The detection of human sapoviruses with universal and genogroup-specific primers.

Sapovirus (SV) causes gastroenteritis in humans and comprises genetically divergent viruses. A nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the capsid-protein-coding region was developed using universal and genogroup-specific primer sets. The universal primers were capable of detecting human SV genogroups I, II, IV and V. Genetic analysis of the amplified products enabled us to phylogenetically determine the genotypes of the viruses. In addition, genogroup-specific primers that amplified different lengths of the amplicon depending on the genogroup were developed. These genogroup-specific primers were also used as inner primers for the nested PCR. These two simple RT-PCR methods are powerful tools for both detection and epidemiological studies of human SV.

Genetic analysis of noroviruses associated with fatalities in healthcare facilities.

Norovirus outbreaks occurred in 236 healthcare facilities for the elderly in Japan during the winter of 2004-2005. Three norovirus strains associated with three fatal clinical courses were isolated from geographically separate facilities and genetically analyzed along with three strains from non-fatal cases in the same season. All six isolates were classified as the GII-4 genotype. No new variant strains like those observed in Europe in 2002 and 2004 were found in fatal cases, and the three outbreaks were deemed to have been caused by genetically close conventional norovirus GII-4 strains.

Genetic diversity of enterovirus 71 isolated from cases of hand, foot and mouth disease in Yokohama City between 1982 and 2000.

Enterovirus 71 (EV71) is known as one of the major causative agents of hand, foot and mouse disease (HFMD) and is also associated with neurological manifestations such as aseptic meningitis, polio-like paralysis and encephalitis. Recently, large HFMD outbreaks, involving severe neurological complications, have been experienced in Malaysia, Taiwan and some other countries in the Western-Pacific region. To investigate the genetic diversity of EV71 isolates in a single community in Japan, nucleotide sequences of the VP4 region of 52 EV71 isolates in Yokohama City from 1982 to 2000 were determined and the phylogenetic relationship was compared with other referential EV71 strains in Japan and in the world. There were two major genotypes of EV71 in Yokohama City through the 1980's and 1990's. Six EV71 isolates in the early 1980's in Yokohama City were closely related to those from HFMD outbreaks in Japan and from outbreaks of polio-like paralysis in Europe in the 1970's. During recent HFMD outbreaks in 1997 and 2000, two distinct genotypes of EV71 were co-circulating in Yokohama City as in HFMD outbreaks in Malaysia and Taiwan. However, the genetic diversity of EV71 in Yokohama City was not directly correlated with the severity of HFMD. The results confirmed the circulation of two distinct genotypes of EV71 over the past 20 years in Japan.

Group C rotavirus NSP4 induces diarrhea in neonatal mice.

Nonstructural glycoprotein NSP4 of group A rotavirus induces diarrhea in neonatal mice by functioning as an enterotoxin. Previously, our laboratory reported that the structural features of group A and group C rotavirus NSP4 proteins are well conserved despite a lack of sequence homology between group A and group C rotavirus NSP4 proteins [Horie Y, et al., Arch Virol (1997) 142: 1865-1872]. To test whether group C rotavirus NSP4 has an enterotoxigenic activity, we expressed in Escherichia coli the carboxy two-thirds (corresponding to amino acid residues 55-150) of the NSP4 protein derived from group C rotavirus strain Ehime 9301. This truncated NSP4 protein was able to induce diarrhea in 5-day-old CD-1 mice when administered intraperitoneally. Thus, group C rotavirus NSP4 acts as an enterotoxin like group A rotavirus NSP4.

Low-fat diet alters intramuscular substrates and reduces lipolysis and fat oxidation during exercise.

