Outbreak and volunteer studies provided significant information on host genetic factors that control susceptibility to norovirus infection. The recent elucidation of the type-specific recognition of histo-blood group antigens (HBGAs) as receptors for noroviruses helped to explain a controversy between clinical illness and immunity of norovirus gastroenteritis that had puzzled the field for many years. Noroviruses are transmitted by the fecal-oral pathway through person-to-person contact, contaminated environmental surfaces, and contaminated water or food, frequently causing large outbreaks. The widespread nature and multiple transmission modes of norovirus gastroenteritis can be explained by the low infectious dose and the high environmental stability of noroviruses and the lack of long-term host immunity. Due to the lack of cell culture methods or an animal model, the minimum infectious dose leading to norovirus gastroenteritis has not yet been thoroughly determined. Norovirus gastroenteritis is relatively mild, lasts for 12 to 48 h, and usually self-resolves. The increasing number of animal noroviruses that are genetically closely related to human isolates has also raised the issues of interspecies transmission of norovirus gastroenteritis and whether animal species can serve as reservoirs for human infections, although direct evidence for this remains lacking. The overall host specificity of noroviruses may not change in the course of long-term infection, but subtle modifications of the capsid surface, particularly the receptor binding interface, may occur, resulting in clones with better fitness. Recent new findings on host-pathogen interactions, molecular evolution, and host immune responses of noroviruses provide valuable information for developing future prevention and intervention strategies.

Human caliciviruses caused 45% of 55 gastroenteritis outbreaks occurring in Santiago, Chile, during 2000–2003. Outbreaks affected ≤99 persons, occurred most commonly in the home, and were associated with seafood consumption. Thirteen outbreak strains sequenced were noroviruses, including 8 GII, 2 GI, and 3 belonging to a novel genogroup.

Norovirus and Sapovirus are two genera of the family Caliciviridae that contain viruses that can cause acute gastroenteritis in humans. Noroviruses (NOR) are genetically highly diverse but limited studies of the genetic diversity of sapoviruses (SAP) have been reported. In this study we characterized twenty-five SAP detected in our laboratory from outbreaks or sporadic cases of acute gastroenteritis in children from different geographical locations and in adults involved in a cruise ship outbreak investigation and a nursing home outbreak. Based on significant differences of partial RNA polymerase sequences (278-286 nt), the 25 strains were grouped into 12 genetic clusters, including 9 potential new clusters. Extended sequence analysis of the capsid gene of selected strains representing five potential new clusters supported this grouping. Four strains (Hou7-1181/90, Mex340/90, Cruise ship/00 and Argentina39) had <84% amino acid (aa) identity to each other and to the published sequences in the GenBank. Mex14917/00 was almost identical to Stockholm/97/SE whose RNA polymerase sequence was unknown. Phylogenetic and distance analyses of the capsid region of the four new strains showed that Hou7-1181/90 and Argentina39 represent two new genogroups and Mex340/90 and Cruise ship/00 belong to two new clusters within the London/92 genogroup. Thus, based on the capsid sequences we propose to classify the currently known SAP into nine genetic clusters within five genogroups, including one genogroup that is represented by an animal calicivirus, the porcine enteric calicivirus (PEC).

Human milk contains a variety of bioactive agents, including oligosaccharides, which are part of the innate defense system (Hanson et al. 1991; Zopf & Roth 1996; Newburg et al. 1998; Erney et al. 2000; Hamosh 2001; Chaturvedi et al. 2001). Oligosaccharides are the third largest solid constituent of human milk after lactose and lipids (Stahl et al. 1994; Zopf & Roth 1996; Erney et al. 2000; Chaturvedi et al. 2001; Hamosh 2001). α 1,2-Linked fucosylated oligosaccharides in human milk have been shown to inhibit host ligand binding to Campylobacter, caliciviruses, and other enteric pathogens (Newburg et al. 1990; Crane et al. 1994; Marionneau et al. 2002; Ruiz-Palacios et al. 2003; Huang et al. 2003). Significant variation exists between mothers in the quantity of α 1,2-linked fucosylated oligosaccharides in their milk. We conducted this study in breastfed infants to determine the clinical relevance of the observed variation in human milk fucosylated oligosaccharides in relation to protection offered infants against diarrhea due to Campylobacter jejuni and caliciviruses.

