over the world; "Pandemic of Asia" in 1957 took up to one million lives, etc. Influenza virus can cause various disorders of the respiratory system: from mild inflammations of upper airways to acute pneumonia that finally results in the patient's death. Scientist Richard E. Shope, who investigated swine flu in 1920, had a suspicion that the cause of this disease might be a virus. Already in 1933, scientists from the National Institute for Medical Research in London - Wilson Smith, Sir Christopher Andrewes, and Sir Patrick Laidlaw - for the first time isolated the virus, which caused human flu. Then scientific community started the exhaustive research of influenza virus, and the great interest in this virus and its unique features is still active even today.

The Matrix protein M1 from influenza C virus induces tubular membrane invaginations in an in vitro cell membrane model.

Matrix proteins from enveloped viruses play an important role in budding and stabilizing virus particles. In order to assess the role of the matrix protein M1 from influenza C virus (M1-C) in plasma membrane deformation, we have combined structural and in vitro reconstitution experiments with model membranes. We present the crystal structure of the N-terminal domain of M1-C and show by Small Angle X-Ray Scattering analysis that full-length M1-C folds into an elongated structure that associates laterally into ring-like or filamentous polymers. Using negatively charged giant unilamellar vesicles (GUVs), we demonstrate that M1-C full-length binds to and induces inward budding of membrane tubules with diameters that resemble the diameter of viruses. Membrane tubule formation requires the C-terminal domain of M1-C, corroborating its essential role for M1-C polymerization. Our results indicate that M1-C assembly on membranes constitutes the driving force for budding and suggest that M1-C plays a key role in facilitating viral egress.

Conformational maturation of the nucleoprotein synthesized in influenza C virus-infected cells.

The conformational maturation of the influenza C virus nucleoprotein (NP) synthesized in infected cells was investigated. Monoclonal antibodies (mAbs) that have previously been characterized [Sugawara, K., Nishimura, H., Hongo, S., Kitame, F., Nakamura, K., 1991. Antigenic characterization of the nucleoprotein and matrix protein of influenza C virus with monoclonal antibodies. J. Gen. Virol. 72, 103-109] enabled this molecular maturation to be detected. Both pulse-labeled and chased NPs could equally retain high reactivity with H31 mAb recognizing a linear epitope on the NP molecule. However, pulse-labeled NP showed three- to four-fold lower reactivity with H27 mAb recognizing a conformational epitope, compared to chased NP. Sedimentation analyses by sucrose gradient centrifugation revealed that the mature NP could readily participate in nucleocapsid formation while the immature NP was free. The immature NP was rapidly transported into the nucleus and its maturation seemed to occur after or during translocation into the nucleus. A single expression of NP cDNA in COS-1 cells demonstrated that the NP maturation was an intrinsic feature of the NP molecule without relation to other viral components.

[Type C influenza].

The influenza C virus genome consists of seven single-stranded RNA segments of negative polarity. The hemagglutinin-esterase (HE) glycoprotein of influenza C virus has three biological activities, i.e. receptor-binding activity for N-acetyl-9-O-acetylneuraminic acid, fusion activity, and receptor-destroying activity, which is a neuraminate-O-acetylesterase. Unspliced mRNA from RNA segment 6 is first translated into a 374-amino-acid protein, P42. P42 is cleaved by signal peptidase, producing M1' and CM2 proteins, composed of the N-terminal 259 amino acids and the C-terminal 115 amino acids, respectively. Xenopus laevis oocytes expressing influenza C virus CM2 protein demonstrated that CM2 protein forms a voltage-activated ion channel permeable to chloride ion.

Hemagglutinin-esterase-fusion (HEF) protein of influenza C virus.

Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion.

Persistent infection with an influenza C virus variant is dominantly established in the presence of the parental wild-type virus.

Two influenza C viruses were used for double-infection experiments to investigate the dominance of their phenotypes. The wild-type virus (C/AA-wt) had been characterized by its short-lived productive cycle, whereas a distinct variant derived from it (C/AA-pi) was demonstrated to persist in long-term passages of infected MDCK cultures. Here we show that the persistent virus C/AA-pi is capable of replicating in the presence of abundant amounts of wild-type virus: the persistent virus could be diluted to 10(-9) within wild-type inoculum, still developing a stable form of persistence. This behaviour was reflected by permanent virus release and by continuous enzymatic activity of the viral HEF glycoprotein in infected cells. All long-term cultures tested remained positive for viral NS protein and vRNA. On the vRNA level, it was shown that viral segments originated from the persistent-type genome, while wild-type vRNAs were not maintained after double-infection. Thus, the genotype of the persistent variant was dominantly selected in serial passages. These results indicate a specific intracellular advantage of persistent influenza C virus over the parental wild-type.

A persistent variant of influenza C virus fails to interact with actin filaments during viral assembly.

C/AA-pi virus, a variant of influenza C/Ann Arbor/1/50 virus, establishes persistent infections in MDCK cells, characterized by low levels of progeny production. During viral assembly, nucleoprotein (NP) was found homogeneously distributed over cytoplasmic and nuclear compartments and matrix (M) protein was likewise localized in a barely structured fashion. In contrast, infections with nonpersistent influenza A, B and C viruses produced cytoplasmic granular structures, which typically consisted of colocalized NP and M proteins. Studies on the in vitro interaction between NP and M proteins revealed identical binding capacities comparing influenza C wild-type virus with the persistent variant. Cytochalasin D treatment of infected cells demonstrated that NP protein of the wild-type virus, but not of the persistent variant, was distinctly associated with cellular actin filaments. Moreover, the assembly characteristics of wild-type virus were modulated in the presence of recombinant persistent-type NP protein towards a behaviour similar to persistent infection. Cell type specificity was particularly illustrated in C/AA-pi virus-infected Vero cells, which did not support viral persistence, but produced granular wild-type-like complexes. Thus, interaction between NP, M and actin proteins (i) is a basic part of the viral assembly process, (ii) is dominantly modulated by NP protein and (iii) is specifically altered in the case of persistent infection.

