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.

Longitudinal antigenic and seroepidemiological analyses of parechovirus A1 in Yamagata, Japan.

To investigate the antigenic changes in parechovirus 1 (PeVA1), seroepidemiological analyses were performed against the Harris strain (Harris), isolated in 1956, and PeVA1/Yamagata.JPN/2021-4785, isolated in 2021, using immune sera and 207 and 237 human serum specimens collected in 2021 and 1976, respectively. Although rabbit immune sera showed the highest neutralization antibody (NT-Ab) titers against the immunized viruses at 1:12 800-1:102 400, they were cross-reactive at 1:400-1:800. All 62 Yamagata isolates obtained between 2001 and 2021 (Yamagata strains), belonging to phylogenetic lineage 1B, reacted more strongly (mostly 4-64 times) to antiserum against PeVA1/Yamagata.JPN/2021-4785 than to antiserum against Harris, belonging to phylogenetic lineage 1 A. Human serum specimens obtained in 2021 showed higher NT-Ab titers against PeVA1/Yamagata.JPN/2021-4785, whereas those obtained in 1976 had similar NT-Ab titers against both strains. These findings suggested that Yamagata strains and Harris were antigenically cross-reactive, although there were differences. There are still high NT-Abs titers present against Harris in 2021 in particular, indicating that PeVA1 has been in circulation with high immunity in the population. In conclusion, this study suggested that PeVA1 has been endemically perpetuated with only minor antigenic changes as well as with high immunity over several decades in the community.

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.

Phylogenetic and antigenic analyses of coxsackievirus A6 isolates in Yamagata, Japan between 2001 and 2017.

Although coxsackievirus A6 (CV-A6) is generally recognized as a causative agent of herpangina in children, CV-A6 infections globally emerged as a new and major cause of epidemic hand-foot-and-mouth-diseases (HFMDs) around 2008. To clarify the longitudinal epidemiology of CV-A6, we carried out sequence and phylogenetic analyses for the VP1 and partially for the VP4-3D regions as well as antigenic analysis using 115 CV-A6 isolates and 105 human sera in Yamagata, Japan between 2001 and 2017. Phylogenetic analysis revealed that CV-A6 isolates were clearly divided into two clusters; strains in circulation between 2001 and 2008 and those between 2010 and 2017. Neutralizing antibody titers of two rabbit antisera, which were immunized with Yamagata isolates in 2001 and 2015, respectively, against 28 Yamagata representative strains as well as the prototype Gdula strain were 1:2560-1:5120 and 1:160-1:640, respectively. The neutralizing antibody titers among residents in Yamagata against the above two strains were similar. Our analyses revealed that there were cross-antigenicities among all analyzed CV-A6 strains, although the newly emerged strains were introduced into Yamagata around 2010 and replaced the previous ones. With regard to control measures, these findings suggest that we can prevent CV-A6 infections through the development of a vaccine that effectively induces neutralizing antibodies against CV-A6, irrespective of genetic cluster.

Parechovirus A3 (PeV-A3)-associated myalgia/myositis occurs irrespective of its genetic cluster: a longitudinal molecular epidemiology of PeV-A3 in Yamagata, Japan between 2003 and 2016.

No longitudinal molecular epidemiology of parechovirus A3 (PeV-A3) over a decade is available and PeV-A3-associated myalgia/myositis has been reported only in Japan. Thus, we aimed to clarify the longitudinal molecular epidemiology of PeV-A3 with a major focus on the strains detected from PeV-A3-associated myalgia/myositis cases. We performed sequence and phylogenetic analysis for the VP1 region of PeV-A3 strains in Yamagata, Japan, between 2003 and 2016. The phylogenetic analysis indicated that PeV-A3 strains caused PeV-A3-associated myalgia/myositis as well as a variety of infectious diseases, ranging from mild to severe, in subjects ranging from neonates to adults, irrespective of genetic cluster or variations. PeV-A3 strains are causative agents of a variety of human diseases, irrespective of their genetic cluster. Furthermore, we consider that PeV-A3-associated myalgia/myositis may occur, not only in Japan, but also in other countries, as closely related PeV-A3 strains have been circulating around the world.

Team-based parent training by child specialists helps maltreated children.