OBJECTIVE:To determine the association between maternal milk levels of 2-linked fucosylated oligosaccharide and prevention of diarrhea as a result of Campylobacter, caliciviruses, and diarrhea of all causes in breast-fed infants. STUDY DESIGN:Data and banked samples were analyzed from 93 breast-feeding mother-infant pairs who were prospectively studied during 1988-1991 from birth to 2 years with infant feeding and diarrhea data collected weekly; diarrhea was diagnosed by a study physician. Milk samples obtained 1 to 5 weeks postpartum were analyzed for oligosaccharide content. Data were analyzed by Poisson regression. RESULTS:Total 2-linked fucosyloligosaccharide in maternal milk ranged from 0.8 to 20.8 mmol/L (50%-92% of milk oligosaccharide). Moderate-to-severe diarrhea of all causes (n=77 cases) occurred less often (P=.001) in infants whose milk contained high levels of total 2-linked fucosyloligosaccharide as a percent of milk oligosaccharide. Campylobacter diarrhea (n=31 cases) occurred less often (P=.004) in infants whose mother's milk contained high levels of 2'-FL, a specific 2-linked fucosyloligosaccharide, and calicivirus diarrhea (n=16 cases) occurred less often (P=.012) in infants whose mother's milk contained high levels of lacto-N-difucohexaose (LDFH-I), another 2-linked fucosyloligosaccharide. CONCLUSION:This study provides novel evidence suggesting that human milk oligosaccharides are clinically relevant to protection against infant diarrhea.

The baculovirus expression system has been widely used to produce the capsid proteins of Norovirus (NV) and the proteins form virus-like particles (VLPs) that are useful in many studies, such as immunology, diagnosis, and host-receptor interaction. We report here the application of the E. coli expression system in the production of recombinant NV capsid proteins. In a direct comparison of a previous well-characterized NV strain (VA387), we have demonstrated that the E. coli-expressed capsid proteins maintain the same antigenicity and receptor binding specificity as that of the baculovirus-expressed capsid, although the E. coli-expressed VA387 proteins did not form VLPs. Using the E. coli-expression system, we characterized the receptor-binding patterns of three additional NV strains (OIF1998, Parris Island and VA115), in which OIF1998 binds to HBGA of nonsecretors but did not bind or binds weakly to the HBGA of secretors, as seen in strain VA207. Parris Island binds to HBGA of types A and B but not type O secretors and nonsecretors. VA115 did not show specific binding to any A, B, O secretor nor nonsecretor, which is also observed when the capsid protein of this strain was expressed in baculovirus. Our data indicate that VLP formation is not required for receptor binding, and that the bacteria expression system offers a simple alternative for large production of NV capsid protein for various research purposes, particularly for strains generating low yields in the insect cells.

OBJECTIVE:To study the epidemiological status on rotavirus diarrhea in Kunming to improve the rotavirus vaccine immunization program. METHODS:A hospital-based sentinel surveillance program for rotavirus was set up among children less than 5 years old with acute diarrhea in Kunming Children's Hospital. Clinical information and fecal specimens were collected and rotavirus were detected by polyacrylamide gel electrophoresis (PAGE) and/or enzyme linked immunosorbent assay (ELISA). Positive specimens were further serotyped or genotyped by ELISA and/or RT-PCR. RESULTS:During the three years of surveillance, 466 specimens were collected. Rotavirus were detected on 246 (52.8%) specimens. 97% of the rotavirus diarrhea cases occurred among children less than 2 years old. There was a peak of admissions for rotavirus diarrhea cases between October and December which accounted for 48% of all the rotavirus hospitalizations each year. Among 204 specimens with G serotyping, the predominant strain was serotype G1 (47.5%) followed by G2 (17.6%), G3 (15.7%), G9 (4.9%) and G4 (1.0%). Mixed infection (2.5%) were rare and 22 specimens (10.8%) remained non-typeable. P genotyping showed P[4], P[8] and P[6] were the most common strains, accounting for 29.3%, 27.6% and 13.8% respectively. P[4]G2 was the most common strain which accounted for 34.1% (14/41) followed by P[8]G1 (29.3%) and P[6]G9 (12.2%). Another 7 uncommon P-G combinations were also identified. CONCLUSION:Rotavirus was the major cause of acute diarrhea in Kunming. An effective rotavirus vaccine for prevention and control of rotavirus diarrhea should be developed.

