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Molecular mimicry is one of the main processes for producing autoantibodies during infections. Although some autoantibodies are associated with autoimmune diseases, the functions of many autoantibodies remain unknown. Previously, we reported that S16, a mouse (BALB/c) monoclonal antibody against the hemagglutinin-esterase fusion glycoprotein of influenza C virus, recognizes host proteins in some species of animals, but we could not succeed in identifying the proteins. In the present study, we found that S16 cross-reacted with acetyl-CoA acyltransferase 2 (ACAA2), which is expressed in the livers of BALB/c mice. ACAA2 was released into the serum after acetaminophen (APAP) administration, and its serum level correlated with serum alanine aminotransferase (ALT) activity. Furthermore, we observed that S16 injected into mice with APAP-induced hepatic injury prompted the formation of an immune complex between S16 and ACAA2 in the serum. The levels of serum ALT (p < 0.01) and necrotic areas in the liver (p < 0.01) were reduced in the S16-injected mice. These results suggest that S16 may have a mitigation function in response to APAP-induced hepatotoxicity. This study shows the therapeutic function of an autoantibody and suggests that an antibody against extracellular ACAA2 might be a candidate for treating APAP-induced hepatic injury.

The antigenicity of the hemagglutinin esterase (HE) glycoprotein of influenza C virus is known to be stable; however, information about residues related to antigenic changes has not yet been fully acquired. Using selection with anti-HE monoclonal antibodies, we previously obtained some escape mutants and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1. To confirm whether the residues identified as the neutralizing epitope possibly relate to the antigenic drift, we analyzed the growth kinetics of these mutants. The results showed that some viruses with mutations in antigenic site A-1 were able to replicate to titers comparable to that of the wild-type, while others showed reduced titers. The mutants possessing substitutions in the A-2 or A-3 site replicated as efficiently as the wild-type virus. Although the mutant containing a deletion at positions 192 to 195 in the Y-1 site showed lower titers than the wild-type virus, it was confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. Then, we revealed that antigenic changes due to substitutions in the A-1, A-3, and/or Y-1 site had occurred in nature in Japan for the past 30 years. These results suggest that some residues (i.e., 125, 176, 192) in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability.

Unlike influenza A and B viruses that infect humans and cause severe diseases in seasonal epidemics, influenza C virus (ICV) is a ubiquitous childhood pathogen typically causing mild respiratory symptoms. ICV infections are rarely diagnosed and less research has been performed on it despite the virus being capable of causing severe disease in infants. Here we report on the isolation of a human ICV from a child with acute respiratory disease, provisionally designated C/Victoria/2/2012 (C/Vic). The full-length genome sequence and phylogenetic analysis revealed that the hemagglutinin-esterase-fusion (HEF) gene of C/Vic was derived from C/Sao Paulo lineage, while its PB2 and P3 genes evolved separately from all characterized historical ICV isolates. Furthermore, antigenic analysis using the hemagglutination inhibition (HI) assay found that 1947 C/Taylor virus (C/Taylor lineage) was antigenically more divergent from1966 C/Johannesburg (C/Aichi lineage) than from 2012 C/Vic. Structure modeling of the HEF protein identified two mutations in the 170-loop of the HEF protein around the receptor-binding pocket as a possible antigenic determinant responsible for the discrepant HI results. Taken together, results of our studies reveal novel insights into the genetic and antigenic evolution of ICV and provide a framework for further investigation of its molecular determinants of antigenic property and replication., (© 2019 Wiley Periodicals, Inc.)

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.

We used Casd1-deficient mice to confirm that this enzyme is responsible for 9-O-acetylation of sialic acids in vivo. We observed a complete loss of 9-O-acetylation of sialic acid on the surface of myeloid, erythroid and CD4+ T cells in Casd1-deficient mice. Although 9-O-acetylation of sialic acids on multiple hematopoietic lineages was lost, there were no obvious defects in hematopoiesis. Interestingly, erythrocytes from Casd1-deficient mice also lost reactivity to TER-119, a rat monoclonal antibody that is widely used to mark the murine erythroid lineage. The sialic acid glyco-epitope recognized by TER-119 on erythrocytes was sensitive to the sialic acid O-acetyl esterase activity of the hemagglutinin-esterase from bovine coronavirus but not to the corresponding enzyme from the influenza C virus. During erythrocyte development, TER-119+ Ery-A and Ery-B cells could be stained by catalytically inactive bovine coronavirus hemagglutinin-esterase but not by the inactive influenza C hemagglutinin-esterase, while TER-119+ Ery-C cells and mature erythrocytes were recognized by both virolectins. Although the structure of the sialoglycoconjugate recognized by TER-119 was not chemically demonstrated, its selective binding to virolectins suggests that it may be comprised of a 7,9-di-O-acetyl form of sialic acid. As erythrocytes mature, the surfaces of Ery-C cells and mature erythrocytes also acquire an additional distinct CASD1-dependent 9-O-acetyl sialic acid moiety that can be recognized by virolectins from both influenza C and bovine coronavirus., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Influenza D virus has been identified in America, Europe, and Asia. We detected influenza D virus antibodies in cattle and small ruminants from North (Morocco) and West (Togo and Benin) Africa. Dromedary camels in Kenya harbored influenza C or D virus antibodies, indicating a potential new host for these viruses.

