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Human parechovirus has rarely been shown to cause clinical disease in adults. During June-August 2008, a total of 22 adults sought treatment at Yonezawa City Hospital in Yamagata, Japan, for muscle pain and weakness of all limbs; most also had fever and sore throat. All patients received a clinical diagnosis of epidemic myalgia; clinical laboratory findings suggested an acute inflammatory process. Laboratory confirmation of infection with human parechovirus type 3 (HPeV3) was made for 14 patients; we isolated HPeV3 from 7 patients, detected HPeV3 genome in 11, and observed serologic confirmation of infection in 11. Although HPeV3 is typically associated with disease in young children, our results suggest that this outbreak of myalgia among adults was associated with HPeV3 infection. Clinical consideration should be given to HPeV3 not only in young children but also in adults when an outbreak occurs in the community. [ABSTRACT FROM AUTHOR]

Abstract Background Human parainfluenza virus type 1 (HPIV1) causes various acute respiratory infections (ARI). Hemagglutinin-neuraminidase (HN) glycoprotein of HPIV1 is a major antigen. However, the molecular epidemiology and genetic characteristics of such ARI are not exactly known. Recent studies suggested that a phylogenetic analysis tool, namely the maximum likelihood (ML) method, may be applied to estimate the evolutionary time scale of various viruses. Thus, we conducted detailed genetic analyses including homology analysis, phylogenetic analysis (using both the neighbor joining (NJ) and ML methods), and analysis of the pairwise distances of HN gene in HPIV1 isolated from patients with ARI in Yamagata prefecture, Japan. Results A few substitutions of nucleotides in the second binding site of HN gene were observed among the present isolates. The strains were classified into two major clusters in the phylogenetic tree by the NJ method. Another phylogenetic tree constructed by the ML method showed that the strains diversified in the late 1980s. No positively selected sites were found in the present strains. Moreover, the pairwise distance among the present isolates was relatively short. Conclusions The evolution of HN gene in the present HPIV1 isolates was relatively slow. The ML method may be a useful phylogenetic method to estimate the evolutionary time scale of HPIV and other viruses.

Abstract Background Oseltamivir is the preferred antiviral drug for influenza, but oseltamivir-resistant A(H1N1) viruses have circulated worldwide since the 2007-2008 influenza season. We aimed to determine the rate of oseltamivir resistance among A(H1N1) isolates from Yamagata, Japan, to compare the virological characteristics between isolates from the 2007-2008 and 2008-2009 seasons, and to evaluate the clinical effectiveness of oseltamivir. Results Oseltamivir resistance, determined by detecting the H275Y mutation in the neuraminidase (NA) gene, was observed in 2.5% (2 of 79) and 100% (77 of 77) of isolates from the 2007-2008 and 2008-2009 seasons, respectively. Antigenic analysis suggested that antigenically different variants of A(H1N1) viruses circulated in the 2008-2009 season. Growth testing demonstrated that the ability of the 2008-2009 isolates to replicate in MDCK cells was similar to those of the oseltamivir-susceptible isolates from the 2007-2008 season. A phylogenetic analysis revealed that two oseltamivir-resistant viruses isolated in the 2007-2008 season were closely related to other oseltamivir-susceptible viruses in Yamagata but were different from oseltamivir-resistant viruses isolated in Europe and North America in the 2007-2008 season. The oseltamivir-resistant viruses isolated in Japan in the 2008-2009 season were phylogenetically similar to oseltamivir-resistant isolates from Europe and North America during the 2007-2008 season. Furthermore, the median duration of fever after the start of oseltamivir treatment was significantly longer in oseltamivir-resistant cases (2 days; range 1-6 days) than in oseltamivir-susceptible cases (1.5 days: range 1-2 days) (P = 0.0356). Conclusion Oseltamivir-resistant A(H1N1) isolates from Yamagata in the 2007-2008 season might have acquired resistance through the use of oseltamivir, and the 2008-2009 oseltamivir-resistant isolates might have been introduced into Japan and circulated throughout the country. Influenza surveillance to monitor oseltamivir-resistance would aid clinicians in determining an effective antiviral treatment strategy.