We characterized the binding of 8 Noroviruses (NORs) to histo-blood group antigens (HBGAs) in human saliva using recombinant NOR (rNOR) capsid proteins. Among the 8 rNORs tested, 6 formed viruslike particles (VLPs) when the capsid proteins were expressed in insect cells, all of which revealed variable binding activities with saliva; the remaining 2 rNORs did not form VLPs, and the proteins did not bind, or bound weakly, to saliva. Four distinct binding patterns were associated with different histo-blood types, defined by Lewis, secretor, and ABO types. Three patterns (VA387, NV, and MOH) recognized secretors, and 1 pattern (VA207) recognized Lewis-positive nonsecretors. The 3 secretor-recognizing patterns were defined as A/B (MOH), A/O (NV), and A/B/O (VA387) binders. Oligosaccharides containing the Lewis and ABH antigenic epitopes were involved in binding. Our findings suggest that different strains of NORs may recognize different human HBGAs on intestinal epithelial cells as receptors for infection.

INTRODUCTION:Norovirus (genera; previously "Norwalk-like viruses") of the family Caliciviridae are an important cause of acute gastroenteritis outbreaks. Epidemics commonly occur in crowded community and spread by fecal-oral route through contaminated food, water and direct contact.AIMS:To determine the incidence and epidemiological role of caliciviruses in nosocomial gastroenteritis outbreaks in hospitals in Hungary.METHODS:Between November 1998 and April 2002, 277 stool samples of 39 nonbacterial hospital outbreaks reported as "enteritis infectiosa" with unknown etiology from 13 of 19 counties in Hungary were examined by reverse transcription-polymerase chain reaction (RT-PCR) for Norovirus and Sapovirus and by a new recombinant enzyme immune assay (rEIA) for Norovirus only.RESULTS:99 (36%) of 277 stool specimens were positive by RT-PCR and 29 (74%) and 38 (97%) of 39 outbreaks were positive by RT-PCR and RT-PCR and/or rEIA for Norovirus, respectively. Norovirus were the most frequently detected agents in the registered nosocomial enteric hospital outbreaks in years 2000 (n = 4; 31%) and 2001 (n = 7; 23%). In the first 4 months of 2002, 25 (66%) of 38 nosocomial outbreaks was caused by Norovirus. Seventeen (44%) outbreaks occurred in single internal wards and eight (21%) in more than one (2-8) ward. An average of 32% (0-80%) of the sick persons, were member of the hospital staff. Twenty four (63%) Norovirus-associated outbreaks occurred between January and March 2002.CONCLUSIONS:This is the first comprehensive country-wide surveillance of nosocomial gastroenteritis outbreaks in hospitals associated with Norovirus. Data showed that Norovirus were the most important cause of these outbreaks in Hungary but the epidemiological role of this virus is probably further underestimated.

Host immune responses to human caliciviruses are difficult to study because of the lack of a clear definition of antigenic or serological types. This report describes antibody responses to several Norwalk-like viruses in large outbreaks of acute gastroenteritis on 2 US Navy ships. Enzyme immunoassays (EIAs) were used to measure antibody responses. To understand the antibody response to a homologous strain causing the outbreaks, the viral capsid gene of one isolate (C59) was expressed in baculovirus and included in the EIAs. Significantly greater seroresponses were detected in patients against the homologous strain than against the heterologous strains. Strains within genogroups reacted more strongly than did strains between genogroups. Significantly higher antibody titers against the outbreak strain were detected in acute serum samples from control subjects than in those from case patients. These results indicate that recombinant EIAs are useful for outbreak investigation and that the homologous antibody might be protective against reinfection.

