Shoko Okitsu

Genetic Diversity of Norovirus and Sapovirus in Hospitalized Infants with Sporadic Cases of Acute Gastroenteritis in Chiang Mai, Thailand

ABSTRACT Stool specimens from hospitalized infants with sporadic gastroenteritis in Chiang Mai, Thailand, between July 2000 and July 2001 were examined for norovirus and sapovirus by reverse transcription-PCR and sequence analysis. These viruses were identified in 13 of 105 (12%) specimens. One strain was found to be a recombinant norovirus.

Isolation and Molecular Characterization of Aichi Viruses from Fecal Specimens Collected in Japan, Bangladesh, Thailand, and Vietnam

ABSTRACT Aichi virus is a new member of the family Picornaviridae, genus Kobuvirus, and is associated with human gastroenteritis. This study detected Aichi virus in 28 of 912 fecal specimens which were negative for rotavirus, adenovirus, norovirus, sapovirus, and astrovirus and were collected in Japan, Bangladesh, Thailand, and Vietnam during 2002 to 2005.

Detection of norovirus and sapovirus infection among children with gastroenteritis in Ho Chi Minh City, Vietnam

Summary.This report describes norovirus (NoV) and sapovirus (SaV) infections in hospitalized children with acute sporadic gastroenteritis in Ho Chi Minh City, Vietnam. Stool specimens collected between December 1999 and November 2000 were examined for NoV and SaV using reverse transcription-PCR and phylogenetic analysis. NoVs were detected in 72 of 448 rotavirus-negative specimens, counted as part of an overall annual detection rate of 5.4% (72 of 1,339 children). This included four NoV genogroup I (GI) strains and 68 NoV GII strains. Only one SaV GI strain was detected in the rotavirus-negative specimens. Over 73% of the NoV sequences belonged to GII/4 (Lordsdale cluster) and were detected in all months except March. We also detected GII/3 strains (Saitama U201 cluster), a naturally occurring recombinant NoV, between January 2000 and March 2000 but not after this period. Other NoV strains belonging to GI/4, GI/8, GII/1, and GII/7 were also detected but were infrequent. In addition, two almost identical NoV GII strains (strains 026 and 0703) collected six months apart were classified into a new genotype that includes the Mc37 strain, which was previously shown to be a recombinant NoV. During this one-year study, the NoV prevailed at the end of the rainy season and the beginning of the dry season. Further epidemiological studies may be necessary to determine whether the GII/4 strains continue to dominant in this region.

Detection and Genetic Characterization of Group A Rotavirus Strains Circulating among Children with Acute Gastroenteritis in Japan

ABSTRACT A total of 603 fecal specimens collected from July 2005 to June 2006 from children with acute gastroenteritis, encompassing five different localities in Japan, were screened for group A rotavirus by reverse transcription-PCR. It was found that 117 fecal specimens (19.4%) were positive for group A rotavirus. Rotavirus infection was detected continuously from November to June, with the highest prevalence in April. The G (VP7 genotypes) and P (VP4 genotypes) types were further investigated. The predominant genotype was G1P[8] (70.1%), followed by G3P[8] (17.9%), G9P[8] (6.8%), and G2P[4] (2.6%). A number of unusual G1P[4] combinations were also detected during this study period. A novel nomenclature for G1 is proposed, in which worldwide rotavirus G1 strains are classified into 11 lineages with 17 sublineages. A wide range of amino acid substitutions (up to 34) in VP7 that are specific for G1 lineages and sublineages were identified. Interestingly, only short amino acid motifs located at positions 29 to 75 and 211 to 213 of VP7 defined correctly the phylogenetic G1 lineages and sublineages. Examination of the deduced sequences of antigenic regions of VP7 also revealed multiple particular amino acid substitutions that correlated with the phylogenetic G1 lineages and sublineages. Of note, at least three distinct clusters of rotavirus G1 isolates were cocirculating in the Japanese pediatric population studied.

Existence of multiple outbreaks of viral gastroenteritis among infants in a day care center in Japan

Summary.A total of 921 fecal specimens collected from 44 infants in a day care center (DCC) in Tokyo, Japan during June 1999 to July 2000 were tested for the presence of rotavirus, norovirus, sapovirus, astrovirus and adenovirus by reverse-transcription-multiplex polymerase chain reaction (RT-multiplex PCR) and sequence analysis. Of 88 fecal specimens from infants with acute gastroenteritis, 51.1% (45) were found to be positive for diarrheal viruses. Astrovirus was the most prevalent (15.9%, 14 of 88), followed by norovirus GII (14.8%, 13 of 88), adenovirus (12.5%, 11 of 88), and sapovirus (2.3%, 2 of 88). Viral mixed infection accounted for 5.7% (5 of 88). Interestingly, 230 of 833 (27.6%) fecal specimens collected from asymptomatic infants were also infected with diarrheal viruses. Of these, astrovirus, norovirus GII, adenovirus and sapovirus were identified in 53, 46, 96 and 22 fecal specimens (23%, 20%, 41.7%, and 9.6%, respectively). Moreover, 13 of 833 (1.6%) normal specimens showed mixed viral infections. Surprisingly, no rotavirus (known as the most common causative agent of acute gastroenteritis in DCCs) was detected in those subjects. Another interesting feature was the demonstration of five separate outbreaks of acute gastroenteritis identified in a single DCC. Outbreak A was associated with both astrovirus serotype 1 and norovirus GII/3 (known as Toronto virus cluster); Outbreak B with adenovirus 12; Outbreak C with norovirus GII/4 (Lordsdale virus cluster); Outbreak D with sapovirus GIV and Outbreak E with astrovirus serotype 1. To our knowledge, this is the first proof of multiple outbreaks of viral gastroenteritis in Japanese infants in a single DCC. Our results confirm the presence as well as the importance of these viruses and warn of the threat they pose.