Background: Schizophrenia often involves persecutory delusions, which cause psychological distress. Some patients use online gaming as a coping tool. However, excessive online gaming has raised concerns about internet gaming disorders (IGD), while any soothing effects of online gaming on psychological distress remain unclear. Herein, we report changes in anxiety and IGD severity, measured using rating scales, in a patient with schizophrenia who used online gaming as a coping strategy for psychological distress., Case Presentation: A 43-year-old woman diagnosed with schizophrenia had worsening persecutory delusions, including that of being targeted by snipers, and had difficulty going out because of anxiety. She coped with her psychological distress using online shooting games. We assessed her state and trait anxiety, social anxiety, avoidance behavior when alone, and IGD severity. There was a notable reduction in the state anxiety score after the introduction of online gaming. The scores for trait anxiety, social anxiety, and avoidance behavior when alone decreased noticeably after the acquisition of coping strategies. This case demonstrates the presence of IGD only during the acquisition of coping strategies., Conclusion: This case highlights the potential of online gaming as a coping strategy for schizophrenia-related anxiety. However, excessive gaming can lead to IGD and thus necessitates caution. Further research should explore the applicability and potential risks of using online gaming to cope with psychological distress among patients with schizophrenia., Competing Interests: The authors declare that they have no conflict of interest., (© 2024 The Author(s). Psychiatry and Clinical Neurosciences Reports published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Psychiatry and Neurology.)

A 6-month prospective study in a hospital setting detected influenza C virus and human metapneumovirus in 10.0% (29/289) and 16.6% (48/289), respectively, of children hospitalized with lower respiratory tract illness. Influenza C virus infection had a similar rate of pneumonia (53.3% vs. 57.1%), significantly lower frequency of wheezing (13.3% vs. 68.6%) and higher values of white blood cell and C-reactive protein than human metapneumovirus infection.

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.

To clarify the epidemiologic features of Mycoplasma pneumoniae, we examined 358 M. pneumoniae strains isolated between 2004 and 2013 in Yamagata, Japan. Analysis of macrolide-resistance-associated 23S ribosomal RNA (rRNA) domain V mutations revealed 6 kinds of mutants (81 A2063G, 43 A2063T, 1 A2063C, 1 A2064C, 4 C2617G and 1 C2617 mutation). There were only two mutants before 2009, but mutants A2063T and A2063G increased in 2009 and from 2010, respectively. The annual ratio of mutants varied from 20.4% to 76.4% between 2009 and 2013. Typing of the p1 gene revealed 4 types; 278 type 1, and 3 kinds of type 2 variant strains (10 type 2a, 5 type 2b and 65 type 2c). Type 1 strains accounted for between 85.2% and 100% of isolates from 2004 to 2011, whereas type 2 variant strains increased by 26.5% and 66.1% in 2012 and 2013, respectively. These results indicate that type 1 strains may have been replaced by type 2 variant strains in 2013. Furthermore, the ratio of type 1 strains with a 23S rRNA mutation was 65.1% in 2012 and 95.2% in 2013, but none of the type 2 variant strains had this mutation. In conclusion, type 1 strains with macrolide-resistant mutations appeared in 2006 and increased from 2009. In contrast, type 2 variant strains, which increased in 2012 and became predominant in 2013, showed no mutations.

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 conducted detailed genetic analyses of the haemagglutinin-neuraminidase (HN) gene in 272 human parainfluenza virus type 3 (HPIV3) isolates from children with acute respiratory illness during the period 2002-2009 in Yamagata prefecture, Japan. A phylogenetic tree reconstructed by the Bayesian Markov chain Monte Carlo method showed that the strains diversified at around 1946 and that the rate of molecular evolution was 1.10×10(-3) substitutions per site per year. Identity was high among the present strains (<90 %) and the pairwise-distances were short. Furthermore, we found four positive selection sites and some key amino acid substitutions in active/catalytic sites of the HN protein. The results suggest that the HN gene of HPIV3 in the present strains evolved rapidly, similarly to other virus genes such as the G gene of respiratory syncytial virus. However, the biological functions and detailed structures of the HN glycoprotein in some of these strains may have been altered.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The measles elimination project led by the World Health Organization (WHO) has been moving toward the target of eliminating measles in the WHO Western Pacific Region. In Japan, prefectural public health institutes play a key role for the laboratory diagnosis of measles virus (MV) infection, which is based on PCR, virus isolation, and genotyping. Microscopic examination of viral-sensitive cell lines during routine virus isolation from nasopharyngeal specimens has been used to detect the morphological changes typical for the growth of respiratory viruses. Here, we describe the unexpected isolation of vaccine-derived MVs from the two unrelated 1-year-old boys with acute respiratory infection. The nasopharyngeal specimens were obtained from one patient in February 2007 and from another in December 2012. Incidentally, the two children had received measles-rubella vaccination 9 or 11 days before the sampling. The isolates from two children induced morphological changes of the viral-sensitive cell lines, such as syncythia formation (cell fusion). We finally identified the isolates as vaccine-derived MVs by sequence analysis and immunological methods with anti-measles nucleoprotein antibodies. As no typical symptoms of MV infection were observed in either patient, the vaccine-derived MVs were isolated not as causative pathogens but by chance. In fact, there was no suspected case of secondary MV infection in either patient, thereby excluding the possibility that vaccine-derived MVs spread from human to human. Our experiences suggest the possibility of vaccine-derived MV isolation by cell cultures and the difficulty in identifying MVs in specimens from patients other than clinically suspected measles cases.

