Search

Influenza C virus (ICV) is an orthomyxovirus related to influenza A and B, yet due to few commercial assays, epidemiologic studies may underestimate incidence of ICV infection and disease. We describe the epidemiology and characteristics of ICV within the New Vaccine Surveillance Network (NVSN), a Centers for Disease Control and Prevention (CDC)-led network that conducts population-based surveillance for pediatric acute respiratory illness (ARI). Nasal or/combined throat swabs were collected from emergency department (ED) or inpatient ARI cases, or healthy controls, between 12/05/2016-10/31/2019 and tested by molecular assays for ICV and other respiratory viruses. Parent surveys and chart review were used to analyze demographic and clinical characteristics of ICV+ children. Among 19,321 children tested for ICV, 115/17,668 (0.7 %) ARI cases and 8/1653 (0.5 %) healthy controls tested ICV+. Median age of ICV+ patients was 18 months and 88 (71.5 %) were ≤36 months. Among ICV+ ARI patients, 40 % (46/115) were enrolled in the ED, 60 % (69/115) were inpatients, with 15 admitted to intensive care. Most ICV+ ARI patients had fever (67.8 %), cough (94.8 %), or wheezing (60.9 %). Most (60.9 %) ARI cases had ≥1 co-detected viruses including rhinovirus, RSV, and adenovirus. In summary, ICV detection was rarely associated with ARI in children, and most ICV+ patients were ≤3 years old with co-detected respiratory viruses., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr Weinberg reported reports honoraria from Merck & Co for writing and revising textbook chapters. Dr Weinberg has served on an Scientific Advisory Board for Inhalon Biopharma. Dr Selvarangan reported receiving grants from Merck & Co, Inc, BioFire, Luminex, Hologic, Abbott, Becton Dickinson, and Cepheid outside the submitted work. Dr Halasa reported receiving grants from Sanofi and Quidel as well as personal fees from Genentech outside the submitted work. Dr Szilagyi reported receiving grants from the University of California, Los Angeles David Geffen School of Medicine BROAD Program during the conduct of the study. Dr Sahni reported receiving grants for her institution from the Centers for Disease Control and Prevention (CDC) outside the submitted work. Dr Martin reported receiving funding from Merck, Sharp and Dohme outside the submitted work. Dr. Williams reported receiving grants from the NIH outside the submitted work. No other conflicts were reported., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Influenza viruses can cause zoonotic infections that pose public health risks. Surveillance of influenza A and B viruses is conducted globally; however, information on influenza C and D viruses is limited. Longitudinal monitoring of influenza C virus in humans has been conducted in several countries, but there has been no long-term monitoring of influenza D virus in humans. The public health risks associated with the influenza D virus therefore remain unknown., Methods: We established a duplex real-time RT-PCR to detect influenza C and D viruses and analyzed respiratory specimens collected from 2144 patients in Japan with respiratory diseases between January 2018 and March 2023. We isolated viruses and conducted hemagglutination inhibition tests to examine antigenicity and focus reduction assays to determine susceptibility to the cap-dependent endonuclease inhibitor baloxavir marboxil., Results: We detected three influenza C viruses belonging to the C/Kanagawa- or C/Sao Paulo-lineages, which recently circulated globally. None of the specimens was positive for the influenza D virus. The C/Yokohama/1/2022 strain, isolated from the specimen with the highest viral RNA load and belonging to the C/Kanagawa-lineage, showed similar antigenicity to the reference C/Kanagawa-lineage strain and was susceptible to baloxavir., Conclusions: Our duplex real-time RT-PCR is useful for the simultaneous detection of influenza C and D viruses from the same specimen. Adding the influenza D virus to the monitoring of the influenza C virus would help in assessing the public health risks posed by this virus., (© 2024 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Antiviral susceptibility of influenza viruses was assessed using a high-content imaging-based neutralization test. Cap-dependent endonuclease inhibitors, baloxavir and AV5116, were superior to AV5115 against type A viruses, and AV5116 was most effective against PA mutants tested. However, these three inhibitors displayed comparable activity (EC 50 8-22 nM) against type C viruses from six lineages. Banana lectin and a monoclonal antibody, YA3, targeting the hemagglutinin-esterase protein effectively neutralized some, but not all, type C viruses., Competing Interests: Andrei A. Ivashchenko and Alexandre V. Ivachtchenko are the cofounders of ChemDiv, managing members of AVISA LCC. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Background: Influenza C virus is a pathogen that causes acute respiratory illness in children. The clinical information about this virus is limited because of the small number of isolated viruses compared to influenza A or B viruses., Methods: A total of 60 influenza C viruses were isolated by clinical tests using cell culture methods conducted in one hospital and one clinic during the 15 years from 2006 to 2020. These 60 cases were retrospectively analyzed by comparing outpatients and inpatients. Moreover, isolated viruses were analyzed for genomic changes during the study period., Results: All were younger than 7 years, and 73% of inpatients (19 out of 26) were under 2 years of age. A significant difference was found in the frequency of pneumonia, accounting for 45% and 4% of inpatients and outpatients, respectively. Most of the viruses isolated from 2006 to 2012 belonged to the S/A sublineage of the C/Sao Paulo lineage, but three sublineage viruses, including the S/A sublineage with K190N mutation, S/V sublineage, and C/Kanagawa lineage, have cocirculated since 2014. Moreover, S/A sublineage viruses were undergoing reassortment since 2014, suggesting significant changes in the virus, both antigenically and genetically. Of the 10 strains from patients with pneumonia, 7 were in the S/A sublineage, which had circulated from 2006 to 2012., Conclusion: Infants under 2 years of age were more likely to be hospitalized with pneumonia. The genomic changes that occurred in 2014 were suggested to affect the ability of the virus to spread., Competing Interests: Declaration of Competing Interest The authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Sentinel surveillance of influenza-like illnesses revealed an increase in the cases of influenza C virus in children and adults in Austria, 2022, compared to previous years, following one season (2020/2021), wherein no influenza C virus was detected. Whole-genome sequencing revealed no obvious genetic basis for the increase. We propose that the reemergence is explained by waning immunity from lack of community exposure due to restrictions intended to limit severe acute respiratory syndrome coronavirus 2 spread in prior seasons, pending further investigation., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Assembly and budding of the influenza C virus is mediated by three membrane proteins: the hemagglutinin-esterase-fusion glycoprotein (HEF), the matrix protein (CM1), and the ion channel (CM2). Here we investigated whether the formation of the hexagonal HEF arrangement, a distinctive feature of influenza C virions is important for virus budding. We used super resolution microscopy and found 250-nm sized HEF clusters at the plasma membrane of transfected cells, which were insensitive to cholesterol extraction and cytochalasin treatment. Overexpression of either CM1, CM2, or HEF caused the release of membrane-enveloped particles. Cryo-electron microscopy of the latter revealed spherical vesicles exhibiting the hexagonal HEF clusters. We subsequently used reverse genetics to identify elements in HEF required for this clustering. We found that deletion of the short cytoplasmic tail of HEF reduced virus titer and hexagonal HEF arrays, suggesting that an interaction with CM1 stabilizes the HEF clusters. In addition, we substituted amino acids at the surface of the closed HEF conformation and identified specific mutations that prevented virus rescue, others reduced virus titers and the number of HEF clusters in virions. Finally, mutation of two regions that mediate contacts between trimers in the in-situ structure of HEF was shown to prevent rescue of infectious virus particles. Mutations at residues thought to mediate lateral interactions were revealed to promote intracellular trafficking defects. Taken together, we propose that lateral interactions between the ectodomains of HEF trimers are a driving force for virus budding, although CM2 and CM1 also play important roles in this process., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