BACKGROUND: This study assessed the effects of the parent training (PT) technique, in which child specialists (CS) such as preschool and school teachers promote secure attachment in children with aberrant social behavior following maltreatment, using a team approach. METHODS: Child specialists confirmed the presence of child abuse, according to Japanese Ministry of Health, Labour and Welfare criteria. CS such as homeroom, special education-related, student guidance-related, nursing teachers and co-workers received a PT course conducted by the authors. A homeroom teacher provided classroom management to model good examples of social life for the target child. A nursing teacher and assistant offered individualized instruction to foster the formation of secure attachments by the target child. RESULT: Behavioral abnormalities in both school and home resolved in seven out of 12 cases. These subjects received the intervention for 2-4 years. In the other cases, behavioral abnormalities disappeared or decreased at school, but continued at home. Almost all children met the alternative criteria of attachment disorder proposed by Boris and Zeanah. One child met the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) criteria for reactive attachment disorder. This intervention is significantly more effective for children who have yet to begin elementary school than those in elementary school. CONCLUSIONS: The PT technique as applied by CS using a team approach may be a useful intervention for fostering secure attachment in children with maltreatment who exhibit behavioral abnormalities. Early detection and intervention are necessary to successfully address the behavioral abnormalities of children with maltreatment.

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.

Chronological changes of mumps virus genotypes in Japan between 1999-2013.

BACKGROUND: The molecular epidemiology of mumps virus (MuV) has been carried out worldwide based on genotyping proposed by the World Health Organisation. However, longitudinal molecular epidemiological studies of MuV are still limited. METHODS: This study carried out genotyping of MuVs isolated in Yamagata prefecture, which is located in northern Japan, between 1999-2013, using standard nomenclature based on the sequence analysis of the entire 316 nucleotides of the small hydrophobic (SH) gene. RESULTS: During this 15-year period, 249 MuVs were isolated, with the majority of them belonging to genotype G. Phylogenetic analysis revealed that genotype G strains were divided into two distinct clusters 1 and 2, consisting of 178 and 47 strains, respectively. The cluster 1 strains were isolated every year since 2001, except for 2012. The cluster 2 strains first appeared in 2011 and were dominant in 2011 and 2012. The epidemic pattern of genotype G strains observed in Yamagata was similar to those in Kanagawa and Hyogo prefectures located in eastern and western Japan, respectively. Only one L, three H and one F genotype strains were isolated in 2001, 2004 and 2010, respectively. Almost every year several genotype B strains related to Japanese vaccine strains were isolated. CONCLUSIONS: These data demonstrated that the genotype G strains have been endemically perpetuating as the major type over a wide area of Japan since 2001, although the genotype G strains that emerged after 2011 differed from the earlier strains.

Detection of the human coronavirus 229E, HKU1, NL63, and OC43 between 2010 and 2013 in Yamagata, Japan.

The available literature on human coronaviruses (HCoVs) in Japan is limited to epidemiological studies conducted over a maximum of 1 year. We conducted a 4-year study of HCoVs by analyzing 4,342 respiratory specimens obtained in Yamagata, Japan, between January 2010 and December 2013. A pan-coronavirus reverse transcription-PCR screening assay was performed, and all HCoV-positive specimens were subsequently confirmed by sequencing of the PCR products. We detected in 332 (7.6%) HCoV strains during the study period, comprising 133 (3.1%) HCoV-NL63, 83 (1.9%) HCoV-HKU1, 78 (1.8%) HCoV-OC43, and 38 (0.9%) HCoV-229E strains. HCoV detection per year ranged from 3.5% to 9.7%. HCoVs were detected mainly in winter, with January (28.5%) and February (25.3%) 2011 and December 2012 (14.6%) being the only months in which HCoV-NL63 detection per month exceeded 10.0%. HCoV-HKU1 displayed clear biennial peaks in January (18.3%) and February (10.7%) 2010 and in February (18.8%) and March (14.7%) 2012. The peak detection of HCoV-OC43 was 13.6% in November 2010, while that of HCoV-229E was 10.8% in March 2013. Our results indicated that there may be annual variations in the circulation of individual HCoV strains. Further long-term surveillance is necessary to clarify HCoV prevalence and circulation patterns in Japan.

Characteristics of Mycoplasma pneumoniae infection identified on culture in a pediatric clinic.