To clarify the longitudinal molecular epidemiology of coxsackievirus A16, phylogenetic analysis based on the VP1 region of 220 isolates in Yamagata, Japan was performed. The resultant phylogenetic tree indicates that the Yamagata isolates and reference strains can be readily genotyped into three genogroups, and 0, 12 and 208 isolates belonged to the first, second, and third genogroups, respectively. The first genogroup includes only the prototype strain, the second strains that had disappeared by the end of the 20th century and the third comprises those that have been circulating since then in local communities, such as Yamagata., (© 2013 The Societies and Wiley Publishing Asia Pty Ltd.)

Intrahepatic eosinophilic proliferative pylephlebitis (EPP) in Japanese Black (JB) cattle generally has been considered to be an atypical form of fascioliasis. However, there are many cases of EPP in which no Fasciola spp. have been detected in the livers of affected cattle. The aims of this study were to ascertain the relationship between EPP and hepatic fascioliasis and to investigate the role of food allergy in the disease. Histologically, EPP lesions were characterised by severe endothelial proliferation of the interlobular veins, accompanied by varying degrees of fibrosis and eosinophilic infiltration in portal areas, which could be differentiated from chronic cholangiohepatitis, the typical lesion of hepatic fascioliasis. In addition to hepatic lesions, all cases of EPP had varying degrees of eosinophilic infiltration in the perilymphoid red pulp of the spleen, whereas both affected and unaffected animals had eosinophilic infiltrates in the mucosa of the small intestine. Antibodies against Fasciola spp. were detected in 1/14 EPP cases by ELISA; the seropositive case had EPP in combination with chronic cholangitis. There was no significant difference in total concentration of IgE between cases of EPP and unaffected cattle. Serum IgE levels specific to curly dock (Rumex crispus) and oats (Avena sativa) were higher in EPP cases than in unaffected cattle by allergen profiling screening testing and ELISA. The results of this study suggest that hepatic fascioliasis is unlikely to be the cause of EPP in JB cattle and that food allergens should be investigated as possible aetiological agents., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: We detected a community outbreak of macrolide-resistant Mycoplasma pneumoniae infection that occurred predominantly among students at 2 schools in Yamagata, Japan., Methods: Throat swab specimens were collected from patients who were clinically suspected to have M. pneumoniae infection after testing negative for influenza virus by a nasopharyngeal swab rapid antigen test. We performed cultures for M. pneumoniae, and all isolates were sequenced for the presence of a mutation of the 23S rRNA gene., Results: Of 96 specimens collected between July 2009 and January 2010, 83 were from students attending junior high school A and primary schools B, C and D. A total of 47 M. pneumoniae isolates were obtained; among them, 25, 15 and 4 were isolated from students attending schools A, B and D, respectively, and M. pneumoniae could not be isolated from students who attended school C. An A2063T mutation in domain V of the 23S rRNA gene, which is associated with macrolide resistance, was identified in 39 (83.0%) isolates. The rates of macrolide resistance at schools A, B and D were 96.0%, 86.7% and 0%, respectively. The minimum inhibitory concentrations for isolates with an A2063T transversion showed high resistance to clarithromycin (minimum inhibitory concentration, 16-64 mg/L), and clarithromycin prescribed initially was clinically ineffective., Conclusions: This school-based cluster of macrolide-resistant M. pneumoniae infections, which was identified in 2 geographically close schools, indicates that the transmission principally occurred by close contact between students at school. Monitoring the spread of macrolide-resistant M. pneumoniae and clinical guidelines for the appropriate medication against such infections would be needed to control outbreaks of M. pneumoniae.