Influenza C is not included in the annual seasonal influenza vaccine, and has historically been regarded as a minor respiratory pathogen. However, recent work has highlighted its potential role as a cause of pneumonia in infants. We performed nasopharyngeal or nasal swabbing and/or serum sampling (n = 148) in Lancaster, UK, over the winter of 2014-2015. Using enzyme-linked immunosorbent assay (ELISA), we obtain seropositivity of 77%. By contrast, only 2 individuals, both asymptomatic adults, were influenza C-positive by polymerase chain reaction (PCR). Deep sequencing of nasopharyngeal samples produced partial sequences for 4 genome segments in one of these patients. Bayesian phylogenetic analysis demonstrated that the influenza C genome from this individual is evolutionarily distant to those sampled in recent years and represents a novel genome constellation, indicating that it may be a product of a decades-old reassortment event. Although we find no evidence that influenza C was a significant respiratory pathogen during the winter of 2014-2015 in Lancaster, we confirm previous observations of seropositivity in the majority of the population. (170 words).

Each year, 5% to 20% of the U.S. population is infected with the influenza virus. The influenza season occurs annually between the months of October and May, with 3,000 to 49,000 influenza-related deaths each year. Since complications delay recuperation, those in high-risk groups need to be monitored carefully. High-risk groups are more vulnerable to severe illness and complications of the disease and include: people older than 65 years of age, children under 2 years of age, pregnant women, obese patients, and patients with other serious comorbid conditions. This article provides home healthcare clinicians with important information on influenza, how it is transmitted, influenza virus types and changes in the virus, signs and symptoms of complications, and measures to prevent the occurrence and transmission of influenza.

Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution.

The nonstructural proteins 1 (NS1) from influenza A and B viruses are known as the main viral factors antagonising the cellular interferon (IFN) response, inter alia by inhibiting the retinoic acid-inducible gene I (RIG-I) signalling. The cytosolic pattern-recognition receptor RIG-I senses double-stranded RNA and 5'-triphosphate RNA produced during RNA virus infections. Binding to these ligands activates RIG-I and in turn the IFN signalling. We now report that the influenza C virus NS1 protein also inhibits the RIG-I-mediated IFN signalling. Employing luciferase-reporter assays, we show that expression of NS1-C proteins of virus strains C/JJ/50 and C/JHB/1/66 considerably reduced the IFN-β promoter activity. Mapping of the regions from NS1-C of both strains involved in IFN-β promoter inhibition showed that the N-terminal 49 amino acids are dispensable, while the C-terminus is required for proper modulation of the IFN response. When a mutant RIG-I, which is constitutively active without ligand binding, was employed, NS1-C still inhibited the downstream signalling, indicating that IFN inhibitory properties of NS1-C are not necessarily linked to an RNA binding mechanism.

Background: To quantify the incidence of flu in different groups of the population of Navarra in the 2004-2005 season, and to evaluate the effectiveness of anti-flu vaccination., Methods: The analysis of influenza cases in the system of compulsory notificable diseases was complemented by the individualised notifications in the network of sentinel doctors that covers a population of 22,339 inhabitants. The coverage and effectiveness of the vaccine was studied., Results: Vaccine coverage in (3)65 year olds reached 62%. The incidence of influenza was 42.6 cases per 1,000 inhabitants. It reached a maximum in mid-January, exceeding 750 weekly cases per 100,000 inhabitants and 1,900 cases per 100,000 children. The highest rate of influenza was observed in under-15 year olds (49.4 cases per 1,000 inhabitants) and the lowest in non-institutionalised (3)65 year olds (2.6 per 1,000 inhabitants), although it was higher in geriatric residences (62.1 per 1,000; p<0.0001). Seventy-nine percent of the cases from 5 to 64 years resulted in absenteeism from school or work. The flu virus was identified in 42/65 (65%) nasopharyngeal smears, 90% being influenza virus A(H3). The incidence of influenza was 3.08% in the unvaccinated and 0.45% in the vaccinated (p<0.001). The global effectiveness of the anti-flu vaccine was 65%, and in (3)65 years old it was 73%., Conclusion: Although its effectiveness is not total, the vaccine is the main measure for preventing influenza. The network of sentinel doctors provide useful information for the coordination of care and public health activities against flu.