From 2014 to week 07/2020 the Centre for Health Protection in Hong Kong conducted screening for influenza C virus (ICV). A retrospective analysis of ICV detections to week 26/2019 revealed persistent low-level circulation with outbreaks occurring biennially in the winters of 2015 to 2016 and 2017 to 2018 (R. S. Daniels et al., J Virol 94:e01051-20, 2020, https://doi.org/10.1128/JVI.01051-20). Here, we report on an outbreak occurring in 2019 to 2020, reinforcing the observation of biennial seasonality in Hong Kong. All three outbreaks occurred in similar time frames, were subsequently dwarfed by seasonal epidemics of influenza types A and B, and were caused by similar proportions of C/Kanagawa/1/76 (K)-lineage and C/São Paulo/378/82 S1- and S2-sublineage viruses. Ongoing genetic drift was observed in all genes, with some evidence of amino acid substitution in the hemagglutinin-esterase-fusion (HEF) glycoprotein possibly associated with antigenic drift. A total of 61 ICV genomes covering the three outbreaks were analyzed for reassortment, and 9 different reassortant constellations were identified, 1 K-lineage, 4 S1-sublineage, and 4 S2-sublineage, with 6 of these being identified first in the 2019-1920 outbreak (2 S2-lineage and 4 S1-lineage). The roles that virus interference/enhancement, ICV persistent infection, genome evolution, and reassortment might play in the observed seasonality of ICV in Hong Kong are discussed. IMPORTANCE Influenza C virus (ICV) infection of humans is common, with the great majority of people being infected during childhood, though reinfection can occur throughout life. While infection normally results in "cold-like" symptoms, severe disease cases have been reported in recent years. However, knowledge of ICV is limited due to poor systematic surveillance and an inability to propagate the virus in large amounts in the laboratory. Following recent systematic surveillance in Hong Kong SAR, China, and direct ICV gene sequencing from clinical specimens, a 2-year cycle of disease outbreaks (epidemics) has been identified, with gene mixing playing a significant role in ICV evolution. Studies like those reported here are key to developing an understanding of the impact of influenza C virus infection in humans, notably where comorbidities exist and severe respiratory disease can develop.

Influenza virus is a persistent threat to human health; indeed, the deadliest modern pandemic was in 1918 when an H1N1 virus killed an estimated 50 million people globally. The intent of this work is to better understand influenza from an RNA-centric perspective to provide local, structural motifs with likely significance to the influenza infectious cycle for therapeutic targeting. To accomplish this, we analyzed over four hundred thousand RNA sequences spanning three major clades: influenza A, B and C. We scanned influenza segments for local secondary structure, identified/modeled motifs of likely functionality, and coupled the results to an analysis of evolutionary conservation. We discovered 185 significant regions of predicted ordered stability, yet evidence of sequence covariation was limited to 7 motifs, where 3-found in influenza C-had higher than expected amounts of sequence covariation., (© 2022. The Author(s).)

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.