We determined whether a low-fat diet reduces intramuscular triglyceride (IMTG) concentration, whole body lipolyis, total fat oxidation, and calculated nonplasma fatty acid (FA) oxidation during exercise. Seven endurance-trained cyclists were studied over a 3-wk period during which time they exercised 2 h/day at 70% of maximum O2 uptake VO(2 max) and consumed approximately 4,400 kcal/day. During the 1st wk, their fat intake provided 32% of energy. During the 2nd and 3rd wk, they were randomly assigned to eat 2 or 22% of energy from fat (2%FAT or 22%FAT). Compared with 22%FAT, 2%FAT lowered IMTG concentration and raised muscle glycogen concentration at rest (P < 0.05). Metabolism was studied during 1 h of exercise at 67% VO(2 max) performed in the fasted state. 2%FAT resulted in a 27% reduction (P < 0.05) in total fat oxidation vs. 22%FAT without altering the stable isotopically determined rates of plasma free fatty acid or glucose disappearance. Therefore, 2%FAT reduced calculated nonplasma FA oxidation by 40% in association with a 19% reduction in whole body lipolysis while increasing calculated minimal muscle glycogen oxidation compared with 22%FAT (all P < 0.05). In summary, an extremely low fat (2% of energy) and high-carbohydrate diet lowers whole body lipolysis, total fat oxidation, and nonplasma FA oxidation during exercise in the fasted state in association with a reduced concentration of intramuscular triglyceride.

Development of simple latex agglutination test for detection of astrovirus serotype 1.

Astroviruses are small RNA viruses associated with pediatric gastroenteritis. A latex agglutination (LA) test is more convenient and rapid than electron microscopy, enzyme immunoassay (EIA) and reverse transcription-polymerase chain reaction (RT-PCR) for detection of astroviruses in stool specimens was developed by using polyclonal antibody against cultured serotype 1 astrovirus, and tested on astrovirus positive- and negative- samples collected between 1985 and 1993 in Ehime, Japan. Cultured serotype 1 astrovirus was detected by the LA test, but serotype 2, 3, 4, 5, 6, and 7 viruses could not be detected, although they yielded high titer of the viruses. When tested on the 27 clinical samples, all 8 serotype 1 astroviruses determined previously by EIA were positive by the LA test, however, 9 other serotype astroviruses and 10 astrovirus negative-samples were all negative. PT-PCR was found to be the most sensitive followed by EIA and LA test. Further development of LA test with other serotypes is necessary.

Phylogenic analysis of echovirus type 30 isolated from a large epidemic of aseptic meningitis in Japan during 1997-1998.

During 1997 to 1998, a nationwide epidemic of aseptic meningitis occurred in Japan. More than 4,500 isolates from patients with aseptic meningitis were identified as echovirus type 30. To investigate the character of these isolates, we examined the nucleotide sequences of thirty-seven geographical representatives and compared them with 50 strains isolated during the past 20 years. The phylogenic analysis used partial sequences from either the VP1 or VP4-VP2 region of the viral capsid. This analysis revealed that the isolates were divided into six genomic groups. All isolates identified during 1997-1998 belonged to only two genomic groups; these two groups are thought to be the causative viral agents involved in the recent epidemic.

Genetic variation in the capsid region of human astrovirus serotype 4 isolated in Japan.

The entire capsid regions of 12 serotype-4 astroviruses from Japan were sequenced and compared with those of other serotypes. Serotype-4 isolates were divided into two new subgroups. The intrasubgroup nucleic acid and deduced amino acid sequences were quite homologous (more than 93%), but slightly less so between subgroups (almost 85%). However, the serotype-4 sequences differed from those of serotypes 1, 2, 3, 5, 6, 7 and 8 (less than 50%). Determining whether these differences significantly alter the epidemiology and antigenicity will require further investigation.

Determination of serotypes of astroviruses by reverse transcription-polymerase chain reaction and homologies of the types by the sequencing of Japanese isolates.

Human standard astroviruses, serotypes 1 to 7, and 35 Japanese isolates were typed by reverse transcription and polymerase chain reaction (RT-PCR) with serotype-specific primers for the first time. The results were identical with those obtained by enzyme immunoassay with serotype-specific polyclonal antibodies, a method which has already been reported. RT-PCR with serotype-specific primers is useful for epidemiological studies of astroviruses where serotype-specific polyclonal antibodies are not available. Two parts of the capsid region, N terminus and C terminus, were sequenced. Serotypes differed in those regions. The N terminus differed less than the C terminus between serotypes. Both the N terminus and C terminus were similar intraserotypically with the exception of serotype-4 isolates which could be divided into A and B subgroups on the basis of their C terminus sequences, which were not known previously.