To identify the vector species for Shimokoshi type Orientia tsutsugamushi, a survey of larval trombiculid mites was conducted in Yamagata Prefecture, Japan from April to May 2012. In all, 2889 larval trombiculid mites were obtained from 21 Apodemus speciosus rodent hosts, 2600 of which were morphologically classified into eight species in three genera. After screening of O. tsutsugamushi DNA in individual larval trombiculid mites using real-time PCR targeting the 16S ribosomal RNA gene, serotype-specific nested PCRs targeting the 56 kDa protein gene were performed, followed by sequencing analysis. As a result, Shimokoshi type O. tsutsugamushi DNA was identified from 3 (1.9%) of 157 Leptotrombidium palpale. This is the first study to identify Shimokoshi type O. tsutsugamushi DNA in L. palpale. The results indicate that L. palpale is a possible vector for Shimokoshi type O. tsutsugamushi., (© 2012 The Societies and Wiley Publishing Asia Pty Ltd.)

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.

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., (© 2012 The Societies and Wiley Publishing Asia Pty Ltd.)

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 hen's 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 hen's 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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (© 2012 The Societies and Blackwell Publishing Asia Pty Ltd.)

Background: Saffold virus (SAFV) is a newly discovered virus belonging to the genus Cardiovirus of the family Picornaviridae. Using molecular techniques, SAFV has been detected, although infrequently, in the stools of both healthy and diarrheic children and in respiratory specimens collected from children with respiratory disease. The epidemiology and pathogenicity of SAFV remain unclear., Methods: Between July 2009 and October 2010, nasopharyngeal specimens were collected from children with acute respiratory infections. The collected samples were used to isolate respiratory viruses, including coxsackievirus, by cell culture and were tested for SAFV by reverse transcription-polymerase chain reaction., Results: SAFV genotype 2 (SAFV2) was detected in 54 (3.5%) of the 1525 children tested. SAFV2 detections showed an epidemic pattern for a 4-month period with a peak in October 2009. The median age of the SAFV2-positive children was 4 years (range: 7 months-16 years). Among the 35 SAFV2-positive children, excluding cases of viral coinfection, 13 (37.1%) had pharyngitis, 12 (34.3%) had tonsillitis, and 8 (22.8%) had herpangina. Bronchitis and gastroenteritis were detected in 1 case each. Fever (temperature, >38°C) was noted in 33 (94.3%) cases. The median duration of fever was 2 days (range: 1-3 days). Diarrhea was observed in 7 (20.0%) children, but watery and frequent diarrhea was not common. The age distribution and clinical diagnoses associated with SAFV2 infections were similar to those observed with coxsackievirus B4 infections, which detections showed an epidemic pattern during the study period., Conclusion: SAFV2 is a cause of upper respiratory tract illness that exhibits a pathogenicity similar to that of coxsackievirus B.

To clarify a longitudinal epidemiology,we isolated 280 hMPV strains from patients with acute respiratory infections in Yamagata, Japan, between 2004 and 2009.We observed that the high season for hMPV was from winter to spring (between January and May) and the low season was in the fall (around September and October). A further molecular analysis revealed that subgenogroup A2 (A2) strains were the most commonly isolated (151/280; 53.9%), followed by B2 (108/280; 38.6%) and B1 (19/280; 6.8%). Our results suggested that A2 and B2 have been endemically in circulation as the major types almost every year, whereas other subgenogroups have appeared less frequently.

Background: Oseltamivir is the preferred antiviral drug for influenza, but oseltamivir-resistant A(H1N1) viruses have circulated worldwide since the 2007-2008 influenza season. We aimed to determine the rate of oseltamivir resistance among A(H1N1) isolates from Yamagata, Japan, to compare the virological characteristics between isolates from the 2007-2008 and 2008-2009 seasons, and to evaluate the clinical effectiveness of oseltamivir., Results: Oseltamivir resistance, determined by detecting the H275Y mutation in the neuraminidase (NA) gene, was observed in 2.5% (2 of 79) and 100% (77 of 77) of isolates from the 2007-2008 and 2008-2009 seasons, respectively. Antigenic analysis suggested that antigenically different variants of A(H1N1) viruses circulated in the 2008-2009 season. Growth testing demonstrated that the ability of the 2008-2009 isolates to replicate in MDCK cells was similar to those of the oseltamivir-susceptible isolates from the 2007-2008 season. A phylogenetic analysis revealed that two oseltamivir-resistant viruses isolated in the 2007-2008 season were closely related to other oseltamivir-susceptible viruses in Yamagata but were different from oseltamivir-resistant viruses isolated in Europe and North America in the 2007-2008 season. The oseltamivir-resistant viruses isolated in Japan in the 2008-2009 season were phylogenetically similar to oseltamivir-resistant isolates from Europe and North America during the 2007-2008 season. Furthermore, the median duration of fever after the start of oseltamivir treatment was significantly longer in oseltamivir-resistant cases (2 days; range 1-6 days) than in oseltamivir-susceptible cases (1.5 days: range 1-2 days) (P = 0.0356)., Conclusion: Oseltamivir-resistant A(H1N1) isolates from Yamagata in the 2007-2008 season might have acquired resistance through the use of oseltamivir, and the 2008-2009 oseltamivir-resistant isolates might have been introduced into Japan and circulated throughout the country. Influenza surveillance to monitor oseltamivir-resistance would aid clinicians in determining an effective antiviral treatment strategy.