Background: Influenza C virus causes mild respiratory diseases in humans. Previous studies suggested that the predominant hemagglutinin-esterase gene lineage circulating in children might be selected among the adult population, yet the prevalence of influenza C virus in adults has not been described., Objectives: To evaluate the frequency of influenza C virus infection in adults., Study Design: We performed hemagglutination inhibition assays of serum samples collected at periodic occupational medical checkups from employees of a hospital. A total of 679 serum samples were collected from 57 subjects who participated in biannual medical checkups between 2011 and 2016 as part of a longitudinal series. Titers of antibodies against the C/Kanagawa and C/Sao Paulo lineage viruses were detected., Results: Ten serum sample pairs from among the 57 subjects showed at least a four-fold increase in influenza C antibody titers. Samples from three subjects exhibited antibody titer increases for both the C/Kanagawa and C/Sao Paulo lineages, four subjects showed an increased titer against the C/Sao Paulo lineage, and three subjects showed an increased titer against the C/Kanagawa lineage. Half of the antibody titer increases for the C/Kanagawa lineage were detected in May 2014, while the increases for the C/Sao Paulo lineage were detected from 2011 to 2016., Conclusion: The 5-year influenza C virus infection rate was estimated at 17.5 %. There were antibodies that cross-reacted with the C/Sao Paulo and C/Kanagawa lineages. The results suggest that C/Sao Paulo was the main lineage in the adult population of this area, with cocirculation of the C/Kanagawa lineage., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge 1 . Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein complexes 2,3 . RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA inefficiently in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity 4 . Host-adaptive mutations, particularly a glutamic-acid-to-lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit, enable avian FluPolA to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here we report cryo-electron microscopy structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain of ANP32A. The C-terminal low-complexity acidic region of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, suggesting a mechanism for how the adaptive PB2(E627K) mutation enables the replication of viral RNA in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a viral ribonucleoprotein complex.

In 2014, the Centre for Health Protection in Hong Kong introduced screening for influenza C virus (ICV) as part of its routine surveillance for infectious agents in specimens collected from patients presenting with symptoms of respiratory viral infection, including influenza-like illness (ILI). A retrospective analysis of ICV detections up to week 26 of 2019 revealed persistent low-level circulation, with two outbreaks having occurred in the winters of 2015 to 2016 and 2017 to 2018. These outbreaks occurred at the same time as, and were dwarfed by, seasonal epidemics of influenza types A and B. Gene sequencing studies on stored ICV-positive clinical specimens from the two outbreaks have shown that the hemagglutinin-esterase (HE) genes of the viruses fall into two of the six recognized genetic lineages (represented by C/Kanagawa/1/76 and C/São Paulo/378/82), with there being significant genetic drift compared to earlier circulating viruses within both lineages. The location of a number of encoded amino acid substitutions in hemagglutinin-esterase fusion (HEF) glycoproteins suggests that antigenic drift may also have occurred. Observations of ICV outbreaks in other countries, with some of the infections being associated with severe disease, indicates that ICV infection has the potential to have significant clinical and health care impacts in humans. IMPORTANCE Influenza C virus infection of humans is common, and reinfection can occur throughout life. While symptoms are generally mild, severe disease cases have been reported, but knowledge of the virus is limited, as little systematic surveillance for influenza C virus is conducted and the virus cannot be studied by classical virologic methods because it cannot be readily isolated in laboratories. A combination of systematic surveillance in Hong Kong SAR, China, and new gene sequencing methods has been used in this study to assess influenza C virus evolution and provides evidence for a 2-year cycle of disease outbreaks. The results of studies like that reported here are key to developing an understanding of the impact of influenza C virus infection in humans and how virus evolution might be associated with epidemics., (Copyright © 2020 Daniels et al.)

Influenza, which is an acute respiratory disease caused by the influenza virus, represents a worldwide public health and economic problem owing to the significant morbidity and mortality caused by its seasonal epidemics and pandemics. Sensitive and convenient methodologies for the detection of influenza viruses are important for clinical care and infection control as well as epidemiological investigations. Here, we developed a multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) with quencher/fluorescence oligonucleotides connected by a 5' backward loop (LF or LB) primer for the detection of two subtypes of influenza viruses: Influenza A (A/H1 and A/H3) and influenza B. The detection limits of the multiplex RT-LAMP assay were 103 copies and 102 copies of RNA for influenza A and influenza B, respectively. The sensitivities of the multiplex influenza A/B/IC RT-LAMP assay were 94.62% and 97.50% for influenza A and influenza B clinical samples, respectively. The specificities of the multiplex influenza A/B/IC RT-LAMP assay were 100% for influenza A, influenza B, and healthy clinical samples. In addition, the multiplex influenza A/B/IC RT-LAMP assay had no cross-reactivity with other respiratory viruses., Competing Interests: The authors have declared that no competing interests exist.

