Masatoshi Tatsumi

Clinical severity of Norwalk virus and Sapporo virus gastroenteritis in children in Hokkaido, Japan

Objective. To clarify the clinical significance and etiologic impact of Norwalk virus (NV) and Sapporo virus (SV) in viral gastroenteritis in Japanese children. Study design. Two outbreaks each of NV gastroenteritis and SV gastroenteritis occurring in an infant home in Sapporo, Japan, as well as 95 hospitalized children with acute gastroenteritis were retrospectively evaluated using a 0- to 20-point clinical severity scoring system. Result. The mean severity scores for NV and SV gastroenteritis outbreaks were 7.9 and 5.2, respectively, as compared with 8.4 for rotavirus A gastroenteritis that occurred in the same infant home. Among 95 hospitalized children with acute gastroenteritis, rotavirus A was detected in 47% followed by NV in 18%. SV was not found. Conclusion. Our data indicate that NV can cause severe gastroenteritis and is an important etiologic agent in hospitalized cases, whereas SV causes mild gastroenteritis in Japanese children.

Detection of enteric viruses in rectal swabs from children with acute gastroenteritis attending the pediatric outpatient clinics in Sapporo, Japan

BACKGROUND Gastroenteritis is a world-wide disorder. Numerous studies to identify causative viral agents have been reported for hospitalized patients but there are only a few for outpatients with mild symptoms who are usually managed in the outpatient clinics. OBJECTIVES Our aim was to clarify the epidemiological and clinical characteristics of acute gastroenteritis in children who visited the outpatient clinics with various complaints suggestive of gastroenteritis. STUDY DESIGN From December 2003 to December 2005, 877 rectal swabs were collected from patients attending outpatient clinics in Sapporo, Japan. Viral genomes of major five enteric viruses (rotavirus, norovirus, adenovirus, astrovirus and sapovirus) and bocavirus were investigated by RT-PCR or PCR. RESULTS At least one viral agent was found in 326 (37.2%) cases of the 877 studied. Rotaviruses were the most prevalent and were detected in 143 (16.3%) followed by norovirus in 116 (13.2%), adenovirus in 42 (4.8%), astrovirus in 40 (4.6%) and sapovirus in 15 (1.7%) cases. Bocavirus was detected in only 4 (0.5%) cases. Frequent diarrhea and frequent vomiting were prominent in rotavirus and norovirus infection, respectively. CONCLUSIONS The prevalence of each enteric virus in outpatients resembled that previously estimated in hospitalized patients, although the detection rate of rotavirus was slightly low. The contribution of bocavirus appears to be small.

Virological, Serological, and Clinical Features of an Outbreak of Acute Gastroenteritis Due to Recombinant Genogroup II Norovirus in an Infant Home

ABSTRACT Norovirus (NV) is an important cause of acute nonbacterial gastroenteritis worldwide. Recently, several sporadic cases due to naturally occurring recombinant NVs have been reported. In January 2000, there was an outbreak of gastroenteritis in an infant home in Sapporo, Japan. Of 34 residents of the home that were less than 2 years old, 23 developed gastrointestinal symptoms and NV infection was confirmed by conventional reverse transcription-PCR to detect the RNA polymerase region of genogroup II NV. In this virus, the RNA polymerase region shared 86% nucleotide identity with Hawaii virus but only 77% with Mexico virus; however, its capsid region shared only 70% identity with Hawaii virus but 90% with Mexico virus. On the other hand, both regions shared a higher 96% nucleotide identity with Arg320 virus, which was found in Mendoza, Argentina, in 1995 and considered to be a recombinant of Hawaii and Mexico viruses. The findings indicate that the virus involved in the outbreak was similar and may have evolved from the Arg320 virus. Clinically the cases were more severe than those of previously reported sporadic or outbreak cases of NV infection.

