Genetic Reassortment in a Child Coinfected with Two Influenza B Viruses, B/Yamagata Lineage and B/Victoria-Lineage Strains.

We identified a child coinfected with influenza B viruses of B/Yamagata and B/Victoria lineages, in whom we analyzed the occurrence of genetic reassortment. Plaque purification was performed using a throat swab specimen from a 9-year-old child, resulting in 34 well-isolated plaques. The genomic composition of eight gene segments (HA, NA, PB1, PB2, PA, NP, M, and NS genes) for each plaque was determined at the lineage level. Of the 34 plaques, 21 (61.8%) had B/Phuket/3073/2013 (B/Yamagata)-like sequences in all gene segments, while the other 13 (38.2%) were reassortants with B/Texas/02/2013 (B/Victoria)-like sequences in 1-5 of the 8 segments. The PB1 segment had the most B/Victoria lineage genes (23.5%; 8 of 34 plaques), while PB2 and PA had the least (2.9%; 1 of 34 plaques). Reassortants with B/Victoria lineage genes in 2-5 segments showed the same level of growth as viruses with B/Yamagata lineage genes in all segments. However, reassortants with B/Victoria lineage genes only in the NA, PB1, NP, or NS segments exhibited reduced or undetectable growth. We demonstrated that various gene reassortments occurred in a child. These results suggest that simultaneous outbreaks of two influenza B virus lineages increase genetic diversity and could promote the emergence of new epidemic strains.

Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin.

(1) Background: Inflammatory responses induce the formation of both anti-tumor and pro-tumor neutrophils known as myeloid-derived suppressor cells (MDSCs). Intermittent intravesical infusion of Bacillus Calmette-Guérin (BCG) is an established cancer immunotherapy for non-muscle-invasive bladder cancer (NMIBC). However, the types of neutrophils induced via the inflammatory response to both tumor-bearing and BCG remain unclear. (2) Methods: We therefore analyzed neutrophil dynamics in the peripheral blood and urine of patients with NMIBC who received BCG therapy. Further, we analyzed the effects of BCG in a mouse intraperitoneal tumor model. (3) Results: BCG therapy induced the formation of CXCL10 and MHC class II-positive neutrophils in the urine of patients with NMIBC but did not induce MDSC formation. CXCL10- and MHC class II-expressing neutrophils were detected in peritoneal exudate cells formed after BCG administration. Partial neutrophil depletion using an anti-Ly6G antibody suppressed the upregulation of CXCL10 and MHC class II in neutrophils and reversed the anti-tumor activity of BCG in mouse models. (4) Conclusions: These results indicated that intracellular MHC class II- and CXCL10-expressing neutrophils indicate the state of anti-tumor activity induced via BCG. The status of neutrophils in mixed inflammation of immunosuppressive and anti-tumor responses may therefore be useful for evaluating immunological systemic conditions.

Epidemiologic, clinical, and genetic characteristics of influenza C virus infections among outpatients and inpatients in Sendai, Japan from 2006 to 2020.

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.

The proton ATPase inhibitor bafilomycin A1 reduces the release of rhinovirus C and cytokines from primary cultures of human nasal epithelial cells.