Influenza C virus is a pathogen that causes acute respiratory illness in children and results in the hospitalization of infants. The antigenicity of the hemagglutinin esterase (HE) glycoprotein is highly stable, and it is not yet known whether antigenic changes contribute to the worldwide transmission and the occurrence of outbreaks of influenza C virus. Here, we performed antigenic analysis of 84 influenza C viruses isolated in Yamagata, Japan, during a 4-year period from 2015 to 2018 and analyzed sequence data for strains of the virus from Japan and many other parts of the world. Antigenic and phylogenetic analyses revealed that 83 strains belonged to the C/Sao Paulo lineage, and two sublineage strains, the Aichi99 sublineage and Victoria2012 sublineage, cocirculated between 2016 and 2018. Aichi99 sublineage strains exhibiting decreased reactivity with the monoclonal antibody YA3 became predominant after 2016, and these strains possessed the K190N mutation. Residue 190 is located in the 190-loop on the top side of the HE protein within a region that is known to show variation that does not impair the biological activity of the protein. The Aichi99 sublineage strains possessing the K190N mutation were detected after 2012 in Europe, Australia, the USA, and Asia as well as Japan. These observations suggest that antigenic variants with K190N mutations have circulated extensively around the world and caused outbreaks in Japan between 2016 and 2018. Our study indicated that the 190-loop is an important antigenic region, and the results suggested that changes in the 190-loop have contributed to the extensive transmission of the virus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The following are the supplementary data related to this article. Supplementary data to this article can be found online at https://doi.org/10.1016/j.meegid.2020.104269., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other mammalian hosts. By using traditional hemagglutination assay coupled with sialoglycan microarray (SGM) platform and functional assays, we demonstrated that IDV is more efficient in recognizing both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac 2 ) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac) than influenza C virus (ICV), a ubiquitous human pathogen. ICV seems to strongly prefer Neu5,9Ac 2 over Neu5Gc9Ac. Since Neu5Gc9Ac is different from Neu5,9Ac 2 only by an additional oxygen in the group at the C5 position, our results reveal that the hydroxyl group in Neu5Gc9Ac plays a critical role in determining receptor binding specificity, which as a result may discriminate IDV from ICV in communicating with 9-O-acetylated SAs. These findings shall provide a framework for further investigation towards better understanding of how newly discovered multiple-species-infecting IDV exploits natural 9-O-acetylated SA variations to expand its host range., Competing Interests: Declaration of competing interest The authors have read the journal's policy and have no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 C virus (ICV) is a common yet under-recognized cause of acute respiratory illness. ICV seropositivity has been found to be as high as 90% by 7-10 years of age, suggesting that most people are exposed to ICV at least once during childhood. Due to difficulty detecting ICV by cell culture, epidemiologic studies of ICV likely have underestimated the burden of ICV infection and disease. Recent development of highly sensitive RT-PCR has facilitated epidemiologic studies that provide further insights into the prevalence, seasonality, and course of ICV infection. In this review, we summarize the epidemiology and clinical characteristics of ICV.

The influenza C virus (ICV) is a human-pathogenic agent, and the infections are frequently identified in children. Compared to influenza A and B viruses, the nucleoprotein of ICV (NPC) has an extended C-terminal region of which the functional significance is ill defined. We observed that the nuclear localization signals (NLSs) found on the nucleoproteins of influenza A and B virus subtypes are absent at corresponding positions on ICV. Instead, we found that a long bipartite nuclear localization signal resides at the extended C-terminal region, spanning from R513 to K549. Our experimental data determined that the KKMK motif within this region plays important roles in both nuclear import and polymerase activity. Similar to the influenza A viruses, NPC also binds to multiple human importin α isoforms. Taken together, our results enhance the understanding of the virus-host interaction of the influenza C virus. IMPORTANCE As a member of the Orthomyxoviridae family, the polymerase complex of the influenza C virus structurally resembles its influenza A and influenza B virus counterparts, but the nucleoprotein differs by possessing an extra C-terminal region. We have characterized this region in view of nuclear import and interaction with the importin α protein family. Our results demonstrate the functional significance of a previously uncharacterized region on Orthomyxoviridae nucleoprotein (NP). Based on this work, we propose that importin α binding to influenza C virus NP is regulated by a long bipartite nuclear localization signal. Since the sequence of influenza D virus NP shares high homology to that of the influenza C virus, this work will also shed light on how influenza D virus NP functions., (Copyright © 2019 American Society for Microbiology.)

Influenza C virus (ICV) was first identified in humans and swine, but recently also in cattle, indicating a wider host range and potential threat to both the livestock industry and public health than was originally anticipated. The ICV hemagglutinin-esterase (HE) glycoprotein has multiple functions in the viral replication cycle and is the major determinant of antigenicity. Here, we developed a comparative approach integrating genetics, molecular selection analysis, and structural biology to identify the codon usage and adaptive evolution of ICV. We show that ICV can be classified into six lineages, consistent with previous studies. The HE gene has a low codon usage bias, which may facilitate ICV replication by reducing competition during evolution. Natural selection, dinucleotide composition, and mutation pressure shape the codon usage patterns of the ICV HE gene, with natural selection being the most important factor. Codon adaptation index (CAI) and relative codon deoptimization index (RCDI) analysis revealed that the greatest adaption of ICV was to humans, followed by cattle and swine. Additionally, similarity index (SiD) analysis revealed that swine exerted a stronger evolutionary pressure on ICV than humans, which is considered the primary reservoir. Furthermore, a similar tendency was also observed in the M gene. Of note, we found HE residues 176, 194, and 198 to be under positive selection, which may be the result of escape from antibody responses. Our study provides useful information on the genetic evolution of ICV from a new perspective that can help devise prevention and control strategies.

We identified influenza C virus (ICV) in samples from US cattle with bovine respiratory disease through real-time PCR testing and sequencing. Bovine ICV isolates had high nucleotide identities (≈98%) with each other and were closely related to human ICV strains (≈95%). Further research is needed to determine bovine ICV's zoonotic potential.

We mapped the hemagglutinin-esterase (HE) antigenic epitopes of the influenza C virus on the three-dimensional (3D) structure of the HE glycoprotein using 246 escape mutants that were selected by a panel of nine anti-HE monoclonal antibodies (MAbs), including seven of the C/Ann Arbor/1/50 virus and two of the C/Yamagata/15/2004 virus. The frequency of variant selection in the presence of anti-HE MAbs was very low, with frequencies ranging from 10 -4.62 to 10 -7.58 for the C/Ann Arbor/1/50 virus and from 10 -7.11 to 10 -9.25 for the C/Yamagata/15/2004 virus. Sequencing of mutant HE genes revealed 25 amino acid substitutions at 16 positions in three antigenic sites: A-1, A-2, and A-3, and a newly designated Y-1 site. In the 3D structure, the A-1 site was widely located around the receptor-binding site, the A-2 site was near the receptor-destroying enzyme site, and the Y-1 site was located in the loop on the topside of HE. The hemagglutination inhibition reactions of the MAbs with influenza C viruses, circulating between 1947 and 2016, were consistent with the antigenic-site amino acid changes. We also found some amino acid variations in the antigenic site of recently circulating strains with antigenic changes, suggesting that viruses that have the potential to alter antigenicity continue to circulate in humans.

