Chieko Abiko

Frequent Importation of Enterovirus 71 from Surrounding Countries into the Local Community of Yamagata, Japan, between 1998 and 2003

ABSTRACT Phylogenetic analysis of 45 enterovirus 71 (EV71) isolates for 6 years in Yamagata, Japan, clarified that the annual outbreak of hand-foot-and-mouth disease was due to four genetically distinct subgenogroups, including a novel “B5.” Our results suggest that the importation of EV71 from surrounding countries has had a major epidemiological impact on the local community used in our study.

Interspecies transmission of influenza C virus between humans and pigs

The antigenic and genetic characteristics of the 18 human strains of influenza C virus isolated in Yamagata and Sendai Cities, Japan between January 1991 and February 1993 were investigated. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase glycoprotein showed that the isolates could be divided into three distinct groups closely related to C/Yamagata/26/81, C/Aichi/1/81 and C/Mississippi/80, respectively. T1-oligonucleotide fingerprinting of total vRNA revealed that the six isolates belonging to the C/Yamagata/26/81 virus group had the genomes greatly similar to one another but considerably different from those of the 1988/1990 isolates (except C/Yamagata/10/89) of the same antigenic group. Comparison of total or partial nucleotide sequences of the seven RNA segments of the three strains (C/Miyagi/3/91, C/Miyagi/9/91 and C/Miyagi/2/92) representative of the 1991/1993 strains of the C/Yamagata/26/81 virus group with those of the previous influenza C isolates obtained from humans and pigs during 1980/1989 showed that the 1991/1993 strains, like C/Yamagata/10/89, are more closely related to viruses isolated from pigs in Beijing, China in 1981/1982 than to any of the isolates from humans. This observation suggests strongly that interspecies transmission of influenza C virus between humans and pigs has occurred in nature, although it is not known whether the virus has been transmitted from pigs to humans or from humans to pigs.

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.

A Nationwide Epidemic of Influenza C Virus Infection in Japan in 2004

ABSTRACT 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.

A two-year survey of the oseltamivir-resistant influenza A(H1N1) virus in Yamagata, Japan and the clinical effectiveness of oseltamivir and zanamivir

BackgroundOseltamivir 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.ResultsOseltamivir 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).ConclusionOseltamivir-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.

Epidemic myalgia in adults associated with human parechovirus type 3 infection, Yamagata, Japan, 2008.

This virus typically causes illness in young children but was found to be associated with illness in adults.

Clinical impact of human metapneumovirus genotypes and genotype-specific seroprevalence in Yamagata, Japan.

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. J. Med. Virol. 80:1084–1089, 2008.

Sequence and phylogenetic analyses of Saffold cardiovirus from children with exudative tonsillitis in Yamagata, Japan.

(Received 15 February 2010 ; accepted 23 May 2010 ) To the Editor, Saffold cardiovirus (SAFV) of the genus Cardiovirus and family Picornaviridae was recently recovered from faecal specimens of an infant with fever of unknown origin [1]. SAFV has also been detected in children with diseases such as gastroenteritis, respiratory tract infection, and non-polio acute fl accid paralysis [2 – 5]. However, the epidemiology and pathogenicity of SAFV is not exactly known. In this study, we detected SAFV in children with exudative tonsillitis and conducted sequence and phylogenetic analyses. We obtained nasopharyngeal swabs from 37 patients with typically exudative tonsillitis between August and December 2009. Informed consent was obtained from the parents of all subjects for the donation of the nasopharyngeal samples used in this analysis. Initially, we sought to isolate or detect pathogens from these samples using cell culture methods, quick immunochromatography (as used to detect Streptococcus), and polymerase chain reaction (PCR; as used to detect Epstein – Barr virus [6]). To isolate various viruses, we used 7 different cell lines (Vero E6, HEp-2, HEL, MDCK, GMK, HMV-II, and RD18S cells) [7,8]. These cells may be sensitive to the various agents of exudative tonsillitis – parainfl uenza viruses, infl uenza viruses, herpes simplex viruses, adenovirus, and respiratory syncytial virus [7,8]. However, these pathogens were not isolated or detected from the samples provided. Next, we attempted to detect SAFV using a nested reverse transcriptase PCR (RT-PCR). We extracted RNA from the samples and amplifi ed the VP1 coding region of SAFV by nested RT-PCR. Primer sets were newly designed by Primer Express ® version 1.5 software (Applied Biosystems LLC, Foster City, CA, USA) [9]. Primer sequences were as follows: 5 ′ -HAA RCA RGR YTG GAR YTT YNT NAT GTT-3 ′ (primer 315F) and 5 ′ DGG BCK DGG RCA RWA VAC YCT CAT-3′ (primer 738R) as outer primers, and 5 ′ -AAR CAR GRY TGG ARY TTY DTH ATG TTY TC-3′ (primer 316F) and 5 ′ -RTT RKK RAA RTY NGM RDA NCY RTT RAA CCA-3 ′ (primer 621R) as inner primers. Reverse transcription was performed for 10 min at 30 ° C, 45 min at 37 ° C, and 5 min at 95 ° C using random hexamers (TAKARA BIO Inc., Otsu, Japan). First and nested PCR conditions were as follows: 5 min at 94 ° C, followed by 40 cycles at 94 ° C for 30 s, 50 ° C for 30 s, and 72 ° C for 1 min, ending with elongation for an additional 10 min at 72 ° C. To prevent carryover contamination of nested-PCR, we took general precautions as previously described [10,11]. As a result, we obtained amplicons from 9 children who showed typical exudative tonsillitis symptoms, including fever ( 38 ° C). They lived in Yamagata Prefecture, Japan and were aged between 2 and 7 y (mean standard deviation, 3.8 1.6 y). Fever lasted for 1 to 3 days (1.8 0.7 days). Amplicons were sequenced and aligned (453 bp) [3,5,12]. Next, we performed phylogenetic LETTER TO THE EDITOR

Outbreak of Human Metapneumovirus Detected by Use of the Vero E6 Cell Line in Isolates Collected in Yamagata, Japan, in 2004 and 2005

ABSTRACT A number of epidemiological studies have shown human metapneumovirus (hMPV) to be one of the most important viral agents associated with acute respiratory infections in humans. However, due to the difficulty in growing the virus, all epidemiological studies of hMPV infection have been performed on the basis of the molecular method. Thus, the development of a cell line suitable for the isolation of hMPV from clinical specimens is a crucial step for further research. Using the Vero E6 cell line, which could be stably maintained for 1 month without passage or medium change, we succeeded in isolating 79 strains from 4,112 specimens obtained in Yamagata, Japan, in 2004 and 2005. The total isolation rate was 1.9% (79/4,112). The monthly distribution revealed that hMPV infections occurred between February and April in 2004 and throughout most of the year in 2005. Phylogenetic analysis indicated that subgenogroup B2 was predominant in 2004, whereas three subgenogroups, A2, B1, and B2, had cocirculated in 2005. Although multiple subgenogroups cocirculated in 2005, each individual subgenogroup strain was found to predominate at specific sites. An infectivity assay of hMPV strains also indicated that the infection efficiency in Vero E6 cells was better than that in LLC-MK2 cells. Finally, we found that Vero E6 cells are useful for the isolation of hMPVs and that this utility might aid further research into hMPVs beyond the epidemiological data shown in this study.

Detailed genetic analysis of hemagglutinin-neuraminidase glycoprotein gene in human parainfluenza virus type 1 isolates from patients with acute respiratory infection between 2002 and 2009 in Yamagata prefecture, Japan.

BackgroundHuman 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.ResultsA 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.ConclusionsThe 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.