Influenza C virus (Inf. C) is one of pathogens of human respiratory tract infection and prevalent throughout the world at an early stage in life. However, Inf. C has been isolated only accidentally and there have been few reports on its clinical and epidemiological features. From November 1999 to March 2000, Inf. C was isolated from clinical specimens (throat swabs) of 4 pediataric patients with respiratory tract illness at Hiroshima Prefectural Hospital and was isolated in 4 peditaric patients at the other medical institutions in Hiroshima prefecture. There were no differences in clinical features including duration of illness, duration of fever, maximum body temperature between 4 patients with Inf. C infection and patients with influenza A (H1N1 and H3N2) and influenza B infection from 1992 to 2000. We investigated geographical distribution of patients with inf. C infection and analyzed for antigenic characteristics with a set of monoclonal antibodies against hemagglutinin-esterase glycoproteins. The data suggested that at least two antigenically different Inf. C prevalented in a region during winter from 1999 to 2000.

In a 9-year survey from December 1990 to December 1999 in Sendai City, Japan, we succeeded in isolating a total of 45 strains of influenza C virus. These 45 strains were isolated in clusters within 4 months in a year, especially from winter to early summer. Previous studies of the hemagglutinin-esterase genes of various influenza C virus isolates revealed the existence of five distinct virus lineages (Aichi/1/81-, Yamagata/26/81-, Mississippi/80-, Sao Paulo/82-, and Kanagawa/1/76-related lineage) in Japan between 1970 and the early 1990s (Y. Matsuzaki, K. Mizuta, H. Kimura, K. Sugawara, E. Tsuchiya, H. Suzuki, S. Hongo, and K. Nakamura, J. Gen. Virol. 81:1447-1452, 2000). Antigenic and genetic analyses of the 45 strains showed that they could be divided into these five virus lineages and a few antigenic groups were cocirculating in Sendai City. In 1990 and 1991 the dominant antigenic group was the Aichi/1/81 virus group, and in 1992 it was Yamagata/26/81 virus group. The Mississippi/80 virus group was isolated from 1993 to 1996, and the Yamagata/26/81 virus group reemerged in 1996 and continued to circulate until 1999. This finding led us to a speculation that the replacement of the dominant antigenic groups had occurred by immune selection within the human population in the restricted area. Phylogenetic analysis of seven RNA segments showed that 44 viruses among the 45 strains isolated in our surveillance work were reassortant viruses that have various genome compositions distinguishable from those of the reference strains of the each lineage. This observation suggests that the reassortment between two different influenza C virus strains occurs frequently in nature and the genome composition of influenza C viruses may influence their ability to spread in humans.

During the 3 years from January 1996 to December 1998, a total of 33 strains of influenza C virus were isolated from 10,726 throat swab specimens collected from children with acute respiratory illness who visited two pediatric clinics in Yamagata City, Japan. These 33 strains were isolated in clusters during two different periods, 20 strains in May to August 1996 and the remaining 13 in March to June 1998. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein and phylogenetic analysis of seven RNA segments showed that the 33 influenza C viruses isolated were antigenically and genetically similar and that they were reassortant viruses which had obtained PB2, PB1, HE, M, and NS genes from a C/pig/Beijing/115/81-like virus and P3 and NP genes from a C/Mississippi/80-like virus. These observations suggest strongly that during the survey period of 3 years, two outbreaks of influenza C occurred in Yamagata City, both of which were caused by a reassortant virus having the genome composition described above.

The hemagglutinin-esterase (HE) glycoprotein of influenza C virus is composed of three domains: a stem domain active in membrane fusion (F), an acetylesterase domain (E), and a receptor-binding domain (R). The protein contains eight N-linked glycosylation sites, four (positions 26, 395, 552, and 603) in the F domain, three (positions 61, 131, and 144) in the E domain, and one (position 189) in the R domain. Here, we investigated the role of the individual oligosaccharide chains in antigenic properties, intracellular transport, and biological activities of the HE protein by eliminating each of the glycosylation sites by site-specific mutagenesis. Comparison of electrophoretic mobility between the wild-type and the mutant proteins showed that while seven of the glycosylation sites are used, one (position 131) is not. Analysis of reactivity of the mutants with anti-HE monoclonal antibodies demonstrated that glycosylation at position 144 is essential for the formation of conformation-dependent epitopes. It was also evident that glycosylation at the two sites in the F domain (positions 26 and 603), in addition to that in the E domain (position 144), is required for the HE molecule to be transported from the endoplasmic reticulum and that mutant HEs lacking one of these three sites failed to undergo the trimer assembly. Removal of an oligosaccharide chain at position 144 or 189 resulted in a decrease in the esterase activity. By contrast, two mutants lacking an oligosaccharide chain at position 26 or 603, which were defective not only in cell surface expression but in trimerization, possessed full-enzyme activity, suggesting that the HE monomers present within the cell have acetylesterase activity. Fusion activity of cells expressing each of mutant HEs was found to be comparable with the ability of the protein to be transported to the cell surface, suggesting that there is no specific oligosaccharide chain that plays a critical role in promoting membrane fusion., (Copyright 2001 Academic Press.)