Influenza virus RNA polymerase (FluPol), a heterotrimer composed of PB1, PB2, and PA subunits (P3 in influenza C), performs both transcription and replication of the viral RNA genome. For transcription, FluPol interacts with the C-terminal domain (CTD) of RNA polymerase II (Pol II), which enables FluPol to snatch capped RNA primers from nascent host RNAs. Here, we describe the co-crystal structure of influenza C virus polymerase (FluPol C ) bound to a Ser5-phosphorylated CTD (pS 5 -CTD) peptide. The position of the CTD-binding site at the interface of PB1, P3, and the flexible PB2 C-terminal domains suggests that CTD binding stabilizes the transcription-competent conformation of FluPol. In agreement, both cap snatching and capped primer-dependent transcription initiation by FluPol C are enhanced in the presence of pS 5 -CTD. Mutations of amino acids in the CTD-binding site reduce viral mRNA synthesis. We propose a model for the activation of the influenza virus transcriptase through its association with pS 5 -CTD of Pol II., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Existing literature suggests that influenza C typically causes mild respiratory tract disease. However, clinical and epidemiological data are limited., Methods: Four outpatient clinics and 3 hospitals submitted clinical data and respiratory specimens through a surveillance network for acute respiratory infection (ARI) from May 2013 through December 2016. Specimens were tested using multitarget nucleic acid amplification for 19-22 respiratory pathogens, including influenza C., Results: Influenza C virus was detected among 59 of 10 202 (0.58%) hospitalized severe ARI cases and 11 of 2282 (0.48%) outpatients. Most detections occurred from December to March, 73% during the 2014-2015 season. Influenza C detections occurred among patients of all ages, with rates being similar between inpatients and outpatients. The highest rate of detection occurred among children aged 6-24 months (1.2%). Among hospitalized cases, 7 required intensive care. Medical comorbidities were reported in 58% of hospitalized cases and all who required intensive care. At least 1 other respiratory pathogen was detected in 40 (66%) cases, most commonly rhinovirus/enterovirus (25%) and respiratory syncytial virus (20%). The hemagglutinin-esterase-fusion gene was sequenced in 37 specimens, and both C/Kanagawa and C/Sao Paulo lineages were detected in inpatients and outpatients., Conclusions: We found seasonal circulation of influenza C with year-to-year variability. Detection was most frequent among young children but occurred in all ages. Some cases that were positive for influenza C, particularly those with comorbid conditions, had severe disease, suggesting a need for further study of the role of influenza C virus in the pathogenesis of respiratory disease.

Background: Influenza C virus (ICV) is associated with acute respiratory illness. Yet ICV remains under recognized, with most previous studies using only culture to identify cases., Objectives: To develop a sensitive and specific real-time RT-PCR assay for ICV that allows for rapid and accurate detection in a clinical or research setting., Study Design: Multiple ICV sequences obtained from GenBank were analyzed, including 141 hemagglutinin-esterase (HE), 106 matrix (M), and 97 nucleoprotein (NP) sequences. Primers and probes were designed based on conserved regions. Multiple primer-probe sets were tested against multiple ICV strains., Results: The ICV M and NP genes offered the most conserved sequence regions. Primers and probes based on newer sequence data offered enhanced detection of ICV, especially for low titer specimens. An NP-targeted assay yielded the best performance and was capable of detecting 10-100 RNA copies per reaction. The NP assay detected multiple clinical isolates of ICV collected in a field epidemiology study conducted in Peru., Conclusions: We report a new real-time RT-PCR assay for ICV with high sensitivity and specificity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Since 2003, India has had a well-established influenza surveillance network, though Influenza C virus was not the focus of study. We therefore retrospectively analyzed clinical samples from Pune, western India collected during January 2009 to August 2015, by real-time RT-PCR. Three of 2530 samples of patients with influenza-like illness (ILI) or severe acute respiratory illness (SARI) showed positivity for Influenza C virus infection, while 105 and 31 samples were positive for Influenza A and B viruses respectively. Influenza C viruses were successfully isolated using the embryonated egg system and whole genomes were sequenced and analyzed phylogenetically. HE gene-based phylogeny showed that two viruses C/India/P119564/2011 and C/India P121719/2012 clustered with the C/Sao Paulo/378/82 (SP82) lineage, whereas C/India/P135047/2013 clustered with the C/Kanagawa/1/76 (KA76) lineage. The internal gene of these viruses grouped in two lineages. The PB1, PB2, M and NS genes of the study viruses grouped with C/Yamagata/26/81 (YA81), while the P3 (PA) and NP genes grouped with C/Mississippi/80 (MS80). Bayesian clock studies conclude that the Indian strains may have emerged through multiple reassortment events., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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).

Infections with the influenza C virus causing respiratory symptoms are common, particularly among children. Since isolation and detection of the virus are rarely performed, compared with influenza A and B viruses, the small number of available sequences of the virus makes it difficult to analyze its evolutionary dynamics. Recently, we reported the full genome sequence of 102 strains of the virus. Here, we exploited the data to elucidate the evolutionary characteristics and phylodynamics of the virus compared with influenza A and B viruses. Along with our data, we obtained public sequence data of the hemagglutinin-esterase gene of the virus; the dataset consists of 218 unique sequences of the virus collected from 14 countries between 1947 and 2014. Informatics analyses revealed that (1) multiple lineages have been circulating globally; (2) there have been weak and infrequent selective bottlenecks; (3) the evolutionary rate is low because of weak positive selection and a low capability to induce mutations; and (4) there is no significant positive selection although a few mutations affecting its antigenicity have been induced. The unique evolutionary dynamics of the influenza C virus must be shaped by multiple factors, including virological, immunological, and epidemiological characteristics., Competing Interests: The authors declare no conflict of interest.

