Shuji Nakata

PROTECTIVE EFFECT OF NATURALLY ACQUIRED HOMOTYPIC AND HETEROTYPIC ROTAVIRUS ANTIBODIES

To assess serotype specificity of immune resistance to rotavirus gastroenteritis, the relation between pre-existing neutralising antibodies to homotypic and heterotypic rotaviruses and protection against infection or clinical illness was investigated. The subjects were 44 orphans exposed once or twice to consecutive outbreaks of gastroenteritis due to type 3 rotavirus in an orphanage in Sapporo. Sera were collected throughout these outbreaks and the serum levels of neutralising antibodies against four different serotypes of group A human rotavirus were measured before and after the outbreaks. Protection against rotavirus gastroenteritis seemed to be serotype specific and to be related to levels of antibody against homotypic virus. A neutralising antibody level of 1/128 or greater seemed to be protective. The protective effect was of short duration, which was probably the explanation for recurrent attacks of gastroenteritis due to a rotavirus of the same serotype. Seroconversions or concomitant antibody responses to type 1 or 4 rotavirus in most children with type 3 rotavirus infection suggested that immunity to heterotypic virus can be induced by a rotavirus vaccine.

Human Caliciviruses in Acute Gastroenteritis of Young Children in the Community

Episodes of acute gastroenteritis in prospectively followed children between 2 months and 2 years of age were examined for rotaviruses, enteric adenoviruses, astroviruses, and human caliciviruses, including both Norwalk-like viruses (NLVs) and Sapporo-like viruses (SLVs), using PCR and reverse transcription (RT)-PCR assays. A virus was identified in 60% (502/832) of all episodes and in 85% of the moderately severe or severe episodes. Human caliciviruses were as common as rotaviruses, both being detected in 29% of the cases. NLVs accounted for a 20% etiologic share of all cases; the clinical picture was a moderately severe disease with vomiting as a predominant symptom. SLVs were detected in 9% of the cases, the clinical picture being a mild diarrheal disease. Astroviruses were found in 10% and enteric adenoviruses in 6% of the cases. Diagnosis with PCR and RT-PCR methods increases the detection of all gastroenteritis viruses, particularly human caliciviruses. As a group, human caliciviruses are common causative agents of gastroenteritis in children <2 years of age in Finland, and, of these, NLVs cause more severe disease than SLVs.

Cross-Reactivity among Several Recombinant Calicivirus Virus-Like Particles (VLPs) with Monoclonal Antibodies Obtained from Mice Immunized Orally with One Type of VLP

ABSTRACT Human caliciviruses (HuCVs) are classified into the Norwalk-like viruses (NLV) and Sapporo-like viruses (SLV) as genera within the family Caliciviridae. The NLV genus is further classified into genogroups I and II, based on sequence similarities. To study the antigenic determinants on the HuCV capsid protein and develop new diagnostic tools for field samples, we established and characterized monoclonal antibodies (MAbs) against baculovirus-expressed recombinant HuCV virus-like particles (VLPs). Hybrid clones producing MAbs were obtained from cultures of PAI myeloma cells fused with spleen or mesenteric lymph node cells from mice immunized orally with either a single type of recombinant Norwalk virus (rNV), Kashiwa 47 virus (rKAV), Snow Mountain agent (rSMA), or Sapporo virus (rSV) VLP or with mixtures of two types of VLPs from different genogroups. Twenty MAbs, obtained as mouse ascites, were characterized and classified into six groups according to their enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) cross-reactivity patterns to VLPs. Five groups of MAbs reacted by both WB and ELISA and were classified as follows: common cross-reactive MAbs for four genogroup I and six genogroup II VLPs (group A), genogroup I-specific MAbs (group B), genogroup II-specific MAbs (group C), and strain-specific MAbs (groups D and E). One MAb group (group F) reacted only by ELISA. The group A MAbs, which showed broad cross-reactivity with VLPs of both NLV genogroups, were obtained from mice immunized orally with a single type of VLP (either rNV or rKAV). Two MAbs, which were obtained from mice immunized with rSV, reacted with rSV but not with any NLV VLP. These are the first MAbs to be reported for any SLV. These strain-, genogroup-, and genus-reactive MAbs will be useful tools for further study of the antigenic and structural topography of the HuCV virion and for diagnostic assays for HuCVs.

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.

Members of the Family Caliciviridae (Norwalk Virus and Sapporo Virus) Are the Most Prevalent Cause of Gastroenteritis Outbreaks among Infants in Japan

Norwalk virus (NV) and Sapporo virus (SV) were approved as type species of the genus Norwalk-like viruses and the genus Sapporo-like viruses, respectively, within the family Caliciviridae. To clarify the importance of NV and SV as causes of gastroenteritis outbreaks in infants, stool samples obtained from 36 outbreaks of nonbacterial gastroenteritis that occurred during 1976-1995 in an infant home in Sapporo, Japan, were examined for diarrhea viruses using electron microscopy, enzyme immunoassays, reverse transcriptase-polymerase chain reaction (PCR), and sequencing of the PCR products. NV and SV were associated with 15 (42%) of the 36 outbreaks and were more prevalent than rotavirus (RV) A, which was associated with 10 (28%) of the 36 outbreaks. Our data indicate that NV and SV were the most common cause of outbreaks of viral gastroenteritis in infants and were indeed more prevalent than RV-A in Sapporo, Japan, during 1976-1995.