Porcine kobuvirus in piglets, Thailand.

To the Editor: To date, the genus Kobuvirus has consisted of 2 officially recognized species, Aichi virus and Bovine Kobuvirus (1). Aichi virus has been shown to be associated with acute gastroenteritis in humans (2–4), and bovine kobuvirus has been detected only in cattle (5,6). Most recently, a third candidate species of Kobuvirus has been described in pigs by 2 different groups of investigators from Hungary and the People’s Republic of China (7,8). This new candidate species was serendipitously recognized in stool specimens from pigs when PCR products (≈1,100 bp) were amplified by using a primer pair for the detection of caliciviruses (7).

Diversity of Human Parechoviruses Isolated from Stool Samples Collected from Thai Children with Acute Gastroenteritis

ABSTRACT A total of 82 fecal specimens which were known to be negative for rotavirus, adenovirus, norovirus, sapovirus, and astrovirus and which were collected from infants and children with acute gastroenteritis in Chiang Mai, Thailand, from January to December 2005 were screened for human parechovirus (HPeV). HPeV was detected by reverse transcription-PCR with a primer pair that amplified the 5′ untranslated region of its genome and was genotyped by sequencing of the VP1 region. HPeV was detected in 12 of 82 specimens tested, and the detection rate was found to be 14.6%. The capsid VP1 gene was successfully sequenced from nine of the HPeV strains detected. The HPeV strains studied clustered into four different genotypes, HPeV genotype 1 (HPeV1) to HPeV4, and the majority of the strains studied (five strains) belonged to HPeV1. This is the first finding of HPeV from children with acute gastroenteritis in Thailand. In addition, the diversity of the Thai HPeV strains was also noted.

A single-tube multiplex PCR for rapid detection in feces of 10 viruses causing diarrhea

A novel multiplex polymerase chain reaction assay was developed to identify 10 viruses in a single tube. The assay was targeted to detect group A and C rotaviruses, adenovirus, norovirus GI, norovirus GII, sapovirus, astrovirus, Aichi virus, parechovirus, and enterovirus. A total of 235 stool samples were collected from infants and children with acute gastroenteritis in Kyoto, Japan, from 2008 to 2009, then tested by this novel multiplex PCR and compared with a multiplex PCR described previously, which used 3 primer sets. The novel multiplex PCR could detect the targeted viruses in 111 of the 235 (47.2%) stool samples. Of these, 9 out of 10 types of viruses were identified, including group A rotavirus, norovirus GII, enterovirus, sapovirus, adenovirus, parechovirus, group C rotavirus, astrovirus, and norovirus GI. In contrast, the multiplex PCR that used 3 sets of primers could detect the targeted viruses in 109 of the 235 (46.4%) stool samples. Among these, 8 types of viruses were identified, including group A rotavirus, norovirus GII, enterovirus, adenovirus, parechovirus, group C rotavirus, sapovirus, and astrovirus. The results suggested that the new multiplex PCR is useful as a rapid and cost effective diagnostic tool for the detection of major pathogenic viruses causing diarrhea.

Detection, genetic characterization, and quantification of norovirus RNA from sera of children with gastroenteritis

BACKGROUND Norovirus (NoV) infection is thought to be confined to the intestines, whereas many reports suggest antigenemia and viremia occur during rotavirus gastroenteritis. OBJECTIVES To detect NoV RNA in sera and cerebrospinal fluids (CSF) from NoV-infected children, and to quantify and genetically characterize the NoV found in these compartments. STUDY DESIGN Semi-nested PCR was conducted on stool, serum and CSF samples from 56 patients with acute gastroenteritis. Positive samples for NoV were analyzed further by sequencing and real-time PCR. RESULTS From 39 patients with NoV RNA in stools, 6 also had NoV RNA in sera and none had NoV RNA in CSF. Genotypes of the NoV in stool and serum from the same patient matched completely. The strains in this study had high homology (98.1-100%) with registered strains in the database. The median viral load in stools of the serum-positive patients was greater than that of the serum-negative patients, but this difference was not statistically significant (9.8 x 10(9)copies/g versus 1.1 x 10(9)copies/g (p=0.117)). CONCLUSIONS NoV RNA appeared in the blood stream in 15% of the patients of NoV gastroenteritis. Although the viral load in stool was not statistically correlated with NoV appearance in serum, genetic analysis indicated that NoV RNA in sera originated from the NoV gastroenteritis.

Characterization of a Broadly Reactive Monoclonal Antibody against Norovirus Genogroups I and II: Recognition of a Novel Conformational Epitope

ABSTRACT Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.