Human caliciviruses (HuCVs) cause sporadic cases and outbreaks of acute gastroenteritis (AGE). Three maior genogrovps of HuCVs have been described including the Norwalk virus (NV)-, the Snow Mountain virus (SMA)-, and the Sapporo-genogroups. This study describes the detection and genetic variation of HuCVs from hospitalized infants with AGE in Korea by RT-PCR and sequencing. The cDNA fragments of 206 to 470bp corresponding to the region of 3 primer pairs (36/35, 35/51 or 3/51) in the polymerase region of NV were generated. Of 185 stools screened, 8% were positive by RT-PCR and their sequences showed that all strains contained the GLPSG and YGDD motifs which are conserved for HuCVs. Amino acid (aa) sequence analysis showed that these strains can be divided into 3 maior genogroups. High conservation was observed in that one strain shares 100% of as sequence with Southampton virus, another shares 99% with the Sapporo virus, and six strains share 90 to 95% with Snow Mountain virus. However, significant sequence variation was also found in other strains. This study indicates that all maior genogroups of HuCVs are circulating in Korea.
Disease Outbreaks ; DNA, Complementary ; Gastroenteritis ; Genetic Variation ; Genotype ; Humans* ; Infant ; Korea* ; Norwalk virus ; RNA Replicase* ; Sapovirus ; Sequence Analysis ; Snow

Norwalk-like virus (NLV), one of the emerging enteric pathogens in sporadic cases and outbreaks of acute gastroenteritis (AG) worldwide, are 30-35 nm in diameter and have a single stranded RNA genome, size 7.6 kb. As genomic sequence data from several isolates of NLVs are being reported, the genetic relationships among these viruses are now being described. Based on the level of sequence similarity in the RNA polymerase region of NLVs, at least three genogroups can be defined: the Norwalk virus (NV)-, the Snow Mountain agent (SMA)-, and the Sapporo-genogroups. A high prevalence of serum antibody to NV-related viruses but no antigenpositve stools (either observed to contain the NLV by immune electron microscopy or suspected of containing the NLV) has suggested that genomically different NLVs are present among Korean children. The purpose of our work therefore was to detect NLVs from Korean infants and children with AG and to characterize the partial genome (RNA-dependent RNA polymerase region) of currently circulating Korean isolates. The cDNA fragments of 110 to 470 bp corresponding to the RNA polymerase region were generated by reverase transcription-polymerase chain reaction (RT-PCR) using three primer pairs (36/35, 36/51, or 36/101) derived from the prototype NV (8FIIaNV/68/US). Of 279 stool samples tested, 25 (9%) specimens were positive by RT-PCR and the amplified productscontained GLPSG and YGDD amino acid motifs, characteristic of positive-strand RNA-dependent RNA polymerases, at the predicted distance from the primers. In 6% of the infectious episodes with diarrhea, NLVs appeared to be the sole pathogen. Double infections occurred mainly with rotavirus and triple infections occured with both rotavirus and adenovirus or with both rotavirus and astrovirus. This study indicates that NLVs are a common cause of sporadic AG in Korean infants.
Adenoviridae ; Amino Acid Motifs ; Child* ; Diarrhea ; Disease Outbreaks ; DNA, Complementary ; DNA-Directed RNA Polymerases ; Gastroenteritis* ; Genome ; Genotype ; Humans ; Infant ; Microscopy, Electron ; Norovirus* ; Norwalk virus ; Prevalence ; RNA ; Rotavirus ; Snow

Sequence comparison of the RNA-dependent RNA polymerase of human caliciviruses (HuCVs) from Korean children with gastroenteritis revealed significant genetic variation among them. cDNA clones were produced from the HuCVs collected from pediatric population during a period of 1987-1994. The application of reverse transcription-polymerase chain reaction (RT-PCR) using primers directed to the RNA-dependent RNA polymerase region within ORF1 of Norwalk virus (NV) showed that 13.7% of HuCVs yielded PCR products of similar size to the NV prototype, NV8Flla/68/US, with exceptions of HuCV185/87/Korea and HuCV1115/90/Korea. Computer analyses showed that the PCR products had a continuous protein encoding frame on the positive strand, and contained GLPSG and YGDD amino acid motifs at the predicted distance from primers. Alignment of the amino acid sequences of HuCVs with previously published sequences for Snow Mountain agent (SMA), NV, and Sapporo/82/Japan indicated that these strains can be divided into four major genogroups. There were 10 (45%) SMA-like CVs, one (4.5%) NV-like HuCVs, two (9%) Sapporo-like HuCVs, and nine (41%) unidentified HuCVs. This fourth genogroup should be investigated further. HuCV185/87/Korea and HuCV1115/90/Korea, Sapporo-like CVs, were genetically distinct from previously characterized HuCVs and more closely related to known animal CVs. One of the animal CV-like strain, HuCV185/87/Korea, showed nucleotide and amino acid homology of only 67% and 73% with the prototype Sapporo/82/Japan. Further characterization of animal and human CV genomes and studies of possible cross-transmission of CVs from animals to humans are likely to be beneficial in understanding the epidemiology of HuCVs.
Amino Acid Motifs ; Amino Acid Sequence ; Animals ; Child ; Clone Cells ; DNA, Complementary ; Epidemiology ; Gastroenteritis ; Genetic Variation ; Genome ; Genotype ; Humans* ; Korea* ; Norwalk virus ; Polymerase Chain Reaction ; RNA Replicase ; Snow

