Yoshitaka Shimotai

Detection and quantification of influenza C virus in pediatric respiratory specimens by real-time PCR and comparison with infectious viral counts

BACKGROUND The epidemiological and clinical impacts of influenza C virus infection may have been underestimated by conventional viral culture screening alone. OBJECTIVE To evaluate a newly developed real-time polymerase chain reaction (PCR) assay as a tool for diagnosing influenza C virus infection. STUDY DESIGN The primers and probe for real-time PCR were designed to amplify the conserved region of the nucleoprotein gene based on the aligned sequences of nine isolates from 1967 to 2010. Respiratory specimens from children collected between January 2010 and August 2010 were examined for the presence of influenza C virus by cell culture and real-time PCR. Specimens that were positive for the virus using real-time PCR were further examined using an infectivity assay with embryonated hens eggs. RESULTS Of the 1203 specimens examined, 34 (2.8%) tested positive for the influenza C virus by cell culture and 51 (4.2%) tested positive by real-time PCR. The mean viral load and infectivity titer in specimens that tested positive using cell culture were 3.97×10(8)copies/ml and 5.43×10(5)EID(50)/ml, respectively, and those in specimens that were negative using cell culture were 2.18×10(6)copies/ml and 3.67×10(2)EID(50)/ml, respectively. In the clinical specimens with viral loads less than 10(5)copies/ml, it was not possible to isolate the virus using embryonated hens eggs. The copy number-to-EID(50) ratio of the clinical specimens was much higher, ranging from 32 to 278,000, than those of culture fluid, ranging from 2.3 to 13.5. CONCLUSION The real-time PCR assay described here can be used as a sensitive method for diagnosing influenza C virus infection.

Epitope Mapping of the Hemagglutinin Molecule of A/(H1N1)pdm09 Influenza Virus by Using Monoclonal Antibody Escape Mutants

ABSTRACT We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb, and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417–427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177, and 180 in the Sa site, residues 170, 173, 202, 206, 210, 211, and 212 in the Sb site, residues 151, 154, 156, 157, 158, 159, 200, and 238 in the Ca2 site, and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb), and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain. IMPORTANCE The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating with swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope.

Role of the CM2 Protein in the Influenza C Virus Replication Cycle

ABSTRACT 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.

Epidemiological information regarding the periodic epidemics of influenza C virus in Japan (1996–2013) and the seroprevalence of antibodies to different antigenic groups

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.

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

ABSTRACT 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.

Serine Proteases and their Inhibitors in Human Airway Epithelial Cells: Effectson Influenza Virus Replication and Airway Serine Proteases and their Inhibitors in Human Airway Epithelial Cells: Effects on Influenza Virus Replication and Airway Inflammation

Influenza virus replication and the production of inflammatory cytokines are associated with symptoms, including fever, and exacerbation of bronchial asthma and chronic obstructive pulmonary disease. Proteolytic activation of influenza viruses by serine proteases that are produced by airway epithelial cells is essential for viral entry and replication. Transmembrane protease serine S1 member (TMPRSS) 2, TMPRSS4 and TMPRSS11D have been detected certain cells, including the human alveolar epithelial cell line A549 and the surface epithelial cells of the human nasal mucosa, the trachea, the distal airways, and the lung. Several protease inhibitors, including aprotinin, reduce influenza virus replication. We previously demonstrated the following: (1) TMPRSSs (TMPRSS2, 4, and 11D) are present in primary cultures of human tracheal epithelial cells; (2) serine protease inhibitors, such as camostat and aprotinin, reduce the influenza virus replication and the release of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α into cell supernatants; and (3) camostat reduces the cleavage of an influenza virus precursor protein, HA0, into the subunit HA1. These findings suggest that serine proteases expressed by human tracheal epithelial cells induce the proteolytic activation of influenza viruses and that serine protease inhibitors may reduce viral replication and the resultant production of inflammatory cytokines. Thus, serine protease inhibitors are potential candidates for anti-influenza virus drugs Here, we review the expression of serine proteases, the role of serine proteases in influenza virus activation, and the effects of serine protease inhibitors. In this review, we aim to introduce the effects of serine proteases and their inhibitors on influenza virus infection of human airway epithelial cells by discussing the findings of previous studies performed by our group and other research groups. Furthermore, the clinical features and virulence of influenza virus infection are reviewed to clarify the association of virus replication and cytokine release with disease severity.

The serine protease inhibitor camostat inhibits influenza virus replication and cytokine production in primary cultures of human tracheal epithelial cells

Abstract Background Serine proteases act through the proteolytic cleavage of the hemagglutinin (HA) of influenza viruses for the entry of influenza virus into cells, resulting in infection. However, the inhibitory effects of serine protease inhibitors on influenza virus infection of human airway epithelial cells, and on their production of inflammatory cytokines are unclear. Methods Primary cultures of human tracheal epithelial cells were treated with four types of serine protease inhibitors, including camostat, and infected with A/Sendai-H/108/2009/(H1N1) pdm09 or A/New York/55/2004(H3N2). Results Camostat reduced the amounts of influenza viruses in the supernatants and viral RNA in the cells. It reduced the cleavage of an influenza virus precursor protein, HA0, into the subunit HA1. Camostat also reduced the concentrations of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the supernatants. Gabexate and aprotinin reduced the viral titers and RNA levels in the cells, and aprotinin reduced the concentrations of TNF-α in the supernatants. The proteases transmembrane protease serine S1 member (TMPRSS) 2 and HAT (human trypsin-like protease: TMPRSS11D), which are known to cleave HA0 and to activate the virus, were detected at the cell membrane and in the cytoplasm. mRNA encoding TMPRSS2, TMPRSS4 and TMPRSS11D was detectable in the cells, and the expression levels were not affected by camostat. Conclusions These findings suggest that human airway epithelial cells express these serine proteases and that serine protease inhibitors, especially camostat, may reduce influenza viral replication and the resultant production of inflammatory cytokines possibly through inhibition of activities of these proteases.

Effect of Phosphorylation of CM2 Protein on Influenza C Virus Replication

ABSTRACT 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 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.

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.