Three influenza C virus strains (C/Yamagata/1/92, C/Yamagata/1/93 and C/Miyagi/5/93) isolated in Yamagata and Sendai Cities, Japan, between June 1992 and May 1993 were found to possess haemagglutinin-esterase glycoproteins that were antigenically indistinguishable from one another but were clearly different from any previous Japanese isolates. To investigate the origin of the 1992/1993 strains, their antigenic and genetic properties were compared with those of eight strains isolated outside Japan between 1967 and 1982. The results showed that the 1992/1993 isolates were closely related to a virus isolated in Brazil in 1982 (C/SaoPaulo/378/82) and that these viruses (including C/SaoPaulo/378/82) are reassortants that had obtained PB1 and NP genes from a C/Yamagata/26/81-like parent and the other genes from another as yet unidentified parent.

The circulation of influenza C viruses in Rio de Janeiro, Brazil, was studied when significant levels of antibodies were detected (56. 7%) with hemagglutination inhibition test, used as a standard methodology for influenza virus studies.

We reported previously that monoclonal antibody S16, which had been raised against the hemagglutinin-esterase (HE) glycoprotein of influenza C/Ann Arbor/1/50 (AA/50) virus, recognizes a linear epitope present on the HE molecules of all influenza C viruses examined except for viruses belonging to a lineage represented by Aichi/1/81 (AI/81). Comparison of the deduced amino acid sequence of HE between viruses on the AI/81-related lineage and those on the others suggests that the epitope recognized by S16 is located in a region containing amino acid residue 403 and that a change from Glu to Lys at this position causes the loss of reactivity with the antibody. To prove it, the wild type (WT) HEs of AA/50 and AI/81 as well as their mutants with an amino acid substitution at residue 403 were expressed in CV-1 cells from the recombinant simian virus 40 (SV40) and tested for reactivity with S16 by immunoprecipitation. The results showed that the AA/50 virus WT and AI/81 virus mutant HEs (both having Glu at residue 403) were reactive with S16 whereas the AI/81 virus WT and AA/50 virus mutant HEs (both having Lys at residue 403) were not. Furthermore, we examined the reactivity of S16 with two synthetic peptides (corresponding to residues 397-409) that possess Glu and Lys at position 403, respectively, by enzyme-linked immunosorbent assays. The results demonstrated that the former peptide but not the latter was reactive with S16. These observations support strongly the notion described above. During this study it was also found that S16 cross-reacts with large T antigen of SV40.

Unspliced mRNA from RNA segment 6 of influenza C virus contains a single open reading frame that potentially encodes a polypeptide of 374 amino acids. This polypeptide, which has not been identified as yet, is predicted to contain the complete amino acid sequence of the matrix protein, M1, as well as that of a small integral membrane protein, CM2. Here, we found that small amounts of two previously unrecognized proteins with apparent molecular masses of 42 (P42) and 44 kDa (P44) were immunoprecipitated with immune serum against CM2. The electrophoretic mobilities of P42 and P44 varied depending on virus strain, indicating that they are virus-coded. Treatment of infected cells with tunicamycin and digestion of immunoprecipitated proteins with various endoglycosidases revealed that P42 is modified by the addition of a high-mannose oligosaccharide chain to generate P44. A monoclonal antibody against M1, like anti-CM2 serum, was able to immunoprecipitate both the P42 and P44 proteins. Furthermore, the tryptic peptide map of either P42 or P44 was indistinguishable from the map of the mixture of M1 and CM2. These results, taken together, suggest strongly that P42 and P44 correspond to the 374 amino acid protein encoded by unspliced RNA segment 6 mRNA and its N-glycosylated form, respectively.

During the winter of 1996 to 1997 two cases of influenza C were confirmed, one by isolation and the second by serology (fourfold increase in hemagglutination inhibition antibodies). The cases of influenza C occurred during an outbreak of influenza A (H3N2) and B viruses. The positive isolation was from one of three throat washings sent to the laboratory, and the other case was from a group of 51 students participating in a study of influenza virus vaccination. It seems, therefore, that influenza C virus should also be considered when examining patients with respiratory infections during the influenza season.

The antigenic and genetic characteristics of the 18 human strains of influenza C virus isolated in Yamagata and Sendai Cities, Japan between January 1991 and February 1993 were investigated. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase glycoprotein showed that the isolates could be divided into three distinct groups closely related to C/Yamagata/26/81, C/Aichi/1/81 and C/Mississippi/80, respectively. T1-oligonucleotide fingerprinting of total vRNA revealed that the six isolates belonging to the C/Yamagata/26/81 virus group had the genomes greatly similar to one another but considerably different from those of the 1988/1990 isolates (except C/Yamagata/10/89) of the same antigenic group. Comparison of total or partial nucleotide sequences of the seven RNA segments of the three strains (C/Miyagi/3/91, C/Miyagi/9/91 and C/Miyagi/2/92) representative of the 1991/1993 strains of the C/Yamagata/26/81 virus group with those of the previous influenza C isolates obtained from humans and pigs during 1980/1989 showed that the 1991/1993 strains, like C/Yamagata/10/89, are more closely related to viruses isolated from pigs in Beijing, China in 1981/1982 than to any of the isolates from humans. This observation suggests strongly that interspecies transmission of influenza C virus between humans and pigs has occurred in nature, although it is not known whether the virus has been transmitted from pigs to humans or from humans to pigs.

