Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus 71.

Human enterovirus species A (HEV-A) consists of at least 16 members of different serotypes that are known to be the causative agents of hand, foot, and mouth disease (HFMD), herpangina, and other diseases, such as respiratory disease and polio-like flaccid paralysis. Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of HFMD. CVA5, CVA6, CVA10, and CVA12 mainly cause herpangina or are occasionally involved with sporadic cases of HFMD. We have previously shown that human scavenger receptor class B, member 2 (SCARB2) is a cellular receptor for EV71 and CVA16. Using a large number of clinical isolates of HEV-A, we explored whether all clinical isolates of EV71 and other serotypes of HEV-A infected cells via SCARB2. We tested this possibility by infecting L-SCARB2 cells, which are L929 cells expressing human SCARB2, by infecting human RD cells that had been treated with small interfering RNAs for SCARB2 and by directly binding the viruses to a soluble SCARB2 protein. We showed that all 162 clinical isolates of EV71 propagated in L-SCARB2 cells, suggesting that SCARB2 is the critical receptor common to all EV71 strains. In addition, CVA7, CVA14, and CVA16, which are most closely related to each other, also utilized SCARB2 for infection. EV71, CVA14, and CVA16 are highly associated with HFMD, and EV71 and CVA7 are occasionally associated with neurological diseases, suggesting that SCARB2 plays important roles in the development of these diseases. In contrast, another group of viruses, such as CVA2, CVA3, CVA4, CVA5, CVA6, CVA8, CVA10, and CVA12, which are relatively distant from the EV71 group, is associated mainly with herpangina. None of these clinical isolates infected via the SCARB2-dependent pathway. HEV-A viruses can be divided into at least two groups depending on the use of SCARB2, and the receptor usage plays an important role in developing the specific diseases for each group.

Molecular epidemiology of Coxsackievirus A16 strains isolated from children in Yamagata, Japan between 1988 and 2011.

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.

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.

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.

Stability of the seven hexon hypervariable region sequences of adenovirus types 1-6 isolated in Yamagata, Japan between 1988 and 2007.

Seven hexon hypervariable regions (HVRs) of adenoviruses (Ads) were identified by comparing the regions among different serotypes; however, no one has compared HVR sequences among the identical serotypes, except for adenovirus type 3 (Ad3). To examine a variability between the HVRs for each serotype, we compared the sequences of Ad1-6 isolates, respectively, isolated between 1988 and 2007 in Yamagata, Japan. We selected 23-43 isolates randomly and sequenced 894-987 bp regions. Except for strains with insertions and deletions, the sequence identities among Ad1-6 were 99-100%, excluding that between the two Ad5 groups (approx. 94%). Even the insertions and deletions were likely to be established, as these changes were repeatedly observed. The obtained phylogenetic tree indicated that Ad isolates and reference strains branched depending on serotype. The Yamagata isolates had similar sequences or amino acid arrangements to the reference strains as well as to other strains isolated in different areas. HVRs have been stably conserved as serotype-specific regions for a long period with only minor genomic variations. Therefore, we herein recommend that these regions be hereafter referred to as "serotype-specific regions", which might be a more appropriate title with which to characterize the epidemiological nature of these sites than the current "HVRs".

Analysis of monthly isolation of respiratory viruses from children by cell culture using a microplate method: a two-year study from 2004 to 2005 in yamagata, Japan.

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.

Prolonged norovirus shedding in infants

BACKGROUND: Noroviruses (NV) are one of the leading causes of gastroenteritis in young children; however, the duration of NV shedding in young children is not well known. METHODS: Fecal specimens were collected from children with acute gastroenteritis at a pediatric clinic during the period from November to December 2002 and tested for NV by reverse transcription-polymerase chain reaction. RESULTS: Of 71 children infected with NV, 60 (84.5%) were less than 3 years old. Among children aged <2 years and those aged 2 to 5 years, the duration of illness was longer (7 days versus 3.5 days, P = 0.0069), the maximum number of stools in a 24-hour period was greater (7 versus 3, P = 0.0078) and a 20-point severity score was higher (11 versus 8, P = 0.0031) in patients aged <2 years than in patients aged 2 to 5 years. Among the 23 children whose follow-up specimens were obtained, the median duration of NV shedding was 16 days (range, 5-47 days). Virus shedding for more than 2 weeks after onset was observed in 75% (6 of 8), 71.4% (5 of 7) and 25% (2 of 8) of children aged <1 year, 1 year and 2 to 3 years, respectively. Three infants aged

Re-emergence of echovirus type 13 infections in 2002 in Yamagata, Japan.