Since influenza C virus was first isolated in 1947, the virus has been only occasionally isolated by cell culture; there are only four strains for which complete genome sequences are registered. Here, we analyzed a total of 106 complete genomes, ranging from the first isolate from 1947 to recent isolates from 2014, to determine the genetic lineages of influenza C virus, the reassortment events, and the rates of nucleotide substitution. The results showed that there are six lineages, named C/Taylor, C/Mississippi, C/Aichi, C/Yamagata, C/Kanagawa, and C/Sao Paulo. They contain both antigenic and genetic lineages of the hemagglutinin-esterase (HE) gene, and the internal genes PB2, PB1, P3, NP, M, and NS are divided into two major lineages, a C/Mississippi/80-related lineage and a C/Yamagata/81-related lineage. Reassortment events were found over the entire period of 68 years. Several outbreaks of influenza C virus between 1990 and 2014 in Japan consisted of reassortant viruses, suggesting that the genomic constellation is related to influenza C virus epidemics. The nucleotide sequences were highly homologous to each other. The minimum percent identity between viruses ranged from 91.1% for the HE gene to 96.1% for the M gene, and the rate of nucleotide substitution for the HE gene was the highest, at 5.20 × 10(-4) substitutions/site/year. These results indicate that reassortment is an important factor that increases the genetic diversity of influenza C virus, resulting in its ability to prevail in humans. IMPORTANCE Influenza C virus is a pathogen that causes acute respiratory illness in children and results in hospitalization of infants. We previously demonstrated (Y. Matsuzaki et al., J Clin Virol 61:87-93, 2014, http://dx.doi.org/10.1016/j.jcv.2014.06.017) that periodic epidemics of this virus occurred in Japan between 1996 and 2014 and that replacement of the dominant antigenic group occurred every several years as a result of selection by herd immunity. However, the antigenicity of the HE glycoprotein is highly stable, and antigenic drift has not occurred for at least 30 years. Here, we analyzed a total of 106 complete genomes spanning 68 years for the first time, and we found that influenza C viruses are circulating worldwide while undergoing reassortment as well as selection by herd immunity, resulting in an increased ability to prevail in humans. The results presented in this study contribute to the understanding of the evolution, including reassortment events, underlying influenza C virus epidemics., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The only spike of influenza C virus, the hemagglutinin-esterase-fusion glycoprotein (HEF) combines receptor binding, receptor hydrolysis and membrane fusion activities. Like other hemagglutinating glycoproteins of influenza viruses HEF is S-acylated, but only with stearic acid at a single cysteine located at the cytosol-facing end of the transmembrane region. Previous studies established the essential role of S-acylation of hemagglutinin for replication of influenza A and B virus by affecting budding and/or membrane fusion, but the function of acylation of HEF was hitherto not investigated. Using reverse genetics we rescued a virus containing non-stearoylated HEF, which was stable during serial passage and showed no competitive fitness defect, but the growth rate of the mutant virus was reduced by one log. Deacylation of HEF does neither affect the kinetics of its plasma membrane transport nor the protein composition of virus particles. Cryo-electron microscopy showed that the shape of viral particles and the hexagonal array of spikes typical for influenza C virus were not influenced by this mutation indicating that virus budding was not disturbed. However, the extent and kinetics of haemolysis were reduced in mutant virus at 37°C, but not at 33°C, the optimal temperature for virus growth, suggesting that non-acylated HEF has a defect in membrane fusion under suboptimal conditions., (© 2015 John Wiley & Sons Ltd.)

Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)-containing domain translocates ∼90 Å to bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

In an earlier study on respiratory infections in Singapore military recruits, four influenza C virus (FLUCV) infections were detected out of the 1354 samples collected. All four isolates were detected in 2006, and their whole genome was completely sequenced and analysed. Phylogenetic analysis of the hemagglutinin esterase fusion (HEF) gene revealed that all four Singapore isolates belonged to the C/Japan-Kanagawa/1/76-related lineage. However, the genes of the four FLUCV isolates had origins from several different lineages, and the genome composition resembles that of the C/Japan-Miyagi/9/96-like strains that had been circulating in Japan between 1996 and 2000., (© 2015 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

From November 2009 to December 2013 in the Philippines, 15 influenza C viruses were isolated, using MDCK cells, from specimens obtained from children with severe pneumonia and influenza-like illness (ILI). This is the first report of influenza C virus isolation in the Philippines. In addition, from January 2008 to December 2013, 7 influenza C viruses were isolated from specimens that were obtained from children with acute respiratory illness (ARI) in Sendai city, Japan. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein showed that 19 strains (12 from the Philippines and 7 from Japan) were similar to the influenza C virus reference strain C/Sao Paulo/378/82 (SP82). Phylogenetic analysis of the HE gene showed that the strains from the Philippines and Japan formed distinct clusters within an SP82-related lineage. The clusters that included the Philippine and Japanese strains were shown to have diverged from a common ancestor around 1993. In addition, phylogenetic analysis of the internal genes showed that all strains isolated in the Philippines and Japan had emerged through reassortment events. The composition of the internal genes of the Philippine strains was different from that of the Japanese strains, although all strains were classified into an SP82-related lineage by HE gene sequence analysis. These observations suggest that the influenza C viruses analyzed here had emerged through different reassortment events; however, the time and place at which the reassortment events occurred were not determined., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Exchange of the non coding regions of the NP segment between type A and C influenza viruses was used to demonstrate the importance not only of the proximal panhandle, but also of the initial distal panhandle strength in type specificity. Both elements were found to be compulsory to rescue infectious virus by reverse genetics systems. Interestingly, in type A influenza virus infectious context, the length of the NP segment 5' NC region once transcribed into mRNA was found to impact its translation, and the level of produced NP protein consequently affected the level of viral genome replication.