Epidemiological study of prevalence of genogroup II human calicivirus (Mexico virus) infections in Japan and Southeast Asia as determined by enzyme-linked immunosorbent assays.

Mexico virus (MXV) is a genogroup II human calicivirus (HuCV). We conducted an epidemiological study to determine the prevalence of MXV infection in infants and adults in Japan and Southeast Asia by enzyme-linked immunosorbent assays (ELISAs) developed by using baculovirus-expressed recombinant MXV (rMXV) capsids. Of 155 stool specimens obtained from children younger than 10 years old with acute clinical gastroenteritis (diarrhea and vomiting) associated with small, round-structured viruses in Japan from 1987 to 1989, only 2 were positive for MXV antigen. In 42 outbreaks of acute gastroenteritis in Japan from 1986 to 1994, 1 in an infant home and 1 among adults were positive for MXV antigen. The pattern of acquisition of antibody to rMXV was different from that of acquisition of antibody to group A rotavirus, the prototype HuCV Sapporo virus, and Norwalk virus. The prevalence of antibody to rMXV remained low for the first 3 years of life, showed a steep rise during nursery school age, reaching a prevalence of 50%, and another steep rise during adolescence, reaching 80%; and steadily increased thereafter. A high prevalence of antibody (82 to 88%) was observed in adult populations in Japan and Southeast Asia, suggesting that MXV infection is common in these areas. The discrepancy between the high prevalence of antibody to MXV and a low rate of detection of MXV antigen may be explained by a high specificity of the antigen ELISA for the prototype and closely related MXV strains while serological responses can detect responses to a broader group of viruses.

Genetic analysis of the capsid region of human astrovirus serotype 3 isolated in Japan.

The total capsid region of astrovirus serotype 3 was analyzed using five isolates including four from Japan and one from the United Kingdom. The nucleic acid and deduced amino acid sequences in the region were homologous (over 97%). However, the sequences of serotype 3 were different from those of serotypes 1, 2, 4, 5, 6 and 8, especially in the C terminus.

Conserved structural features of nonstructural glycoprotein NSP4 between group A and group C rotaviruses.

The nonstructural glycoprotein NSP4 of group C human rotavirus strain Ehime 9301 was determined to be 150 amino acids in length and 96% identical with the NSP4 of another group C human rotavirus strain Bristol. Both NSP4 sequences were virtually unrelated to group A rotavirus NSP4s. However, the structural features of group A and group C rotavirus NSP4s were similar with hydrophobic domains being in the amino terminus and a coiled coil domain after the membrane-spanning domain, although group C rotavirus NSP4 lacked one amino-terminal hydrophobic domain.

Detection of bacterial antigens using immuno-PCR.

A new and very sensitive antigen detection technique, immuno-polymerase chain reaction (immuno-PCR), was developed. This method is basically similar to the enzyme-linked immunosorbent assay which detects an antigen-antibody reaction, but instead of an enzyme being conjugated to an antibody, a DNA fragment is used and this DNA can be amplified by PCR. We applied this method to the detection of the fish pathogen, Pasteurella piscicida, in naturally infected yellowtail. Using immuno-PCR, 3.4 cfu ml-1 of bacteria could be detected. In comparison, ELISA detected only 3.4 x 10(4) cfu ml-1. Immuno-PCR is a powerful method for detection of pathogens in host tissues.

Isolation of group B porcine rotavirus in cell culture.

While group A and C rotaviruses have been grown in cell culture, group B rotavirus has never been cultured. In this study we successfully isolated porcine group B rotavirus in swine kidney cells. Pancreatin treatment is essential for the propagation of group B rotavirus.