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.

150 dog and 240 pig sera were tested for antibodies against influenza C virus in the hemagglutination inhibition test and confirmed by immunofluorescence and Western blotting tests. Virus strain C/JHB/1/66 was utilized as a standard antigen. It was found that 50.6% of the total number of dogs in the age between one month and 14 years possessed antibody titers of 1:20 or more, which was set as the general cut-off. In comparison, 24.0% of pigs in the age between one day and one month (n = 90) and 25.9% of pigs in the age between one month and one year (n = 150) were found to be positive. The highest hemagglutination inhibition titer was determined as 1:320 for of the dog sera and 1:160 for of the pig sera, respectively.

The nucleotide sequences of the haemagglutinin-esterase (HE) genes of 18 influenza C virus strains isolated in Japan during the period from 1964 to 1988 (11 published and 7 new sequences) were analysed to examine their evolutionary relationships. The phylogenetic tree constructed by the maximum parsimony method revealed the existence of four discrete lineages (I to IV), one of which (lineage III) may have died out in the late 1970s. Sequential evolution was demonstrated within seven strains of lineage I, which allowed estimation of an evolutionary rate of 0.49 x 10-3 nucleotide substitutions per site per year, a value corresponding to about one-ninth of the rates of human influenza A virus haemagglutinin genes. In the previously proposed immunodominant region on HE protein (positions 178 to 217), there was little or no amino acid sequence divergence among viruses on the same lineage although considerable divergence was seen among those on different lineages, raising the possibility that immune selection may not have played a significant role in the evolution of the glycoprotein, at least not after separation into lineages occurred. It was also found that the HE genes of the seven isolates obtained outside Japan during 1966-1983 could be each assigned to one of lineages I, II and IV, which suggests that influenza C virus is capable of spreading worldwide.

The antigenic and genetic relationships among 15 strains of influenza C virus isolated between August, 1988, and May, 1990, in Yamagata City, Japan, were investigated. Based on the results of antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoproteins and oligonucleotide mapping of total vRNA, the isolates were divided into two distinct groups closely related to either C/Yamagata/26/81 or C/Aichi/1/81. Antigenic differences between two groups could be detected clearly with heterogeneous antiviral sera. Comparison of the HE gene sequences between the representative Yamagata isolates and the previous isolates from other countries showed that C/Yamagata/26/81-like and C/Aichi/1/81-like viruses had high degrees of nucleotide sequence homology with C/pig/Beijing/115/81 and C/Johannesburg/1/66, respectively. These observations suggest that two lineages of influenza C virus, markedly different from each other in both antigenic and genetic structures, were simultaneously present in Yamagata City during 1988-1990 and that viruses belonging to each of the two lineages may also be prevalent in other areas of Japan and even in other countries.

From an overall point of view, the epidemiological situation of influenza C virus infections in western Europe is hardly known. In some countries like Spain, no epidemiological survey has been carried out to determine whether influenza C virus does or does not circulate and cause infection in the considered geographical area. We thus decided to perform such a study. A total of 191 serum samples was collected from people (from 1.5 to 80 years old) living in Spain in October 1990. These sera were tested for the presence of antibodies to influenza C virus by hemagglutination-inhibition (HI) tests. Significant HI activity was found in 59.3 to 64.9% of the 191 tested sera and titres ranged from 20 to 320. The high prevalence of antibody as well as the highly significant titres indicate an intense circulation of influenza C virus in Spain. A significant difference was found between children/teenagers and adults.

Two seroepidemiological surveys carried out so far, one in Japan, the other in France, gave a strong indication that dogs may be naturally infected by influenza C virus, considered to be exclusively human until recently. In this work, 101 serum samples were collected during winter 1989/1990 from dogs in Castilla y León, Spain. Sera were tested for the presence of antibodies to influenza C virus by Hemagglutination Inhibition (HI) test. Using antibody absorption by staphylococcal protein A, we demonstrated the specificity of the results. Significant HI activity was found in 56.3% of the 101 tested sera and titres ranged from 25 to 200.

Four different antigenic sites (A-1, A-2, B-1, B-2) have been identified previously on the haemagglutinin-esterase (HE) glycoprotein of influenza C/Ann Arbor/1/50 virus with seven monoclonal antibodies (MAbs). In this study we produced 30 additional anti-HE MAbs, nine of which demonstrated at least one of the following activities: haemagglutination inhibition, receptor-destroying enzyme inhibition, haemolysis inhibition, and neutralization (group A). The remaining had none of these activities (group B). These antibodies, and those previously isolated, were used to construct a more complete antigenic map of the HE molecule. Operational and topological analyses showed that a minimum of nine non-overlapping or partially overlapping antigenic sites were present on the HE protein, five recognized by group A MAbs (A-1 to A-5) and four by group B (B-1 to B-4). Among these antigenic sites, site A-5 was unique in that MAbs to this site inhibited the receptor-destroying activity without influencing the receptor-binding activity, which supports the idea that the sites responsible for these two functions are separate. It was also found that several group B MAbs were cross-reactive with host cell antigens.