Rhinovirus species C (RV-C) causes more severe asthma attacks than other rhinovirus species. However, the modulation of RV-C replication by drugs has not been well studied. Primary human nasal epithelial (HNE) cells cultured on filter membranes with air-liquid interface methods were infected with RV-C03, and the levels of RV-C03 RNA collected from the airway surface liquid (ASL) of HNE cells were measured with a SYBR Green assay. Pretreatment of HNE cells with the specific vacuolar H+-ATPase inhibitor bafilomycin A1 reduced the RV-C03 RNA levels in the ASL; inflammatory cytokines, including interleukin (IL)-1ß, IL-6 and IL-8, in the supernatant; the mRNA expression of the RV-C receptor cadherin-related family member 3 (CDHR3) in the cells; and the number of acidic endosomes where RV-B RNA enters the cytoplasm. The levels of RV-C03 RNA in the ASL obtained from HNE cells with the CDHR3 rs6967,330 G/A genotype tended to be higher than those obtained from HNE cells with the G/G genotype. Pretreatment with the Na+/H+ exchanger inhibitor ethyl-isopropyl amiloride or either of the macrolides clarithromycin or EM900 also reduced RV-C03 RNA levels in the ASL and the number of acidic endosomes in HNE cells. In addition, significant levels of RV-A16, RV-B14 and RV-C25 RNA were detected in the ASL, and bafilomycin A1 also decreased the RV-C25 RNA levels. These findings suggest that bafilomycin A1 may reduce the release of RV-Cs and inflammatory cytokines from human airway epithelial cells. RV-Cs may be sensitive to drugs, including bafilomycin A1, that increase endosomal pH, and CDHR3 may mediate virus entry through receptor-mediated endocytosis in human airway epithelial cells.

TMPRSS2 Activates Hemagglutinin-Esterase Glycoprotein of Influenza C Virus.

Influenza C virus (ICV) has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein. HE functions similarly to hemagglutinin (HA) and neuraminidase of the influenza A and B viruses (IAV and IBV, respectively). It has a monobasic site, which is cleaved by some host enzymes. The cleavage is essential to activating the virus, but the enzyme or enzymes in the respiratory tract have not been identified. This study investigated whether the host serine proteases, transmembrane protease serine S1 member 2 (TMPRSS2) and human airway trypsin-like protease (HAT), which reportedly cleave HA of IAV/IBV, are involved in HE cleavage. We established TMPRSS2- and HAT-expressing MDCK cells (MDCK-TMPRSS2 and MDCK-HAT). ICV showed multicycle replication with HE cleavage without trypsin in MDCK-TMPRSS2 cells as well as IAV did. The HE cleavage and multicycle replication did not appear in MDCK-HAT cells infected with ICV without trypsin, while HA cleavage and multistep growth of IAV appeared in the cells. Amino acid sequences of the HE cleavage site in 352 ICV strains were completely preserved. Camostat and nafamostat suppressed the growth of ICV and IAV in human nasal surface epithelial (HNE) cells. Therefore, this study revealed that, at least, TMPRSS2 is involved in HE cleavage and suggested that nafamostat could be a candidate for therapeutic drugs for ICV infection. IMPORTANCE Influenza C virus (ICV) is a pathogen that causes acute respiratory illness, mostly in children, but there are no anti-ICV drugs. ICV has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein on the virion surface, which possesses receptor-binding, receptor-destroying, and membrane fusion activities. The HE cleavage is essential for the virus to be activated, but the enzyme or enzymes in the respiratory tract have not been identified. This study revealed that transmembrane protease serine S1 member 2 (TMPRSS2), and not human airway trypsin-like protease (HAT), is involved in HE cleavage. This is a novel study on the host enzymes involved in HE cleavage, and the result suggests that the host enzymes, such as TMPRSS2, may be a target for therapeutic drugs of ICV infection.

Growth Kinetics of Influenza C Virus Antigenic Mutants That Escaped from Anti-Hemagglutinin Esterase Monoclonal Antibodies and Viral Antigenic Changes Found in Field Isolates.

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.

Longitudinal epidemiology of human coronavirus OC43 in Yamagata, Japan, 2010-2017: Two groups based on spike gene appear one after another.