Molecular characterization of morphologically typical human calicivirus Sapporo

SummaryHuman calicivirus Sapporo (SV) has typical calicivirus morphology and causes acute gastroenteritis in children. The nucleotide sequence of 3.2 kb of the 3′ end of SV was determined from a cloned cDNA. The 3′ end of the SV genome is predicted to encode the RNA-dependent RNA polymerase region, the capsid protein and two small open reading frames. The nonstructural and capsid protein coding sequences in the SV genome are fused in a single open reading frame. The organization of these proteins in the SV sequence is similar to that of rabbit hemorrhagic disease virus and the recently described Manchester virus, and distinct from the genome organization of the prototype human calicivirus, Norwalk virus, that lacks typical calicivirus morphology and has been described as a small round structured virus (SRSV). Sequence analysis of the predicted capsid region showed that the SV capsid is longer by ∼30 amino acids than the capsid of any of the SRSVs, and multiple sequence alignments showed that these additional amino acids are located in the variable region of the capsid protein. Expression of the capsid protein of SV in insect cells resulted in the self-assembly of virus-like particles that have a morphology similar to that of the native virus. This result shows that calicivirus morphology is determined by the primary sequence of the capsid protein.

OUTBREAK OF INFANTILE GASTROENTERITIS DUE TO TYPE 40 ADENOVIRUS

Genetic and antigenic characterisation was performed on a strain of adenovirus (EAd) isolated from an outbreak of gastroenteritis which occurred in an orphanage in the City of Sapporo, in the room housing the eldest children, who ranged in age from 14 to 22 months. 7 of the 11 children housed in that room had diarrhoea between July 11 and July 22, 1982. All 7 shed adenoviruses detectable by electron microscopy in their stools. Immune electron microscopy showed that all patients as well as the healthy contacts sharing the room underwent seroconversion to EAd. There was no homology, or very slight homology, between DNA of EAd and those of adenoviruses belonging to subgroups A to E. Antigenically EAd was closely related to type 40 adenovirus, so far the sole member of the newly identified subgroup F. This outbreak of gastroenteritis is the first in which the causative agent has been identified as being a member of subgroup F adenoviruses.

Sapporo-like human caliciviruses are genetically and antigenically diverse.

SummaryThe Sapporo-like human caliciviruses (HuCVs) comprise one of three genogroups of HuCVs associated with acute gastroenteritis. Phylogenetic analysis has shown that Sapporo-like HuCVs are related more closely to animal caliciviruses than to other known HuCVs. We produced 3.2 kb cDNA fragments from the 3′ end to three Sapporo-like HuCVs that were associated with acute gastroenteritis in children (Houston/86, Houston/90, and London/92). Sequence analysis of the 3.2 kb cDNAs showed that two of the three viruses had a genomic organization similar to that of other Sapporo-like strains and the third strain (London/92) lacked an open reading frame overlapping the 5′ end of the capsid gene. Alignment of the capsid sequences of these three strains showed 44–78% amino acid identity among the three strains. Phylogenetic analysis of the aligned sequences indicated the three strains are related but each belongs to a distinct genetic cluster. The genetic differences are associated with antigenic differences in that an enzyme immune assay (EIA) specific for the prototype Sapporo/82 strain detected the Houston/86 strain, but not the Houston/90 and London/92 strains. In vitro transcription and translation of viral cDNA containing the predicted capsid gene of Houston/90 resulted in a protein of 63 K, which is immunoprecipitated by sera from children infected with the strain. Genetically and antigenically distinct strains in the Sapporo-like HuCVs have not been described previously and the occurrence of such diverse strains in the same community likely increases the importance of these strains as a cause of illness in children.

Human Calicivirus-Associated Diarrhea In Children Attending Day Care Centers

Abstract We investigated human calicivirus (HCV)-associated diarrheain children attending day care centers by using stool specimens collected in 1981–1983. We used a screening enzyme linked immunosorbent assay (ELISA) derived from reagents prepared against the Sapporostrain ofHCV and confirmed positive results with a blocking ELISA an dimmunosorbent electron microscopy. HCV was detected in 11 (2.9%) of 375 diarrheal stools and in none of 86 stools from asymptomatic contacts. This incidence ratewas half that noted for rotaviruses and higher than that noted for Campylobaeter, Salmonella, and Shigella in the original study. HCV was found in stool specimens from children in nine day care centers; HCV-associated diarrhea was sporadic, occurred with greater frequency in young children, and had a summer-fall predominance. Our results indicate that HeV is an important cause of diarrheain day care centers and that frozen stool samples can yield epidemiological data on HCV infection.

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.