A large outbreak of acute gastroenteritis occurred among three different groups of guests and the employees of a Virginia hotel within a 2-week period in November 2000. At least 76 of the hotel's guests and 40 hotel employees had acute gastroenteritis during this period. All tested ill persons were infected with the same strain of Norwalk-like virus, as shown by cloning and sequencing of virus detected in stool specimens from the three guest groups and the employees. Epidemiologic investigation suggested food as the probable source for the guests. Most of the employees, including those sick, did not eat in the hotel, suggesting that environmental contamination and person-to-person transmission could have contributed to the outbreak. The disease continued to spread in the hotel, passing from one guest group to another, by food, environmental contamination, and/or by person-to-person transmission through infected employees and guests. The study describes procedures implemented to control the outbreak and makes recommendations for future outbreak control.

UNLABELLED:Sapporo-like viruses (SLVs) are members of the family Caliciviridae. The etiologic role of these viruses is evident but little is known about the incidence of acute gastroenteritis caused by them. Sapporo-like viruses have not been detected in Hungary before.METHODS:Between October and December 2000, 72 sporadic diarrhoeal stool samples from Baranya County, Hungary, from infants and young children (under 12 years) with acute gastroenteritis were collected. Common enteric bacterial pathogens, adeno- and rotaviruses were not found in these stool samples. Reverse transcription-polymerase chain reaction with specific primer pairs for human caliciviruses were used to detect Sapporo-like viruses.RESULTS:The amplicons of expected size were cloned and subsequently sequenced. Seven (9.7%) of 72 stool samples were found to be positive for Sapporo-like viruses. Comparative sequence analysis confirmed that all strains belonged to the London/92/UK cluster.CONCLUSION:This is the first molecular detection and molecular sequence analysis of Sapporo-like viruses in Hungary.

Three viral gastroenteritis (VGE) outbreaks that occurred in 1998-1999, in Hungary were investigated for the presence of human caliciviruses (HuCVs). HuCVs in stool specimens were detected by reverse transcription-polymerase chain reaction (RT-PCR) using primer pair 289/290, which was designed based on the RNA-dependent RNA polymerase (RdRp) sequence. RT-PCR results were confirmed by sequencing showing that all three outbreak strains belonged to genogroup II of "Norwalk-like viruses" (NLVs). Two strains had high sequence identity with strains in known genetic clusters (Hawaii and Lordsdale clusters). The third strain (MOH) had distinct RdRp sequence, sharing 77/86% (nt/aa) identity with Snow Mountain virus (SMV), the closest genogroup II virus. To characterize MOH further, we cloned, sequenced, and expressed in baculovirus its capsid gene. It had 75/79% (nt/aa) identity with SMV, but 97/98% (nt/aa) identity with NLV/Hillingdon/90/UK, a recently identified genetic cluster of HuCVs. The recombinant MOH (rMOH) capsid protein self-assembled into virus-like particles (VLPs), which is antigenically distinct from other recombinant HuCV capsid antigens available in our laboratory. Further study of this VLP will have important applications in antigenic characterization and diagnosis of HuCVs.

人类杯状病毒(human calicivirus,HuCV)是引起儿童和成人非菌性胃肠炎的主要病原之一.为了掌握HuCV在我国的流行情况,1998年7月至2001年6月,从长春市儿童医院2343例5岁以下腹泻患儿中共收集粪便标本1264份,其中1056份来自2135例住院患儿.对轮状病毒检测为阴性的588份标本,经多价酶免疫试验(EIA)和两组引物反转录-聚合酶链反应(RT-PCR)检测HuCV,202份为阳性,其中住院患儿标本178份,HuCV检出率为16.9%.HuCV腹泻以2岁以下儿童为主(占96%),流行高峰季节为11月至次年3月.选择17株HuCV进行分子鉴定,15株属GⅡ-4群,1株属GⅡ-3群,另1株属GⅠ-2群,表明GⅡ-4群HuCV是我国流行的优势株.根据HuCV住院患儿的监测资料初步估计,HuCV腹泻住院率约为0.5‰～2.4‰.讨论了长春地区HuCV的流行趋势和疾病负担.以上结果为我国HuCV腹泻的预防和控制提供了科学依据.