Development and Validation of DNA Microarray for Genotyping Group A Rotavirus VP4 (P[4], P[6], P[8], P[9], and P[14]) and VP7 (G1 to G6, G8 to G10, and G12) Genes

ABSTRACT Previously, we reported the development of a microarray-based method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) (V. Chizhikov et al., J. Clin. Microbiol. 40:2398-2407, 2002). The expanded version of the rotavirus microarray assay presented herein is capable of identifying (i) five clinically relevant human rotavirus VP4 genotypes (P[4], P[6], P[8], P[9], and P[14]) and (ii) five additional human rotavirus VP7 genotypes (G5, G6, G8, G10, and G12) on one chip. Initially, a total of 80 cell culture-adapted human and animal reference rotavirus strains of known P (P[1] to P[12], P[14], P[16], and P[20]) and G (G1-6, G8 to G12, and G14) genotypes isolated in various parts of the world were employed to evaluate the new microarray assay. All rotavirus strains bearing P[4], P[6], P[8], P[9], or P[14] and/or G1 to G6, G8 to G10, or G12 specificity were identified correctly. In addition, cross-reactivity to viruses of genotype G11, G13, or G14 or P[1] to P[3], P[5], P[7], P[10] to P[12], P[16], or P[20] was not observed. Next, we analyzed a total of 128 rotavirus-positive human stool samples collected in three countries (Brazil, Ghana, and the United States) by this assay and validated its usefulness. The results of this study showed that the assay was sensitive and specific and capable of unambiguously discriminating mixed rotavirus infections from nonspecific cross-reactivity; the inability to discriminate mixed infections from nonspecific cross-reactivity is one of the inherent shortcomings of traditional multiplex reverse transcription-PCR genotyping. Moreover, because the hybridization patterns exhibited by rotavirus strains of different genotypes can vary, this method may be ideal for analyzing the genetic polymorphisms of the VP7 or VP4 genes of rotaviruses.

The VP7 Genes of Two G9 Rotaviruses Isolated in 1980 from Diarrheal Stool Samples Collected in Washington, DC, Are Unique Molecularly and Serotypically

ABSTRACT In a retrospective study of archival diarrheal stool samples collected from 1974 to 1991 at Childrens Hospital National Medical Center, Washington, DC, we detected three genotype G9P[8] viruses in specimens collected in 1980, which represented the earliest human G9 viruses ever isolated. The VP7 genes of two culture-adapted 1980 G9 viruses were phylogenetically related closely to the lineage 2 G9 virus VP7 gene. Unexpectedly, however, the VP7s of the 1980 G9 viruses were more closely related serotypically to lineage 3 VP7s than to lineage 2 VP7, which may be supported by amino acid sequence analyses of the VP7 proteins.

Phylogenetic and computational structural analysis of VP7 gene of group a human rotavirus G1P[8] strains obtained in Sapporo, Japan from 1987 to 2000

Many studies indicate that G1P[8] genotypes are the most prevalent rotavirus strains worldwide. Although two vaccines have been licensed and their value proven in many countries, continuous surveillance for genetic evolution of circulating rotavirus strains before and after the introduction of the vaccines is desirable. G and P typing were carried out on all field strains isolated during 1987–2000 in Sapporo, Japan. Phylogenetic analysis for the VP7 gene of rotavirus G1P[8] strains was performed. Amino acid substitutions were mapped on the predicted three‐dimensional VP7 protein image. G1P[8] genotype predominated. One hundred thirteen strains with G1P[8] genotype were analyzed. Phylogenetic studies of the VP7 gene classified these strains into three lineages. The mean estimated substitution rate was 7.25 × 10−4 nucleotide substitutions per site per year. One predominant lineage contained the mutant strains which had VP7 amino acid substitutions at residue 91 and 212 that is in the neutralization domains. They were estimated to locate in or near intersubunit boundary of VP7 trimer. It is suggested that the most prevalent G1P[8] lineage strains in Sapporo obtained some survival advantages by changing the neutralization domains of VP7. J. Med. Virol. 84:832–838, 2012.

Characterization of the NSP4 gene of group A human rotavirus G1P[8] strains circulating in Sapporo, Japan from 1987 to 2000.