The present study was designed to estimate the seroprevalence of NLVs among diarrheagenic children and in healthy adults in Seoul and its vicinity with the use of an EIA and an Western blot (WB) based on recombinant Norwalk virus capsid protein (rNV) and crude virus preparations as antigen. Seroconversion was observed in 34 (83%) of 41 tested using the EIA and in 21 (54%) of 39 using the WB, suggesting that the NLVs with epitopes common to rNV are prevalent in Seoul area. Diarrheal children who were known to have been infected with several other strains of the NLVs showed no significant antibody response to the rNV. Infection with rNV occurred earlier in life: primary infections with rNV were common before the age of 6 months and over 91% of children had evidence of infection by that age by the EIA. Since the amount of the NLV antigens available for seroepidemiologic surveys is limited, we tried to detect NLV antibody by using crude virus preparations as antigen. One crude virus preparation of a child whose stool yielded genetically distinct NLV revealed the presence of the plural number of bands upon SDS-PAGE, but precipitated only one band (62 kDa) after the WB with a serum (collected 10 days after the onset of symptoms) of another diarrheal child. The WB assay we present in this report revealed that the NLVs are prevalent among Korean population and that the sera contained antibody to a single major structural protein, with molecular sizes of 58 to 62 kDa, compatible with the sizes reported for the Norwalk virus and Snow Mountain agent proteins, respectively. When the results of the WB were compared with those obtained by the EIA, the EIA antibody assay was sensitive enough to detect an antibody rise of as much as 4096-fold but not as specific as the WB. The WB assay presented in this paper will provide a powerful tool to elucidate not only antigenic structures of the NLVs but also seroepidemiology of the NLV infection. The availability of an unlimited source of antigen will enable a large scale serologic studies that will greatly increase our understanding of the role of NLVs in human enteric illness.
Adult ; Antibody Formation ; Blotting, Western ; Capsid Proteins ; Capsid* ; Child ; Electrophoresis, Polyacrylamide Gel ; Epitopes ; Humans ; Norovirus* ; Norwalk virus* ; Seoul* ; Seroepidemiologic Studies* ; Snow

To develop a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of Norwalk GII. 4 primers which recognized 6 distinct regions on the RNA-dependent RNA polymerase gene of Norwalk GII were designed and used for LAMP assay. Norwalk GII RNA was amplified under isothermal conditions (65 degrees C) for 120 min, and LAMP results were then judged with naked eye, SYBR Green I staining, electrophoretic analysis and restriction digestion. To evaluate the specificity of the RT-LAMP, 48 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses were tested. To compare the sensitivity of the RT-LAMP with that of conventional RT-PCR, Norwalk GII RNA was serially diluted and amplified by RT-LAMP and RT-PCR, respectively. With 46 fecal specimens of Norwalk GII, observation with naked eyes, SYBR Green I staining and electrophoretic analysis were able to detect the PCR products in the RT-LAMP assay. The specificity of RT-LAMP products was also confirmed by digestion of the RT-LAMP products with restriction enzymes. No RNA amplification was observed in 2 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses. The specificity of the RT-LAMP assay with regard to RT-PCR were 100% for Norwalk GII. The detection limits of RT-LAMP was 15.6 pg/tube for Norwalk GII and similar to that of a RT-PCR assay. Compared to RT-PCR, the RT-LAMP assay has been proven to be a rapid, sensitive, specific and accurate method for detection of the Norwalk GII in fecal specimens, and that RT-LAMP assay is potentially useful for the rapid detection of Norwalk GII from fecal specimens in outbreaks of infectious diarrhea.
Caliciviridae Infections ; virology ; Feces ; virology ; Humans ; Norwalk virus ; genetics ; isolation & purification ; RNA Replicase ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Proteins ; genetics

OBJECTIVETo clone, express and characterize the capsid protein of human Norwalk virus Guangzhou strain NVgz01.

METHODSOn the basis of successful construction of full-genome clones and sequence analysis of human norovirus Guangzhou strain NVgz01, the full capsid gene was ligated into pET28a (+) for expression. After IPTG induction, the recombinant protein was purified through metal (Ni(2+)) chelating affinity chromatography. Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to determine the antigenicity of the recombinant protein.