Human coronavirus OC43 (HCoV-OC43) is divided into genotypes A to H based on genetic recombination including the spike (S) gene. To investigate the longitudinal transition of the phylogenetic feature of the HCoV-OC43 S gene in a community, phylogenetic analysis of the S1 region of the S gene was conducted using 208 strains detected in Yamagata during 2010 to 2017 with reference strains of the genotype. The S1 sequences were divisible into four groups: A to D. All Yamagata strains belonged to either group B or group D. In group B, 46 (90.2%) out of 51 Yamagata strains were clustered with those of genotype E reference strains (cluster E). In group D, 28 (17.8%) and 122 (77.7%) out of 157 Yamagata strains were clustered, respectively, with genotype F and genotype G reference strains. In cluster G, 28 strains formed a distinct cluster. Monthly distributions of HCoV-OC43 in Yamagata in 2010 to 2017 revealed that group B and group D appeared one after another. In group B, the cluster E strains were prevalent recurrently. In conclusion, epidemics of HCoV-OC43 in Yamagata, Japan might be attributable to two genetically different groups: group B showed a recurrent epidemic of strains belonging to a single phylogenetic cluster and group D showed epidemic strains belonging to multiple clusters.

The clinically used serine protease inhibitor nafamostat reduces influenza virus replication and cytokine production in human airway epithelial cells and viral replication in mice.

The effects of the clinically used protease inhibitor nafamostat on influenza virus replication have not been well studied. Primary human tracheal (HTE) and nasal (HNE) epithelial cells were pretreated with nafamostat and infected with the 2009 pandemic [A/Sendai-H/108/2009/(H1N1) pdm09] or seasonal [A/New York/55/2004(H3N2)] influenza virus. Pretreatment with nafamostat reduced the titers of the pandemic and seasonal influenza viruses and the secretion of inflammatory cytokines, including interleukin-6 and tumor necrosis factor-α, in the supernatants of the cells infected with the pandemic influenza virus. HTE and HNE cells exhibited mRNA and/or protein expression of transmembrane protease serine 2 (TMPRSS2), TMPRSS4, and TMPRSS11D. Pretreatment with nafamostat reduced cleavage of the precursor protein HA0 of the pandemic influenza virus into subunit HA1 in HTE cells and reduced the number of acidic endosomes in HTE and HNE cells where influenza virus RNA enters the cytoplasm. Additionally, nafamostat (30 mg/kg/day, intraperitoneal administration) reduced the levels of the pandemic influenza virus [A/Hyogo/YS/2011 (H1N1) pdm09] in mouse lung washes. These findings suggest that nafamostat may inhibit influenza virus replication in human airway epithelial cells and mouse lungs and reduce infection-induced airway inflammation by modulating cytokine production.

Antigenic changes among the predominantly circulating C/Sao Paulo lineage strains of influenza C virus in Yamagata, Japan, between 2015 and 2018.

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.

Inhibitory effects of glycopyrronium, formoterol, and budesonide on coronavirus HCoV-229E replication and cytokine production by primary cultures of human nasal and tracheal epithelial cells.

BACKGROUND: Coronavirus 229E (HCoV-229E), one of the causes of the common cold, exacerbates chronic obstructive pulmonary disease (COPD) and bronchial asthma. Long-acting muscarinic antagonists and ß2-agonists and inhaled corticosteroids inhibit the exacerbation of COPD and bronchial asthma caused by infection with viruses, including HCoV-229E. However, the effects of these drugs on HCoV-229E replication and infection-induced inflammation in the human airway are unknown. METHODS: Primary human nasal (HNE) and tracheal (HTE) epithelial cell cultures were infected with HCoV-229E. RESULTS: Pretreatment of HNE and HTE cells with glycopyrronium or formoterol decreased viral RNA levels and/or titers, the expression of the HCoV-229E receptor CD13, the number and fluorescence intensity of acidic endosomes where HCoV-229E RNA enters the cytoplasm, and the infection-induced production of cytokines, including IL-6, IL-8, and IFN-ß. Treatment of the cells with the CD13 inhibitor 2'2'-dipyridyl decreased viral titers. Pretreatment of the cells with a combination of three drugs (glycopyrronium, formoterol, and budesonide) exerted additive inhibitory effects on viral titers and cytokine production. Pretreatment of HNE cells with glycopyrronium or formoterol reduced the susceptibility to infection, and pretreatment with the three drugs inhibited activation of nuclear factor-kappa B p50 and p65 proteins. Pretreatment with formoterol increased cAMP levels and treatment with cAMP decreased viral titers, CD13 expression, and the fluorescence intensity of acidic endosomes. CONCLUSIONS: These findings suggest that glycopyrronium, formoterol, and a combination of glycopyrronium, formoterol, and budesonide inhibit HCoV-229E replication partly by inhibiting receptor expression and/or endosomal function and that these drugs modulate infection-induced inflammation in the airway.