The seroepidemiological study of the circulation of influenza C virus was achieved by testing HI antibodies in human sera collected from healthy subjects in Bucharest belonging to various age groups. The investigations were carried out over a two years period (October 1988--September 1990), using 3 influenza C virus strains: C/Taylor/1233/47, C/USSR/0303/77 and C/Moscow/1/84, the last two being antigenically similar. The analysis of the distribution of HI antibody values against the three influenza C virus strains showed that over October 1988--June 1989 the reported circulating viruses belonged to two distinct antigenic groups, one similar to the prototype C/Taylor/47 strains and the other to C/USSR/and/C/Moscow strains, whilst during the July 1989--September 1990 time interval viruses belonging solely to the C/USSR and C/Moscow antigenic group circulated. Similarly, one should note that circulation of influenza C viruses is not seasonal, it can appear anytime and even several times per year; this accounts for the relatively high and permanent antibody level in the population belonging to all age groups.

Five strains of influenza C virus were isolated and passaged in the amniotic sacs of embryonated hens' eggs, or in the HMV-II line of human malignant melanoma cells, and were tested for reactivity with a panel of monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein. It was observed with two strains (C/Yamagata/4/88, C/Yamagata/7/88) that the HE of virus passaged in HMV-II cells was antigenically distinguishable from that of virus cultivated in eggs. Virus clones obtained after repeated passages of these two strains in HMV-II cells all showed a significant increase in the ability to replicate in the cell culture compared to clones derived from viruses grown in eggs. No difference was seen, by contrast, in the ability to grow in eggs between HMV-II- and egg-derived virus clones. It was also found that HMV-II-grown viruses but not egg-grown viruses could agglutinate glutaraldehyde-fixed chicken erythrocytes at 23 degrees. These observations, taken together, suggest that isolation and passage of influenza C virus in HMV-II cells sometimes result in selection of antigenically distinct variants which have an advantage in binding to the cell surface receptors. Sequence analyses of the HE genes revealed that compared to egg-grown viruses, HMV-II-adapted variant of the Yamagata/4/88 strain had a single amino acid substitution in the HE molecule at position 283 (Asp----Asn) and that of the Yamagata/7/88 strain had two substitutions at positions 212 (Glu----Lys) and 519 (Asn----Asp).

Neutralization-resistant variants of influenza C/Ann Arbor/1/50 virus were selected with monoclonal antibodies against four different antigenic sites on the hemagglutinin-esterase (HE) glycoprotein, and their HE genes were sequenced to identify amino acid residues important for the integrity of each site. Twelve different amino acid substitutions in a total of 18 antigenic variants were all located on the HE1 subunit. Although variants for antigenic site A-2 had a change at position 367, all substitutions in the variants for sites A-1, A-3, and A-4 occurred in the central region of the HE1 spanning amino acid positions 178 to 283. Furthermore, it was found that many of the substitutions in the variants selected with antibodies to sites A-1 and A-3 were clustered within or near one of the three variable regions revealed previously by comparing amino acid sequences of the HEs among various influenza C isolates (Buonagurio, D. A., Nakada, S., Fitch, W. M., and Palese, P., Virology 146, 221-232, 1985). The antigenic variants were also examined for their ability to agglutinate chicken and human erythrocytes in order to obtain information concerning the receptor-binding site on the HE molecule. The results suggested that the amino acid changes at residues 178, 186, 187, 190, 206, 212, and 226 decreased the hemagglutinating activity whereas those at residues 245, 266, and 283 produced an opposite effect.

An improved binding assay for detection of ganglioside receptors for influenza A, B, and C viruses was developed. In this system, the virions bound to gangliosides that were developed on a silica gel thin-layer plate were detected by mouse monoclonal antibody against viral hemagglutinin and peroxidase-conjugated anti-mouse immunoglobin. No hydrolysis of the gangliosides by viral receptor-destroying enzyme was detected in the present condition. The reactivity of the viruses to gangliosides depended on the amount of developed gangliosides (10 pmols-10 nmols), the molecular species of sialic acid, and their sugar sequences. Human influenza A (PR/8/34), B (Lee/40), and C (Ann Arbor/1/50) viruses bound different receptor epitopes of sialo-sugar chains of gangliosides. The A/PR/8 virus bound most effectively to Neu5Ac-containing lacto-series gangliosides carrying type I and type II sugar chains, followed by ganglio-series and hematoside-series gangliosides. The A/PR/8 virus weakly bound to Neu5Ac alpha 2,6lactotetraosylceramide [IV6(Neu5Ac)Lc4Cer] and Neu5Ac alpha 2,6paragloboside [IV6(Neu5Ac)nLc4Cer] carrying Neu5Ac alpha 2,6Gal sequence, although their Neu5Ac alpha 2,3Gal derivatives were the most potent gangliosides tested. B/Lee/40 bound restrictively to IV6(Neu5Ac)Lc4Cer and IV6(Neu5Ac)nLc4Cer, which carry Neu5Ac alpha 2,6Gal sequence, and type I and type II lacto-series sugar chain, respectively. C/Ann Arbor/1/50 reacted only with 9-O-Ac-Neu5Ac-carrying sugar chains in all the gangliosides tested. This method also allowed the microanalysis of receptor gangliosides of unknown samples. ESK cells, sensitive to the influenza A viruses infection, expressed several kinds of receptor active gangliosides, while those from ESK-R cells, resistant to the virus infection, were undetectable.