The clinical impact of human metapneumovirus (hMPV) genotypes and the relation between the hMPV genotype in circulation and genotype-specific seroprevalence are yet to be clarified. We determined the genotypes of 93 hMPV strains that were isolated between 2004 and 2006 in Yamagata, Japan, and identified 35 genotype A2, 14 genotype B1, and 44 genotype B2 isolates. Children infected with genotype A2 hMPV were significantly older than those infected with genotype B1 hMPV. Diagnosis of laryngitis was more common in children with genotype B1 hMPV infection and wheezing was more prevalent in children with genotype B1 and B2 hMPV infection than in those with genotype A2 hMPV infection. We then examined genotype-specific seroprevalence by neutralization assay. The higher seropositive rate for the B2 genotype among the children aged 1-2 years is likely to reflect the outbreak of B2 genotype strains in the previous year in this community. The low seropositive rate for the B1 genotype among children aged 1-2 years appears to be associated with a finding that more than 70% of children infected with the B1 genotype were less than 3 years old. In conclusion, we found that the different clinical characteristics of hMPV infection may be associated with hMPV genotype, and the predominant genotype during a season and the affecting age may be closely related to genotype-specific immune status within a community.

Although well over 200 viral agents have been implicated in acute respiratory infections (ARIs) among children, no system able to detect such a wide range of viruses has been established. Between January 2004 and December 2005, a modified microplate method, including HEF, HEp-2, Vero E6, MDCK, RD-18S, and GMK cell lines (HHVe6MRG plate), was adopted to isolate viruses. A total of 1,551 viruses were isolated, representing both outbreaks and sporadic cases, from 4,107 nasopharyngeal specimens, at monthly isolation rates of 22.3 to 52.6%. Influenza, parainfluenza, respiratory syncytial (RS), and mumps viruses, and human metapneumovirus, enterovirus, parechovirus, rhinovirus, adenovirus, herpesvirus, and cytomegalovirus were all isolated. The use of multiple cell lines increased the isolation rates of most of these viruses. The findings showed that ARIs due to a number of respiratory viruses occurred across all seasons in succession and/or concurrently in children in the community. These data will help clinicians determine in which seasons and for which age groups they should use the rapid diagnostic test kits available for influenza virus, RS virus, and adenovirus. In conclusion, we verified that the modified microplate method was able to clarify the etiology and epidemiology of numerous viruses isolated from children with ARI.

During the period from January to July 2004, a total of 131 influenza C viruses were detected by cell culture or reverse transcription-PCR (RT-PCR) from specimens that were obtained from children with acute respiratory symptoms in 10 prefectures across Japan. Influenza C virus was identified most frequently in the Miyagi (1.4%, 45 of 3,226 specimens) and Yamagata (2.5%, 31 of 1,263 specimens) prefectures, and the frequency in this year was the highest since 1990. Phylogenetic analysis of the hemagglutinin esterase gene of the 13 strains isolated in nine prefectures revealed that genetically similar strains belonging to the Kanagawa/1/76-related lineage dominantly spread throughout Japan. During the 2004 influenza season, influenza C virus coexisted with epidemics of influenza A virus (H3 strain), and 12 cases were identified from patients who had been diagnosed with influenza-like illness (7 were detected by RT-PCR, and 5 were detected by culture). A comparison of specimens that were found positive by culture with those found positive only by RT-PCR shows that the amount of virus in PCR-positive specimens tended to be lower than in isolation-positive specimens. Although the mean peak temperature in patients in the PCR-positive group was slightly lower, there were no significant differences in characteristics between specimens (i.e., kind of specimen, period from onset to specimen collection, age distribution of patients, and severity of illness). These results suggest that an epidemic of influenza C virus occurred on a national scale during this period and that RT-PCR can be an effective supplemental tool for the evaluation of clinical and epidemiological information.