Polyclonal spread of multiple genotypes of Mycoplasma pneumoniae in semi-closed settings in Yamagata, Japan.

PURPOSE: To clarify the spread of Mycoplasma pneumoniae infections in semi-closed settings such as schools and family homes using molecular typing methods. METHODOLOGY: We retrospectively searched for school- and family-based clusters of M. pneumoniae infections based on information regarding patients from whom M. pneumoniae strains had been isolated between 2011 and 2013 in Yamagata, Japan. The molecular typing profile, including the P1 type and the four-locus (Mpn13, 14, 15 and 16) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) type, was obtained from our previous study. RESULTS: We identified 11 school-based clusters involving 71 patients and 16 family-based clusters involving 38 patients, including 14 duplications between these types of clusters. A total of 95M. pneumoniae strains isolated from those patients were divided into 4 genotypes: 33 strains of type 4-5-7-2, 1; 31 of type 4-5-7-3, 1; 24 of type 3-5-6-2, 2c; and 7 of type 3-5-6-2, 2a. Of the 11 school-based clusters, 6 clusters (54.5%) consisted of multiple genotypes, and the remaining 5 clusters consisted of a single genotype. Moreover, the presence of multiple genotypes was identified in three classrooms of a school. On the other hand, in 14 (87.5%) of the 16 family-based clusters, the genotypes of the M. pneumoniae strains isolated from each family member were identical. CONCLUSION: The spread of M. pneumoniae infection in schools is likely polyclonal, since M. pneumoniae strains are brought into schools from various sites, such as family homes, which are important sites of disease transmission.

Effects of high temperature on pandemic and seasonal human influenza viral replication and infection-induced damage in primary human tracheal epithelial cell cultures.

High temperature reduces influenza viral replication; however, the treatment of fevers is thought to be necessary to improve patients' conditions. We examined the effects of high temperature on viral replication and infection-induced damage to human tracheal epithelial cells. Cell viability and dome formation were reduced, the number of detached cells was increased and lactate dehydrogenase (LDH) levels tended to be increased from 72 h to 120 h in uninfected cells cultured at 40 °C. Long-term (72 h and/or 120 h) exposure to high temperatures (39 °C and/or 40 °C) decreased RNA levels and/or viral titers of eight influenza virus strains. Cell viability and dome formation were reduced, and the number of detached cells and LDH levels were increased to a similar extent after infection with the A/H1N1 pdm 2009 virus at 37 °C and 40 °C. High temperature increased the endosomal pH, where the viral RNA enters the cytoplasm, in uninfected cells. High temperature reduced the production of IL-6, which mediate viral replication processes, and IL-1ß and IL-8 in uninfected and infected cells. Based on these findings, high temperature may cause similar levels of airway cell damage after infection to cells exposed normal temperatures, although high temperature reduces viral replication by affecting the function of acidic endosomes and inhibiting IL-6-mediated processes.

Neutralizing Epitopes and Residues Mediating the Potential Antigenic Drift of the Hemagglutinin-Esterase Protein of Influenza C Virus.

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.

Multiple-Locus Variable-Number Tandem-Repeat Analysis of Mycoplasma pneumoniae Isolates between 2004 and 2014 in Yamagata, Japan: Change in Molecular Characteristics during an 11-year Period.