The genetic diversity of the NSP4 gene of rotavirus G1P[8] strains obtained in Sapporo was analyzed, Japan from 1987 to 2000. Sixty‐four strains, which were distributed across the whole study period, were included. All G1P[8] NSP4 genes detected in this study belonged to genotype E1, which divided into at least three lineages. The Sapporo rotavirus G1P[8] isolates were found in each lineage. The mean estimated substitution rate was 1.40 × 10−3 nucleotide substitutions per site per year, which was comparable to that of the G1P[8] VP7 gene. Comparison of the deduced NSP4 amino acid sequences showed genetic diversity at the center of antigenic site II, but not in the enterotoxic domain. This report represents the first investigation of the genetic diversity and evolution of group A rotavirus NSP4 genes in Asia. J. Med. Virol. 86:354–359, 2014.

Full sequence analysis of the original Sapporo virus

In this study, the full‐length genome sequence of the prototype of sapovirus, namely Sapporo virus (SV82), was identified. Sapporo virus RNA was extracted from a fecal sample, amplified by RT‐PCR and the PCR products sequenced directly and analyzed. Sequence analysis showed that Sapporo virus consists of 7433 nucleotides and has three open reading frames. The Sapporo strain shows 91.7% nucleotide sequence identity to the Manchester virus. Phylogenic analysis has also revealed the closeness of Sapporo virus to other sapovirus/genogroup I strains. Basic information on the evolutionary history of sapovirus analysis is provided here.

Neonatal apnea with Chlamydia pneumoniae infection

A male neonate weighing 2650 g was born at 37 weeks 6 days of gestation. There was no history of maternal infection during pregnancy or medical problems in the family. On day 24 of life, he developed cough and sputum; these symptoms continued for 3 days. On day 26, he developed persistent apnea and was admitted to hospital. On admission, vital signs were normal and he showed no signs of respiratory distress. Respiratory rate was 38 breaths/ min and oxygen saturation on pulse oximeter was 97%. Physical examination indicated slight rhonchi bilaterally on his back, but 2 h after admission the patient developed severe apnea, which then recurred two to three times per day, during which the oxygen saturation dropped to 60–70% for each episode. Laboratory data including biochemistry and hematology were normal, except for increase in eosinophil count of 10% with 8740 white blood cells. On computed tomography broad faint interstitial inflammation was seen in the lower lung field. Atypical pneumonia or viral pneumonia was suspected. We investigated Chlamydia pneumoniae, C. trachomatis and respiratory syncytial virus (RSV). Repeated immunochromatography test for RSV antigen was negative. Serum obtained at admission and 1 month after admission was negative for C. pneumoniae enzyme immunoassay (EIA; IgG, IgA, IgM) and C. trachomatis EIA (IgG, IgA) antibodies. Throat swab culture collected at admission, however, showed C. pneumoniae inclusion body (Fig. 1). In addition, polymerase chain reaction (PCR) for C. pneumoniae was positive in the same throat swab. Consequently, the patient was diagnosed with pneumonia due to C. pneumoniae infection. The patient was treated with clarithromycin (15 mg/kg/ day) for 28 days. On day 4 after the start of treatment, the apnea was resolved. The patient was discharged on day 14 after admission. In this case, we could not specify the route of infection but, considering his environment, it is doubtful that the infection came from the mother or father. Discussion

Longitudinal analysis of VP7 gene of group A human rotavirus G2P[4] strains circulating in the pre‐vaccine era in Sapporo, Japan from 1991 to 2011

Sequence analysis of the VP7 gene in 23 group A human rotavirus G2P[4] strains obtained during 1991–2011, that is, the pre‐vaccine era, in Sapporo, Japan showed considerable genetic diversity, mainly in variable regions. Recent G2P[4] epidemic strains were located in sublineage IVa with a distinctive substitution of D96N. This study provides background data on the genetic variability of G2P[4] rotavirus‐VP7 gene prior to the widespread use of rotavirus vaccines in Japan.