We have continued the epidemiological study on adenovirus type 7 (Ad7), which re-emerged in 1995 in Yamagata, Japan. Between 1999 and 2004, we isolated only four strains from 10,778 throat swab specimens among children with acute respiratory infections. A serological survey of 303 specimens revealed the antibody-positive rate against Ad7 to be 0-7.4% in children under 10 years of age in 2005, although it was 3.3-16.7% in 1997 and 0% in 1993. Our results suggest that a re-emergence does not always provoke a sudden major outbreak, even if the antibody-positive rate against Ad7 is low in the local community.

We investigated a measles virus (MV) outbreak that occurred at a junior high school in Yamagata, Japan between January and February, 2004. We received throat swab specimens from three patients at this school and carried out virus isolation with Vero/hSLAM cells and virus genome detection by reverse-transcription polymerase chain reaction. As a result, we isolated the virus from one patient and succeeded in amplifying the MV genome from the others. Further sequence analysis of the N gene revealed that these viruses were completely identical, and that their genotype could be characterized as type D9, which has not been reported in Japan previously. We also identified D9 viruses in two students at other junior high schools in Yamagata. These results suggested that D9 strains were imported from a region outside Japan. The genotypes of MVs found in Yamagata have changed in recent years, with D5 predominating in 2001 and H1 predominating in 2002 and 2003 as reported as national surveillance data. Therefore, we should monitor carefully to be sure that D9 strains do not become the next predominant virus. The more the number of measles cases decrease, the more important become the roles of public health laboratories, which genotype MVs and monitor their circulation and transmission pathways.

Objectives: To analyze the prevalence of echovirus type 13 (Echo13) in Yamagata, Japan., Methods: Virus isolation was performed from 6514 clinical specimens using six cell lines between January 1999 and December 2002. We also carried out a seroepidemiological study against Echo13, using 234 serum samples collected in 2001., Results: In 2002, we isolated a total of 50 Echo13 strains, which had not been detected from 1981 until 2001 in Japan. The antibody positive rate was higher (57.2-62.0%) in subjects 50 years or over than in those under 50 years (0-14.4%)., Conclusions: Serological study suggested that Echo13 had been present in Yamagata until around 1960, at which time the antibody positive persons were exposed to Echo13 in their childhood. Furthermore, results of virus isolation demonstrated that Echo13 re-emerged in around 2002 after a hiatus of several decades.

Objective: To evaluate a modified microplate method, utilizing HEF, HEp-2, Vero, MDCK and newly introduced RD-18S and GMK cell lines, for virus isolation., Methods: From June to October 2001, 723 throat swab specimens taken from children with acute respiratory infections (ARIs) were inoculated onto these cells. To analyze cell sensitivity, we also inoculated 20 serotypes of stocked enteroviruses., Results: During the period, we isolated 40 Coxsackie A2 (CoxA2), 13 CoxA4, 16 CoxA16, 1 CoxB2, 11 CoxB3, 2 CoxB5, 54 echo16, 2 entero71 and 1 polio2. By observing a cell sensitivity pattern with HEF, HEp-2, Vero, RD-18S, and GMK, we could finally differentiate five enterovirus groups: CoxA except for CoxA16, CoxA16/entero71, CoxB, echovirus, and poliovirus., Conclusions: With this system, the RD-18S cell line enabled us to isolate CoxA virus, except for CoxA16, for the first time. Differentiation of five enterovirus groups by cell sensitivity simplified the specific identification by neutralization test as a presumptive identification. A modified microplate method may be an appropriate cell combination for virus isolation, especially for enteroviruses, and is expected to be used routinely for virologic diagnosis and to clarify the epidemiology of ARI in children.

To find a new influenza subtype A(H1N2), 383 isolates identified as H1 by hemagglutination inhibition test between the 1998-1999 and 2001-2002 seasons in Yamagata, Japan, were screened by reverse transcription polymerase chain reaction. As a result, 3 strains from the 1999-2000 season were identified as possibly being A(H1N2). Although several of their clones were found to be A(H1N2), A(H1N1) and A(H3N2), we could not confirm the origin of the A(H1N2) clones without the original specimens. These results suggest that a reassortment to produce A(H1N2) does not readily occur even when A(H1N1) and A(H3N2) co-circulate in a community such as Yamagata.