BACKGROUND: The appropriate choice of antibiotics against Mycoplasma pneumoniae infection has become difficult, as the prevalence of macrolide-resistant M. pneumoniae has increased. METHODS: Throat swab specimens were collected from children with clinically suspected M. pneumoniae infection while visiting an outpatient clinic. Cultures for M. pneumoniae were done, and all isolates were sequenced for the presence of a mutation in 23S rRNA. RESULTS: Of the 80 specimens collected between February 2012 and March 2013, 27 (34%) were positive for M. pneumoniae on culture. Macrolide-resistant mutation was detected in 24 isolates (89%): 23 isolates had an A2063G transition, and one had a C2617G mutation. Both the median age and the prevalence of pneumonia were significantly higher in M. pneumoniae-positive than in M. pneumoniae-negative children (median, 7 years vs 4 years; 88.9% vs 60.4%, respectively). The percentage of serum samples with particle agglutination titer ≥ 1:160 was 69.6% in M. pneumoniae-positive cases and 17.6% in M. pneumoniae-negative cases when the serum was collected ≥ 4 days after the onset of fever. Defervescence within 72 h after the initiation of macrolides never occurred in M. pneumoniae-positive children and also did not occur in 54% of M. pneumoniae-negative children. Switching to either minocycline or tosufloxacin resulted in fever resolution within 48 h in M. pneumoniae-positive children. CONCLUSIONS: The described clinical and laboratory characteristics of M. pneumoniae infection may be useful in guiding appropriate treatment in an outpatient clinic.

Molecular epidemiology of enterovirus 71 strains isolated from children in Yamagata, Japan, between 1990 and 2013.

Enterovirus 71 infections have become a major public issue in the Asia-Pacific region due to the large number of fatal cases. To clarify the longitudinal molecular epidemiology of enterovirus 71 (EV71) in a community, we isolated 240 strains from children, mainly with hand-foot-and-mouth diseases, between 1990 and 2013 in Yamagata, Japan. We carried out a sequence analysis of the VP1 region (891 bp) using 223 isolates and identified six subgenogroups (B2, B4, B5, C1, C2 and C4) during the study period. Subgenogroups C1 and B2 were found only between 1990 and 1993 and have not reappeared since. In contrast, strains in subgenogroups C2, C4 and B5 appeared repeatedly with genomic variations. Recent reports from several local communities in Japan have suggested that identical predominant subgenogroup strains, which have also been found in the Asia-Pacific region, have been circulating in a wide area in Japan. However, it is likely that there is a discrepancy between the major subgenogroups circulating in the Asia-Pacific region and those in Europe. It is necessary to continue the analysis of the longitudinal epidemiology of EV71 in local communities, as well as on regional and global levels, to develop strategies against severe EV71 infections.

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.

Interaction of PICK1 with C-terminus of growth hormone-releasing hormone receptor (GHRHR) modulates trafficking and signal transduction of human GHRHR.

Release of growth hormone (GH) from the somatotroph is regulated by binding GH-releasing hormone (GHRH) to its cognate receptor (GHRHR), one of the members of the G protein-coupled receptor (GPCR) superfamily. Proteins bound to the carboxy (C)-terminus of GPCR have been reported to regulate intracellular trafficking and function of the receptor; however, no functionally significant protein associated with GHRHR has been reported. We have identified a protein interacting with C-kinase 1 (PICK1) as a binding partner of GHRHR. In vitro binding assay revealed the PDZ-domain of PICK1 and the last four amino acid residues of GHRHR were prerequisite for the interaction. Further, in vivo association of these proteins was confirmed. Immunostaining data of a stable cell line expressing GHRHR with or without PICK1 suggested the C-terminus of GHRHR promoted cell surface expression of GHRHR and PICK1 affected the kinetics of the cell surface expression of GHRHR. Furthermore, cAMP production assay showed the C-terminus of GHRHR is involved in the regulation of receptor activation, and the interaction of GHRHR with PICK1 may influence intensities of the signal response after ligand stimulation. Thus, the interaction of the C-terminus of GHRHR with PICK1 has a profound role in regulating the trafficking and the signaling of GHRHR. [Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.12287FP].

Epidemic myalgia associated with human parechovirus type 3 infection among adults occurs during an outbreak among children: findings from Yamagata, Japan, in 2011.

BACKGROUND: Based on our findings in Yamagata, Japan, in 2008, we reported that human parechovirus type 3 (HPeV3) could be associated with epidemic myalgia among adults, although HPeV3 is generally associated with infectious diseases in children. OBJECTIVES: To clarify the relationship between community outbreaks among children and myalgia through the continued surveillance of HPeV3 infections. STUDY DESIGN: In the summer season (June-August) of 2011, we collected 586 specimens from children with infectious diseases, and throat swabs, and stool and serum specimens from 5 patients with myalgia. We detected HPeV3 using virus isolation and reverse-transcription PCR, and carried out phylogenetic analysis. We also performed screening for HPeV3 using 309 stocked frozen specimens collected in 2008 for a comparison between 2008 and 2011 strains. RESULTS: We detected HPeV3 in 59 children and isolated HPeV3 from all myalgia patients. Phylogenetic analysis indicated that the HPeV3 strains circulating in 2008 and 2011 could be clearly distinguished, apart from two strains. Further, we detected HPeV3 strains with identical nucleotide sequences from children and adults in 2008 and 2011, respectively. Two children belonging to one myalgia patient had upper respiratory infections prior to the onset of their father's illness, and the HPeV3 isolates from these three patients had identical nucleotide sequences. CONCLUSIONS: These findings suggest that HPeV3, circulating among children in the community, infects their household, including parents, a portion of whom may subsequently show symptoms of myalgia. Our observations in 2008 and 2011 strongly suggest that clinical consideration should be given to HPeV3 in children as well as in adults during summer seasons in which an HPeV3 outbreak occurs among the children in the community.