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.

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.

Endemicity of human metapneumovirus subgenogroups A2 and B2 in Yamagata, Japan, between 2004 and 2009.

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.

Extreme Tall Stature in a Japanese Boy with a 48,XXYY Karyotype.

We report an 18-yr-old Japanese boy with a 48,XXYY karyotype and extreme tall stature (194 cm). A GnRH test at 12.5 yr of age showed hypergonadotropism (LH, 4.2 → 72.2 mIU/mL; FSH, 28.9 → 61.7 mIU/mL), and an hCG test at 15.5 yr of age revealed a normal testosterone response (1.67 → 4.08 ng/mL). The tall stature is remarkable, because the mean adult height of Caucasian 48,XXYY patients is 181 cm. Although the underlying factors for the tall stature are unknown, this report indicates an association of the 48,XXYY karyotype with marked tall stature.

A slow spread of adenovirus type 7 infection after its re-emergence in Yamagata, Japan, in 1995.

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.

Clinical features of influenza C virus infection in children.

BACKGROUND: Seroepidemiological studies have revealed that influenza C virus is widely distributed globally. However, because the isolation of this virus is difficult, there have been few reports on its clinical features. METHODS: Between December 1990 and November 2004, 84,946 respiratory-tract specimens were obtained from patients < or = 15 years old. On the basis of the results of isolation of virus, we examined the clinical data on children infected with influenza C virus. RESULTS: Of 170 children infected with influenza C virus, 157 (92.4%) were < 6 years old. Fever (frequency, 90.0%), cough (frequency, 74.1%), and rhinorrhea (frequency, 61.8%) were the most frequent symptoms. The mean duration of fever was 2.88 days (standard deviation, 1.66 days). Of the 170 children, 29 were hospitalized, and 21 (72.4%) of these 29 had lower-respiratory-tract illness such as pneumonia, bronchitis, and bronchiolitis. The rate of hospital admission was significantly higher in children < 2 years old than in children 2-5 years old (30.4% vs. 11.9%; P = .0043). CONCLUSIONS: Influenza C virus is a significant cause of upper-respiratory-tract illness in children < 6 years old, and the risk of complications with lower-respiratory-tract illness is particularly high in children < 2 years old.

A rare appearance of influenza A(H1N2) as a reassortant in a community such as Yamagata where A(H1N1) and A(H3N2) co-circulate.

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.

Genetic diversity of influenza B virus: the frequent reassortment and cocirculation of the genetically distinct reassortant viruses in a community.

To characterize the genetic diversity of influenza B viruses isolated during one influenza season, the antigenic and genetic relationships among 20 strains of influenza B virus isolated in February and March 2001 at one pediatric clinic in Yamagata City, Japan, were investigated. The HA gene and seven other gene segments were phylogenetically divided into three distinct sublineages (Harbin/7/94-, Tokyo/6/98-, and Shiga/T30/98-related lineage) of the Yamagata/16/88-like lineage. The NS genes of the viruses belonging to the Harbin/7/94-related lineage have additional three nucleotides at positions 439-447, and were phylogenetically distinguishable from those of the currently circulating Yamagata/16/88- and Victoria/2/87-like lineages, but were closely related to that of the Yamagata/16/88-like lineage isolated before 1994. Moreover, four strains of influenza B virus isolated in the same community between 2002 and 2003 were further examined. Phylogenetic analysis revealed that a virus of Victoria/2/87-like lineage isolated in 2003 had acquired the NA, NS, M, and PA gene segments from a Shiga/T30/98-like virus, and two strains of Harbin/7/94-related lineage had acquired the various gene segments from Shiga/T30/98-like virus through a reassortment event. These results indicate that genetically distinct multiple viruses can combine to cause an influenza B epidemic in a community and that the frequent reassortment among these viruses plays a role in generating the genetic diversity of influenza B viruses.