Background: Although influenza C virus is widely distributed throughout the world, epidemiological information, based on long-term surveillance, has not yet been acquired., Objectives: To clarify the epidemiological features of influenza C virus infection, and to examine whether the prevalence of the antibodies against the influenza C virus is associated with the epidemics., Study Design: Between 1996 and 2013, 36,973 respiratory specimens were collected from two pediatric outpatient clinics in Yamagata, Japan. The specimens were examined for the presence of influenza C virus using cell culture methods. Isolated viruses were antigenically analyzed. The differences in seropositivity, with respect to the different antigenic groups, were examined using serum samples collected in 2001 and 2011 by a hemagglutination inhibition assay., Results: Influenza C viruses were isolated from 190 specimens during an 18-year period. Most influenza C viruses were isolated from winter to early summer in even-numbered years, and the frequency of virus isolation per year ranged from 0.43% to 1.73%. An antigenic analysis revealed that the dominant antigenic groups were the C/Yamagata/26/81 from 1996 to 2000, the C/Kanagawa/1/76 in 2002 and 2004, and the C/Sao Paulo/378/82 from 2006 to 2012. When compared to the other antigenic groups, the seroprevalence of the C/Sao Paulo/378/82 group was lower in 2001 for individuals older than 5 years and was higher in 2011 in individuals younger than 40 years., Conclusions: The results from our study suggest that epidemics of influenza C virus infection periodically occur and the replacement of the dominant antigenic group may be caused by immune selection within older children and/or adults in the community., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We recently described the isolation of a novel influenza virus from swine exhibiting respiratory disease in the United States that is distantly related to human influenza C virus. Based on genetic, biochemical and morphological analysis, the virus was provisionally classified as C/swine/Oklahoma/1334/2011 (C/OK). To further understand the genetics and evolution of this novel pathogen, we performed a comprehensive analysis of its sequence and phylogeny. The results demonstrated that C/OK and human influenza C viruses share a conserved array of predicted functional domains in the viral RNA genome replication and viral entry machinery but vary at key functional sites. Furthermore, our evolutionary analysis showed that homologous genes of C/OK and human influenza C viruses diverged from each other an estimated several hundred to several thousand years ago. Taken together, the findings described in this study support and extend our previous observations that C/OK is a genetically and evolutionarily distinct influenza virus in the family Orthomyxoviridae.

Background: Influenza B and C are single-stranded RNA viruses that cause yearly epidemics and infections. Knowledge of RNA secondary structure generated by influenza B and C will be helpful in further understanding the role of RNA structure in the progression of influenza infection., Findings: All available protein-coding sequences for influenza B and C were analyzed for regions with high potential for functional RNA secondary structure. On the basis of conserved RNA secondary structure with predicted high thermodynamic stability, putative structures were identified that contain splice sites in segment 8 of influenza B and segments 6 and 7 of influenza C. The sequence in segment 6 also contains three unused AUG start codon sites that are sequestered within a hairpin structure., Conclusions: When added to previous studies on influenza A, the results suggest that influenza splicing may share common structural strategies for regulation of splicing. In particular, influenza 3' splice sites are predicted to form secondary structures that can switch conformation to regulate splicing. Thus, these RNA structures present attractive targets for therapeutics aimed at targeting one or the other conformation.

Reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR were used to detect 14 (6.6%) influenza C virus (InfC) among 213 clinical samples collected from children with respiratory symptoms in Mie Prefecture, Japan, between January 2012 and December 2012. Virus isolation using Madin-Darby canine kidney cells and/or embryonated chicken eggs was also successful for 3 of the 14 PCR-positive samples. Eleven patients (78.6%) were aged <3 years. Phylogenetic analysis of the hemagglutinin-esterase gene showed that the InfC detected in Mie Prefecture belonged to the C/Sao Paulo/82-related lineage. To determine the seroprevalence of InfC, a total of 575 serum samples from patients aged 1 month to 69 years in Mie Prefecture were screened by hemagglutination inhibition test using the C/Mie/199/2012 (C/Sao Paulo/82-related lineage) strain as the antigen. The samples with an antibody titer of ≥1:16 were designated as antibody-positive. The results showed that 53.7% of the 296 serum samples collected in 2011 and 85.3% of the 279 samples collected in 2012 were positive for antibodies against InfC, suggesting that an outbreak of InfC infection occurred in Mie Prefecture in 2012. Therefore, continuous and proactive monitoring is important to determine the number of InfC-infections and to better understand the epidemiology.

Exchange of the extremities of the NS segment of type A and C influenza viruses in reverse genetics systems was used to assess their putative role in type specificity. Restoration of each specific proximal panhandle was mandatory to allow the rescue of viruses with heterotypic extremities. Moreover, the transcription level of the modified segment seemed to be directly affected by the distal panhandle strength.