Seasonal patterns of respiratory syncytial virus, influenza A virus, human metapneumovirus, and parainfluenza virus type 3 infections on the basis of virus isolation data between 2004 and 2011 in Yamagata, Japan.

Most acute respiratory infections (ARIs) are thought to be associated with respiratory viruses that cause similar symptoms. Therefore, assessment of clinical and epidemiologic features of these viruses is important for diagnosing a viral infection. We collected 13,325 nasopharyngeal specimens from patients with ARIs and isolated the virus using a microplate method involving 7 cell lines between 2004 and 2011 in Yamagata, Japan. We isolated a total of 5,483 viruses. Respiratory syncytial virus (RSV), influenza A virus (FluA), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (hPIV3) showed clear yearly seasonal patterns; generally, RSV infections peaked at the end of the year, FluA infections peaked between January and March, hMPV infections peaked between March and April, and hPIV3 showed seasonal outbreaks between May and July. Further, RSV, hMPV, and hPIV3 were commonly isolated in 12.0-13.1% of specimens from children aged less than 4 years, whereas FluA was isolated in 7.3-8.2% of specimens from school-aged children. A generalized view of seasonality and age distribution, particularly on the basis of longitudinal epidemiological data, will be helpful for medical decision-making, including decisions related to the use of rapid test kits, selection of antiviral treatments, restriction of antibiotic therapy, and implementation of infection control strategies.

Epidemiology of parainfluenza virus types 1, 2 and 3 infections based on virus isolation between 2002 and 2011 in Yamagata, Japan.

To clarify the epidemiology of viral acute respiratory infections (ARIs), 305 human parainfluenza virus types 1 (HPIV1), 154 HPIV2 and 574 HPIV3 strains were isolated from 16,962 nasopharyngeal swabs obtained between 2002 and 2011 at pediatric clinics in Yamagata, Japan. The total isolation frequency for HPIV1-3 was 6.1%. Unlike HPIV1 infections, HPIV3 showed clear seasonality with yearly outbreaks in the spring-summer season. HPIV2 tended to appear biannually in autumn-winter. Although no reliable techniques for the laboratory diagnosis of these infections have been established, the present results suggest that HPIV1-3 are an important causative agent of ARIs in children.

Acute respiratory infections due to enterovirus 68 in Yamagata, Japan between 2005 and 2010.

To clarify the epidemiology of enterovirus 68 (EV68), which is one of the most rarely identified enteroviruses, virus isolation and molecular screening using RT-PCR was performed on 6307 respiratory specimens collected at pediatric clinics in Yamagata, Japan between 2005 and 2010. In the years 2005-2009, 10, 1, 2, 0, and 2 (40) EV68-positive cases, respectively, were identified by RT-PCR. In 2010, 40 cases were identified altogether: 2 by isolation only, 26 by RT-PCR only, and 12 by both isolation and RT-PCR. Phylogenetic analysis indicated that plural genetically distinct clusters co-circulated. These results suggest that that difficulty in EV68 isolation leads to an underestimation of the prevalence of EV68 infections.

Supernumerary impacted teeth in a patient with SOX2 anophthalmia syndrome.

SOX2 anophthalmia syndrome characteristically presents as anophthalmia or microphthalmia, with various extraocular symptoms, such as hypogonadotropic hypogonadism, brain anomaly, and esophageal abnormalities. In this report, we describe a patient with SOX2 anophthalmia syndrome complicated with a dental anomaly, multiple supernumerary impacted teeth, and persistence of deciduous teeth. Multiple supernumerary teeth are usually not solitary symptoms, but indicate systemic syndrome such as cleidocranial dysplasia. In odontogenesis, many transcriptional factors, such as BMPs, FGFs, and Wnts, play significant roles and SOX2 is known to interact with some of them. The role of SOX2 in dental development remains unknown, however, multiple supernumerary teeth can be considered as extraocular symptoms of SOX2 anophthalmia syndrome, rather than the coincidence of two rare diseases.