Multiple-locus variable-number tandem-repeat analysis (MLVA) typing was performed for Mycoplasma pneumoniae strains isolated between 2004 and 2014 in Yamagata, Japan. The results were examined by considering the combination of the P1 type and prevalence of macrolide resistance-associated mutations. Four-locus (Mpn13-16) MLVA classified 347 strains into 9 MLVA types, including 3 major types: 3-5-6-2, 4-5-7-2, and 4-5-7-3. All type 3-5-6-2 strains (77 strains) were P1 type 2 variants (2a or 2c), while types 4-5-7-2 (181 strains) and 4-5-7-3 (75 strains) were P1 type 1. MLVA type 4-5-7-2 strains circulated and were dominant until 2010, accounting for 88.4% of the 121 strains isolated between 2004 and 2010. The prevalence of types 4-5-7-3 and 3-5-6-2 strains increased rapidly in 2011 and 2012, respectively, resulting in cocirculation of 3 MLVA types, including type 4-5-7-2, between 2011 and 2013. The prevalence of macrolide resistance-associated mutations in MLVA types 4-5-7-2, 4-5-7-3, and 3-5-6-2 strains was 59.7% (108/181), 25.3% (19/75), and 0% (0/77), respectively. Because the prevalence of macrolide resistance-associated mutations differed by current MLVA types in Yamagata, continued surveillance combined with molecular typing and identification of macrolide resistance-associated mutations is necessary.

Development of macrolide resistance-associated mutations after macrolide treatment in children infected with Mycoplasma pneumoniae.

PURPOSE: To determine the timing of the emergence of macrolide-resistant mutations after macrolide treatment in individuals with Mycoplasma pneumoniae infections. METHODOLOGY: Between October 2011 and December 2013, serial pharyngeal swab specimens were collected before and after macrolide treatment from 21 otherwise healthy children infected with M. pneumoniae without macrolide-resistant mutations. The copy numbers of a M. pneumoniae gene and the proportion of clones showing macrolide-resistance mutations were determined for each specimen. RESULTS: After macrolide treatment (10-15 mg kg-1 day-1 clarithromycin for 5-10 days or 10 mg kg-1 day-1 azithromycin for 3 days), fever resolved in 19 (90 %) of 21 children within 1 to 2 days, and the M. pneumoniae gene copy number decreased in all but one specimen in the second set of specimens relative to the number in the corresponding initial specimens. None of the second specimens, which were collected 2-4 days after initiation of macrolide treatment, showed mutations in the 23S rRNA gene. However, the proportion of mutant clones with A2063G and A2064G mutations in the specimens collected 7-24 days after initiation of treatment increased to 100 %. We identified a family in which three members had M. pneumoniae infections. The analysis of transmission in this household indicated that the M. pneumoniae harbouring a macrolide-resistant mutation that developed in the index patient after macrolide treatment was not transmitted to the family members. CONCLUSION: A macrolide-resistant population might develop in individual patients up to 24 days after initiation of macrolide treatment. However, the decrease in M. pneumoniae load after macrolide administration effectively reduces interpersonal transmission.

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

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

Development of an endpoint genotyping assay to detect the Mycoplasma pneumoniae 23S rRNA gene and distinguish the existence of macrolide resistance-associated mutations at position 2063.

The prevalence of macrolide-resistant Mycoplasma pneumoniae harboring a mutation in the 23S rRNA gene is increasing, and rapid detection assays are needed for clinical management. We developed an endpoint genotyping assay to detect the M. pneumoniae 23S rRNA gene and determine the existence of macrolide resistance-associated mutations at position 2063 (A2063G, A2063T and A2063C mutations). This A2063B genotyping assay detected more than 50 copies/reaction of the M. pneumoniae gene in every nucleotide mutation at position 2063. Of 42 clinical specimens, 3 were positive without mutation, 6 were positive with the A2063G mutation, and 33 were negative. The results were confirmed using nested PCR with the sequencing of the M. pneumoniae 23S rRNA gene, and a high sensitivity (90%), specificity (100%), and coincidence ratio (kappa coefficient=0.93) were obtained. Therefore, the A2063B genotyping assay is useful for the rapid discrimination of macrolide resistance mutations at position 2063.

Genetic Lineage and Reassortment of Influenza C Viruses Circulating between 1947 and 2014.

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.