To date, only one seroepidemiological survey, carried out in Japan, gave a strong indication that dogs may be naturally infected by the influenza C virus, long considered to be exclusively human. In the present work, 134 serum samples were collected during the winter of 1988/89 from dogs aged 6 months to 16 years in northern France. Samples were tested for the presence of antibodies to influenza C virus by both haemagglutination inhibition (HI) and ELISA. Using antibody absorption by staphylococcal protein A, we demonstrated the specificity of the results. In 62% of cases, the results were identical using the two methods. Significant HI activity was found in 32% of the 134 tested sera and titres ranged from 20 to 320. Of the sera tested, 42% were positive by ELISA and titres ranged from 500 to 8,000. The discordant results are discussed. The population tested was divided into five age groups: less than 4 years, 4 to 6 years, 7 to 9 years, 10 to 11 years and greater than 12 years. The distribution of antibodies in the tested canine population, in contrast to that of humans, did not show a significant degree of association with age.

Eight strains of influenza C virus isolated in various areas of Japan between January 1985 and January 1989 were compared using monoclonal antibodies to the haemagglutinin-esterase (HE) glycoproteins and by oligonucleotide mapping of total vRNA. Five of six strains isolated during 1986-9 were closely related to one another and also resembled the virus, C/Aichi/1/81, isolated in 1981 in Aichi prefecture. This suggests that the C/Aichi/1/81-related viruses had an epidemiological advantage over any co-circulating viruses at least during that period. One of two 1985 isolates (C/Nara/1/85) was antigenically indistinguishable from the C/Mississippi/1/80 strain though their oligonucleotide patterns were markedly different from each other. This raises the possibility that C/Nara/1/85 may be a recombinant virus which receives its HE gene from the C/Mississippi/1/80-related parent.

Little is known of the epidemiology of influenza C virus infections in western Europe and of the exact role of this agent in acute viral respiratory infections. Several tests may be used for detecting antibodies against this agent but the significance of their respective results is not clear. A total of 301 samples of serum was collected from persons aged from 4 months to 88 years living in France in 1988. The samples were tested for the presence of antibodies to influenza C virus by haemagglutination-inhibition (HI) tests and ELISA. The specificity of the results was checked by immunoblotting and by antibody absorption with staphylococcal protein A. Significant HI activity was found in 61% of the 301 samples tested, titres ranging from 20-320; 70% were positive by ELISA with titres ranging from 500 to 32,000. The population tested was divided into four age groups: 0-15 years; 16-30 years; 31-50 years and 51-88 years. The highest rates for positive samples were found in the 16-30 year group (76 and 79% by HI tests and ELISA respectively) as well as significant HI and ELISA geometric mean titres. Positive samples were less common in young children (46 and 50% by HI tests and ELISA respectively) and in the oldest group (44 and 54% respectively). The 31-50 years age group formed an intermediate class. The high prevalence of antibody as well as the significant titres indicate intense circulation of influenza C virus, especially among young adults.

The effects of serum antibody on the replication of influenza C virus in the nose and lung were evaluated in rats challenged with the virus by the intranasal and endotracheal routes, respectively. Convalescent rat serum administered intraperitoneally prior to infection suppressed virus replication significantly in both the nasal and pulmonary tissues. Resistance achieved was however much greater in the lung than in the nose. Rats with a serum neutralizing antibody titer of 1:800 showed almost complete resistance to pulmonary virus infection, and virus yield from the lung was reduced 10- to 100-fold in animals with the antibody titer of 1:80-160 or less. In contrast, significant decrease in virus shedding from the nose was observed only in animals with a serum antibody titer of 1:800 or greater. The effect of adoptive transfer of monoclonal antibodies (MAbs) to haemagglutinin-esterase (HE) glycoprotein and matrix (M) protein on pulmonary virus replication was also examined. Anti-HE MAbs with neutralization activity prevented virus shedding from the lung almost completely whereas non-neutralizing anti-HE MAb and anti-M Mab showed no inhibitory effect.