Reverse transcription-polymerase chain reaction detection and sequence analysis of small round-structured viruses in Japan.

Between 1985 and 1995, mass outbreaks of acute gastroenteritis caused by small round-structured virus (SRSV), occurred in eight prefectures in Japan. Fecal samples from 59 patients ill during these outbreaks were recently examined in our laboratory by electron microscopy (EM) and by reverse transcription-polymerase chain reaction (RT-PCR). For RT-PCR, we prepared two sets of primers, a set corresponding to the polymerase region of open reading frame 1 (ORF-1) and a set corresponding to the capsid region of ORF-2 of Norwalk virus (NV). The SRSV nucleic acid detection rate with these primers was more than double that achieved with EM. Most samples found by EM to contain virus particles were also positive by PCR. When the two sets of primers were used separately, the virus detection rate differed depending on the primer used, suggesting that the viral strains examined were not genetically not homogeneous. We then selected nine strains of the virus, cloned their PCR products and analyzed their base sequences. The base sequences of these strains were compared with those of reference strains including prototype NV and Snow Mountain agent (SMA). This comparison yielded the following findings: (1) SRSVs that cause mass outbreaks of gastroenteritis in Japan are genetically variable; (2) SRSV strains that are genetically similar to SMA and SRSV-OTH 25/89/J(OTH25) are dominant in Japan, but strains similar to NV are also present in this country; and (3) a strain (MI1/94) which is genetically identical to Southampton virus (SHV) was detected. Detection of SRSV using sensitive RT-PCR and analysis of the sequences of the amplification products seems to provide a useful means of studying the molecular epidemiology of SRSV.

Sequence conservation and expression of the gene encoding the outer capsid glycoprotein among human group C rotaviruses of global distribution.

Group C rotaviruses have been identified recently from fecal samples of children with diarrhea in the United States. Using reverse transcriptasepolymerase chain reaction and sequence analysis, we sequenced gene 8s encoding VP7 from two U.S. strains (RI-1 and RI-2), and eight other strains isolated from patients on four continents, and compared these with the sequences of four published strains. The gene 8s of the 14 strains were remarkably conserved in size and in predicted primary and secondary structures. When the sequences of the human VP7s were compared with that of the prototype porcine Cowden strain, six regions were found variable in both deduced primary and predicted secondary structures, four of which were predicted to be hydrophilic and might determine serotype specificity. Gene 8 of the human S-1 strain was further characterized by expression in recombinant baculoviruses. The expressed product was immunogenic but failed to elicit neutralizing antibodies. Our sequence analysis indicates that all the human strains characterized to date belong to a single G genotype, which may constitute a single G serotype, pending further antigenic analysis. Whether the human strains and the Cowden strain are the same serotype remains to be determined.

[Epidemiological studies on enteric adenovirus gastroenteritis in children].

Enteric adenoviruses (EAd), adenovirus (Ad) types 40 and 41, have been established as causative agents of gastroenteritis. By electron microscopic (EM) survey of acute gastroenteritis in children in the Matsuyama area, Ad were detected in 275 of 6476 fecal samples obtained from 1980 to 1993. Two-hundred-thirteen Ad-positive samples were tested for serotyping by the enzyme-linked immunosorbent assay (ELISA) using three monoclonal antibodies, Ad group-specific, Ad 40 type-specific and Ad41 type-specific antibody. Of 199 samples serotyped by ELISA, 65 were identified as Ad40, 73 as Ad41, 1 as double infection with Ad40 and Ad41, and 60 as Non-EAd. About 70% of Adenovirus detected by EM were suggested to be EAd. Other epidemiological feature was as follows: EAd were detected throughout the year. The predominant serotype was Ad40 during 1980-1985, while Ad41 were observed after 1986. EAd were detected most frequently from the children aged 0-3 years. The incidence of fever in EAd positive group was lower (30%) than that (67%) in the Non-EAd positive group. The incidence of vomiting, nausea and respiratory symptoms was higher in Ad41 associated infections than Ad40.