To evaluate the impact of influenza C (ICV) infection in children with community-acquired pneumonia (CAP), all of the children consecutively seen during 4 influenza seasons with respiratory symptoms and radiographically confirmed CAP were prospectively evaluated. ICV was identified in the respiratory secretions of five of 391 patients (1·3%). In children with ICV-associated CAP, clinical data were similar to those observed in children with IAV-associated CAP and worse than those observed in children with IBV-associated. The phylogenetic tree showed that the sequenced strains clustered in two of the six ICV lineages. These findings highlight that ICV can be a cause of CAP of children and that this can be severe enough to require hospitalization.

In tropical countries of Asia, like India, approximately 0.5 million children of <5 years of age die annually due to acute respiratory illness (ARI). Of common respiratory pathogens, influenza viruses (A & B) are associated with annual worldwide epidemics; while the information on influenza C virus is inadequate. During January 2011 through December 2012, 2737 nasal and/or throat swabs were collected from patients reporting at outpatient department of hospitals in eastern India with ARI. Nucleotide sequencing was carried out using gene specific primers followed by pair-wise sequence alignments, multiple alignments, construction of phylogenetic tree and analysis of deduced amino acid sequences. Study reveals that, out of 2737 samples, 1616 (59.04%) were positive for one or more respiratory viruses; of which 23.72% were positive for influenza A and B viruses. From influenza A & B negative samples, influenza C virus was screened and detected with a frequency of 0.18%. Phylogenetic analysis showed that the HE, matrix, NS, PB1 and PB2 gene of the studied strain (C/Eastern-India/1202/2011) possessed a close relatedness to C/Yamagata/26/81 like strains. The P3 gene shows proximity with C/Mississipi/80 like strains whereas NP gene revealed similarity with C/Miyagi/1/93 like strains. The outcome of the whole genome analysis of the strain C/Eastern-India/1202/2011 provided useful information regarding genetic diversity of influenza C strains in India., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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 influenza C virus is spread worldwide and causes diseases of the upper and (less frequently) lower respiratory tract in human. The virus is not pandemic, but it circulates together with pandemic influenza A and B viruses during winter months and has quite similar clinical manifestations. The influenza C virus is also encountered in animals (pigs and dogs) and is known to override the interspecific barriers oftransmssion. The immune system of mammals often fails to recognize new antigenic variants of influenza C virus, which invariably arise in nature, resulting in outbreaks of diseases, although the structure of antigens in influenza C virus in general is much more stable than those of influenza viruses A and B. Variability of genetic information in natural isolates of viruses is determined by mutations, reassortment, and recombination. However, recombination events very rarely occur in genomes of negative-strand RNA viruses, including those of influenza, and virtually have no effect on their evolution. Unambiguous explanations for this phenomenon have thus far not been proposed. There is no proof of recombination processes in the influenza C virus genome. On the contrary, reassortant viruses derived from different strains of influenza C virus frequently appear in vitro and are likely to be common in nature. The genome of influenza C virus comprises seven segments. Based on the comparison of sequences in one of its genes (HEF), six genetic or antigenic lineages of this virus can be distinguished (Yamagata/26/81, Aichi/1/81, Mississippi/80, Taylor/1233/47, Sao Paulo/378/82, and Kanagawa/1/76). However, the available genetic data show that all the seven segments of the influenza C virus genome evolve independently.

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.

CM2 is the second membrane protein of influenza C virus. The significance of the posttranslational modifications of CM2 remains to be clarified in the context of viral replication, although the positions of the modified amino acids on CM2 have been determined. In the present study, using reverse genetics we generated rCM2-C65A, a recombinant influenza C virus lacking CM2 palmitoylation site, in which cysteine at residue 65 of CM2 was mutated to alanine, and examined viral growth and viral protein synthesis in the recombinant-infected cells. The rCM2-C65A virus grew as efficiently as did the parental virus in cultured HMV-II cells as well as in embryonated chicken eggs. The synthesis and biochemical features of HEF, NP, M1 and mutant CM2 in the rCM2-C65A-infected HMV-II cells were similar to those in the parental virus-infected cells. Furthermore, membrane flotation analysis of the infected cells revealed that equal amount of viral proteins was recovered in the plasma membrane fractions of the rCM2-C65A-infected cells to that in the parental virus-infected cells. These findings indicate that defect in palmitoylation of CM2 does not affect transport and maturation of HEF, NP and M1 as well as CM2 in virus-infected cells, and palmitoylation of CM2 is dispensable to influenza C virus replication., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although particular attention is paid to influenza A and B virus isolates during influenza surveillance, influenza C virus (FLUCV) coexisted during the first influenza A (H1N1) 2009 pandemic wave during the 2009-2010 season. From 27 April 2009 to 9 May 2010, 12 strains of FLUCV were detected in specimens collected from 1713 nonhospitalized patients with upper respiratory tract illness using a molecular method. Half of the patients with FLUCV infection were older than 14 years. The most frequent symptoms were cough and fever, similar to other viral respiratory infections. Phylogenetic analysis of the hemagglutinin-esterase gene revealed that the strains belonged to the C/Kanagawa/1/76-related and C/Sao Paulo/378/82-related lineages, demonstrating their co-circulation in Catalonia. In addition to regular virological surveillance that provides information about the incidence and the exact role of FLUCV in acute viral respiratory infections in the general population, the genetic lineage identification offers additional data for epidemiological purposes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

The occurrence of homologous recombination in influenza viruses has been under some debate recently. To determine the extent of homologous recombination in influenza C virus, recombination analyses of all available gene sequences of influenza C virus were carried out. No recombination signal was found. With the previous evidence in influenza A and B viruses, it seems that homologous recombination has minimal or no effect on influenza virus evolution.