During the interepidemic periods of 34 years (1955-1988) the National Influenza Centre of Bulgaria with the aid of 11 supporting stations investigated a total of 27240 nasopharyngeal washings and 42530 paired sera of patients with acute respiratory diseases. Eighty strains of subtype A (H1N1) influenza virus, 60 strains of subtype a (H2N2), 148 strains of subtype A (H3N2), 3 strains of subtype A (Heg2, Neg2), 2 strains of subtype A (Hsw1N1), 14 strains of type B, and 6 strains of type C were isolated. Serologically positive results for influenza were observed in 12.65% for influenza type A virus, 3.83% for type B, and 1.78% for type C (on the whole in 18.26%). The data obtained enable to recommend that practicing physicians should not hesitate to make the diagnosis of influenza during the interepidemic periods on the basis of sufficient clinical and especially epidemiological data and should actively try to get the laboratory verification of the preliminary diagnosis.

The unique glycoprotein of influenza C virus, designated hemagglutinin (HEF), exhibits three functions: hemagglutination, esterase activity, and fusion factor. As the virus uses 9-O-acetylated sialic acid as a high-affinity receptor determinant for attachment to cells, its binding activity was used to reveal O-acetylated sialic acid residues after polyacrylamide gel electrophoresis and transfer onto nitrocellulose sheets of proteins and thin-layer chromatography of lipids. The specificity of the binding for O-acetylated sialoglycoconjugates was investigated. Our results showed that influenza C virus could detect the different forms of the two murine glycophorins which are known to be O-acetylated sialoglycoconjugates. The virus also bound to O-acetylated gangliosides isolated from embryonic chicken brain such as purified O-acetylated NeuAc alpha (2-8)NeuAc alpha (2-8)NeuAc alpha (2-3)Gal beta (1-4)Glc beta (1-1)ceramide (GT3). The esterase activity of the HEF protein of influenza C virus was used to unmask the sialic acid. After its deacetylation by the virus enzyme, the O-acetylated GT3 was recognized by a monoclonal antibody which binds only to the nonacetylated derivative. The results presented here show that influenza C virus is a discriminating analytical probe for identifying O-acetylated sialoglycoconjugates directly after Western blotting of proteins and thin-layer chromatography of lipids, thus providing a new analytical tool.

The prevalence of influenza C virus among pigs in Hyogo Prefecture, Japan, was investigated by serological techniques. Out of 240 sera tested, 45 (19%) showed haemagglutination inhibition (HI) to influenza C virus. Pig sera with high HI titres also scored high in neutralization tests and ELISAs. When fractionated by sucrose density gradient ultracentrifugation, the HI/ELISA reactivities corresponded to antibodies of the IgM and IgG classes. Radioimmunoprecipitation tests revealed that some, but not all, of the pig sera with high HI activities precipitated HEF glycoprotein of influenza C virus. These results suggested that the HI activities of pig sera in Hyogo Prefecture were due to the presence of antibody to influenza C virus. Sera with IgM class antibody to influenza C virus were found throughout the year. However, the question of whether or not pigs serve as a natural reservoir for human influenza C virus still remains to be solved.

Monoclonal antibodies against the nucleoprotein (NP) and matrix (M) protein of influenza C virus were prepared and characterized. At least two non-overlapping or partially overlapping antigenic sites were delineated on each of the proteins by competitive binding assays. Western blot analysis showed that two antigenic sites on the M protein were highly resistant to conformational changes whereas two sites on NP were sensitive. No antigenic variation was seen in either the NP or the M protein when the reactivity of monoclonal antibodies with 23 different influenza C strains isolated over a 41 year period was studied by radioimmunoprecipitation and enzyme-linked immunosorbent assay. Immunofluorescence analysis with the monoclonal antibodies revealed that both the NP and M proteins migrated to the cell nucleus during the replication cycle of influenza C virus.

Virus-infected cells were analyzed using multiparameter flow cytometry. Two virus-cell systems were investigated: HSV-1-infected VF cells and influenza C virus JHB/1/66-infected MDCK cells. Analysis included the measurement of the appearance of virus specific antigens. On individual cells, with polyclonal antibodies, antigens were first detected at 12 h p.i., and the numbers of labeled cells were followed up to 96 h p.i. The efficacy of four antiviral agents was tested with this system. The results were in good agreement with those of plaque reduction tests and indicated that this new method may be extremely useful for the correlation of viral and cellular events with antiviral activity. Finally, it was demonstrated that infected cells in both systems have a considerably greater volume than non-infected cells.

The distribution of the antibody to influenza C virus in the dogs and pigs in Yamagata prefecture, Japan was investigated by using three different serological methods: hemagglutination-inhibition (HI), radioimmunoprecipitation (RIP), and immunoblotting. The antibody against influenza C virus glycoprotein (gp88) was detected in 5 out of 112 sera collected from mongrel dogs, three by RIP test and two by any of the three methods, suggesting that the virus can cause natural infection in dogs. Significant levels of HI activity were found in 58 out of 269 sera collected from domestic pigs, but none of them showed positive reaction in the more sensitive method, RIP, which suggests that the inhibitors against the hemagglutination by influenza C virus rather than the antibody to gp88 are responsible for the observed HI activity. It appears, therefore, that at least in the Yamagata area, pigs do not play significant roles in the spread of influenza C virus in humans.