RESULTSThe recombinant capsid gene was overexpressed in E.coli, yielding the recombinant protein with relative molecular mass of 62x10(3) that was highly purified through metal (Ni(2+)) chelating affinity chromatography. IDEIA Norovirus Kit and immunoassay showed that the recombinant protein had good antigenicity.

CONCLUSIONThe capsid gene of norovirus Guangzhou strain has been cloned and expressed, which can be useful for developing diagnostic reagents or vaccine of norovirus.

Blotting, Western ; Capsid Proteins ; analysis ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Norwalk virus ; genetics ; Plasmids ; genetics

PURPOSE: The hospital-acquired outbreak of acute gastroenteritis caused by a virus is currently one of major health problems among newbron nurseries in Korea. We report an outbreak of acute gastroenteritis with metabolic acidosis over a eight-month period in many nurseries in Gyeonggi-do, Korea. METHODS: We retrospectively evaluated 73 neonates who were admitted in Ansan Hospital, Korea University Medical Center, presenting with symptoms of acute viral gastroenteritis from March, 2001 to September, 2003. Epidemiologic, clinical and laboratory data for these neonates were reviewed. Enzyme-linked immunosorbent assay (ELISA) for rotavirus and enzyme immunoassay (EIA) for astrovirus, norwalk virus and enteric adenovirus were performed in 54 and 16 neonates, respectively. RESULTS: The mean age at admission was 9.5 +/- 3.9 days. The mean body weight was significantly decreased, compared with mean birth weight. The numbers of severe dehydration were 37 (50.7%) and severe metabolic acidosis (pH < 7.2) were 40 (54.8%). Although the symptoms and laboratory findings were all compatible with acute gastroenteritis caused by a virus, the positive results for ELISA for rotavirus and EIA for astrovirus, norwalk virus and enteric adenovirus were 8 (14.8%) for 54 neonates and none for 16 neonates, respectively. CONCLUSION: Recognizing an outbreak of viral gastroenteritis necessitates notification of local health officials, collection of appropriate specimen for diagnosis and prompt institution of infection control measures.
Academic Medical Centers ; Acidosis* ; Adenoviridae ; Birth Weight ; Body Weight ; Cross Infection ; Dehydration ; Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Gastroenteritis* ; Gyeonggi-do* ; Humans ; Immunoenzyme Techniques ; Infant, Newborn ; Infection Control ; Korea* ; Norwalk virus ; Nurseries* ; Retrospective Studies ; Rotavirus

Adenovirus Infections, Human ; epidemiology ; virology ; Adenoviruses, Human ; isolation & purification ; Caliciviridae Infections ; epidemiology ; virology ; Child, Preschool ; China ; epidemiology ; Diarrhea ; epidemiology ; virology ; Diarrhea, Infantile ; epidemiology ; virology ; Enzyme-Linked Immunosorbent Assay ; Feces ; virology ; Female ; Gastroenteritis ; epidemiology ; virology ; Humans ; Incidence ; Infant ; Male ; Norwalk virus ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Rotavirus ; isolation & purification ; Rotavirus Infections ; epidemiology ; virology

OBJECTIVETo investigate the epidemiological features of human calicivirus( HuCV) infection in children with diarrhea in a hospital in Guangzhou.

METHODSStool specimens were collected from children with viral diarrhea diagnosed between October, 2003 and January, 2004 and between October, 2004 and January, 2005. HuCV was detected by means of RT-PCR and sequence analysis of the PCR products.

RESULTSEighty specimens positive for Norwalk-like viruses (NLVs) were identified from 648 stool specimens, with a positivity rate of 12.35%, and sapporo-like viruses (SLVs) were identified in 2 specimens (0.31%). The monthly NLV positivity rate was 11.74% (Oct.), 14.16% (Nov.), 9. 09% (Dec.) and 13.95% (Jan.), respectively, showing no significant variation in these months. NLVs mainly infected children below 2 years old. Twenty-two strains of NLVs were sequenced and analyzed, and 21 of them were identified as GII and the genotype of 1 strain could not be determined. The prevalent viral population were GII-3 and GII-4 in 2003 and was GII-4 in 2004, and both of the SLVs belong to GI-1.

CONCLUSIONNLVs is one of the important pathogens causing sporadic acute gastroenteritis in children admitted in the hospital in Guangzhou, and the prevalent strains are GII-3 and GII-4 , but different prevalent strains are possible in different periods.

Caliciviridae ; classification ; genetics ; isolation & purification ; Caliciviridae Infections ; epidemiology ; virology ; Child, Preschool ; China ; epidemiology ; Diarrhea ; epidemiology ; virology ; Diarrhea, Infantile ; epidemiology ; virology ; Feces ; virology ; Female ; Genotype ; Hospitals ; statistics & numerical data ; Humans ; Infant ; Male ; Molecular Epidemiology ; methods ; Molecular Sequence Data ; Norwalk virus ; genetics ; isolation & purification ; Phylogeny ; Prevalence ; RNA, Viral ; genetics ; Seasons ; Sequence Analysis, DNA