Inhibition of influenza C viruses by human MxA protein.

Human MxA protein was analyzed for its ability to inhibit the replication of different influenza C viruses. Three laboratory derivatives of viral strain C/Ann Arbor/1/50 were investigated, namely the parental wild-type virus C/AA-wt, the persistent variant C/AA-pi and the highly cytopathogenic variant C/AA-cyt. In addition, strain C/Paris/214/91 isolated from an influenza patient was used. Multiplication of all four viruses was suppressed in MxA-expressing Vero cells, as indicated by a decrease in viral RNA synthesis, viral protein synthesis, virion production and induction of a cytopathic effect. Inhibition correlated with the level of MxA expression. Furthermore, inhibition was independent of cell clone-specific differences in expression of virus receptors, as demonstrated by receptor reconstitution experiments. Thus, human MxA protein has antiviral activity against influenza C viruses.

The effect of influenza C virus on the Purkinje cells of chick embryo cerebellum.

Intra-amniotic inoculation of influenza C virus resulted in observable and quantitatively measurable changes in the Purkinje cells of chick embryo cerebellum. Purkinje cells were visualized by the Golgi-Cox procedure and prepared for statistical and computer evaluation from camera lucida drawings. Four computer-generated measurements (the area of the dendritic arbor, the perimeter of the dendritic tree, and the height and width of the cell's arborization) and two manually counted measurements (total number of branches and the number of first order branches) were made. Analysis of Purkinje cells from influenza C virus-infected embryos showed disturbances in dendritic arborization patterns and misalignment in the arrangement of the cells in the Purkinje cell layer compared to control cells. Statistical evaluation of Purkinje cell arborization showed significant decreases in all measured parameters for the influenza C virus-infected members when compared with the members of the uninfected control group.

Polarized entry of bovine coronavirus in epithelial cells.

Epithelial cells are highly polarized cells divided into an apical and a basolateral plasma membrane. The two domains are composed of a distinct set of proteins and lipids. Concerning virus infection of epithelial cells, the polarity of host cell receptor distribution defines the domain from which infection may be mediated. We were interested to analyze the infection of polarized cells by bovine coronavirus (BCV). The entry of BCV into MDCK I cells was investigated by growing the cells on a permeable support. Cell were infected with BCV from either the apical or basolateral domain. The efficiency of infection was determined by measuring the hemaglutinating activity of the virus released into the apical compartment. Virus replication was only detectable after inoculation from the apical surface. Therefore, infection of MDCK I cells with BCV is restricted to the apical side.

Experimental infection with a persistent influenza C virus variant leads to prolonged genome detection in the chicken lung.

A persistent variant of influenza C virus was used to infect chickens by intraamniotic (i.a.) inoculation. The infected hatchings were analyzed for virus production in different tissues and for the continuous presence of viral RNA genomes. The permissiveness for infection was demonstrated primarily for the chicken lung, besides other organs. Viral antigens could be detected by indirect immunofluorescence staining for a period of 8 days and reisolates were obtained mainly at early time points post infection (p.i.). Nested reverse transcription-polymerase chain reaction (RT-PCR) directed to 3 genomic sequences was positive at least until day 53, whereby no distinct end point was determined. These experiments provide first evidence for the long-term stability of influenza C virus RNA segments in vivo.

[The effect of different factors on the reproduction of influenza viruses and reassortants in cell cultures]. / Vliianie razlichnykh faktorov na reproduktsiiu virusov grippa i reasortantov v kletochnykh kul'turakh.

The influence of the maintenance medium, polyethylene glycol (PEG), DEAE-dextran, and low temperature on reproduction of influenza A, B, and C viruses and their reassortants in diploid and continuous cell cultures was determined. Lowering of pH in the maintenance medium to 6.5 was found to decrease reproduction of influenza A (H1N1) and A (H3N2) viruses and increase that of influenza B viruses. Treatment of cells with PEG solution increased the yield of influenza B and C but not A viruses. However, influenza A virus strains proved to be capable of producing infectious progeny in nonpermissive cell lines treated with PEG. Addition of DEAE-dextran to the medium exerted no effect on the infectivity of influenza A and B reassortants. Moreover, infection of MDCK cells after a "cold shock" led to an increase in hemagglutinin titres in influenza A reassortants.

[Structure and function of the hemagglutinin of influenza viruses].

The hemagglutinin(HA) of influenza virus is a major glycoprotein and plays a crucial role in the early stage of virus infection: HA is responsible for binding of the virus to cell surface receptors, and it mediates liberation of the viral genome into the cytoplasm through membrane fusion. The essential component of the receptor for influenza viruses has been considered to be the sialic acid. Influenza A and B viruses recognize N-acetylneuraminic acid, whereas influenza C virus specifically recognizes N-acetyl-9-O-acetylneuraminic acid as the receptor. Influenza A viruses are subdivided into 15 subtypes by their antigenic differences, but several amino acid residues composing functional domains (receptor binding site and fusion peptide) are shown to be conserved among HAs.