Interaction between norovirus and Histo-Blood Group Antigens: A key to understanding virus transmission and inactivation through treatments?

Human noroviruses (HuNoVs) are a main cause of acute gastroenteritis worldwide. They are frequently involved in foodborne and waterborne outbreaks. Environmental transmission of the virus depends on two main factors: the ability of viral particles to remain infectious and their adhesion capacity onto different surfaces. Until recently, adhesion of viral particles to food matrices was mainly investigated by considering non-specific interactions (e.g. electrostatic, hydrophobic) and there was only limited information about infectious HuNoVs because of the absence of a reliable in vitro HuNoV cultivation system. Many HuNoV strains have now been described as having specific binding interactions with human Histo-Blood Group Antigens (HBGAs) and non-HBGA ligands found in food and the environment. Relevant approaches to the in vitro replication of HuNoVs were also proposed recently. On the basis of the available literature data, this review discusses the opportunities to use this new knowledge to obtain a better understanding of HuNoV transmission to human populations and better evaluate the hazard posed by HuNoVs in foodstuffs and the environment.

Qualitative and quantitative analysis of the binding of GII.4 norovirus variants onto human blood group antigens.

Noroviruses (NoVs) are one of the leading causes of gastroenteritis in children and adults. For the last 2 decades, genogroup II genotype 4 (GII.4) NoVs have been circulating worldwide. GII.4 NoVs can be divided into variants, and since 2002 they have circulated in the population before being replaced every 2 or 3 years, which raises questions about the role of their histo-blood group antigen (HBGA) ligands in their evolution. To shed light on these questions, we performed an analysis of the interaction between representative GII.4 variants and HBGAs, and we determined the role of selected amino acids in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the amino acids, directly implicated in interactions with HBGAs. However, the ablation of the threonine residue at position 395 (ΔT395), an epidemiological feature of the post-2002 variants, was not deleterious to the binding of the virus-like particle (VLP) to the H antigen, while binding to A and B antigens was severely hampered. Nevertheless, the ΔT395 VLPs gained the capacity to bind to the Lewis x and sialyl-Lewis x antigens in comparison with the wild-type VLP, demonstrating that amino acid residues outside the HBGA binding site can modify the binding properties of NoVs. We also analyzed the attachment of baculovirus-expressed VLPs from six variants (Bristol, US95/96, Hunter, Yerseke, Den Haag, and Osaka) that were isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs. We showed that the six variants could all attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, Den Haag and Osaka variants additionally bound to carbohydrates present in the saliva of Lewis-positive nonsecretors. The carbohydrate binding profile and the genetic and mutagenesis analysis suggested that GII.4 binding to Lewis x and sialyl-Lewis x antigens might be a by-product of the genetic variation of the amino acids located in the vicinity of the binding site. Analysis of the binding properties for the six variants by surface plasmon resonance showed that only post-2002 variants (i.e., Hunter, Yerseke, Den Haag, and Osaka) presented strong binding to A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post-2002 variants. The combination of increased affinity for ABH antigens and of a newly acquired ability to recognize glycans from Lewis-positive nonsecretors could have contributed to the epidemiological importance of strains such as the Den Haag GII.4 subtype.

Evidence of the co-circulation of enteric viruses in sewage and in the population of Greater Cairo.

AIMS: To characterize major enteric viruses (enterovirus, rotavirus, norovirus, astrovirus and adenovirus) in the sewage of Greater Cairo and to compare the results with clinical data collected during the same period. METHODS AND RESULTS: Seventy-two sewage samples from two waste water treatment plants were collected from April 2006 through February 2007. Enteroviruses, noroviruses (NoVs) and rotaviruses (RVs) were detected by RT-PCR in 22%, 18% and 8.3% of the samples, respectively. No adenovirus and astrovirus was detected. G2P[8], G9P[8], G1P[8], G2P[4] and rare G12 RV isolates were detected in the environment as well as a bovine RV. The environmental NoV strains mostly belonged to genogroup I (84%). Rotaviruses and some of the NoVs were similar to those found in the clinical samples at the same time. CONCLUSIONS: The comparison of environmental and clinical data suggests that similar RV and NoV isolates were circulating in the environment and in the population during the same period. SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies have investigated the prevalence and the epidemiology of RVs and NoVs in Cairo. This work is the first to establish a correlation between viral gastroenteritis and the concomitant presence of enteric viruses in the environment for Greater Cairo where combined environmental and clinical surveys should help to prevent infections caused by these major pathogens.

Relationship between GII.3 norovirus infections and blood group antigens in young children in Tunisia.

Noroviruses (NoVs) constitute a major cause of gastroenteritis in Tunisia. One hundred and fourteen matched saliva and stool samples were collected from children (n = 114) suffering from acute gastroenteritis at the hospital of Monastir during the winter season 2011-2012. For 98 of 114 children, blood samples were collected for secretor genotyping. NoVs were associated with 36.8% (n = 42/114) of the gastroenteritis cases. The GII.3 genotype was the most common (69% of all NoVs). For patients who were phenotyped (n = 114) for human blood group antigens (HBGAs), the secretor and non-secretor phenotypes represented 79% and 21%, respectively. Of the NoV infections, 83% were detected in all ABO groups. Five GII.3 isolates, one GII.1 isolate and one GII.7 isolate were detected in Lewis-positive non-secretors, confirmed by genotyping of the FUT2 gene. Even though our data showed that GII.3 NoVs could infect non-secretors, no binding was observed with saliva and GII.3 baculovirus-expressed virus-like particles from the same symptomatic non-secretor individual. This suggests that other factors might also participate in NoV attachment in children and newborns.

Capsid protein composition of reference strains and wild isolates of human astroviruses.

Astroviruses are small RNA viruses that are frequently associated with gastroenteritis in humans and animals. Despite much work on the genetic analysis of astrovirus strains, little progress has been made in the characterization of the proteins composing mature virions. We have analyzed the capsid protein composition of the reference strains and several wild isolates of human astroviruses using high-resolution polyacrylamide gradient gels. For reference strains of the seven serotypes analyzed, a consistent pattern of three infection-specific proteins--designated P1, P2, and P3 -was generally observed. The strains could be divided into two groups, based upon the reactivity of these proteins in immune precipitation assays that used homologous rabbit serum. One group included reference types 1 4 for which all three proteins were precipitated by homologous rabbit sera; for the other group, types 5 7, only proteins P2 and P3 were precipitated. When wild isolates from around the world were compared to the reference strains, a correlation between genetic type and the pattern of protein sizes and immune reactivity was observed for strains of the common types (1-4). Strains of types 2 and 4 consistently exhibited P3 proteins larger than those of types 1 and 3. Unusual patterns of proteins or immune reactivity were detected in strains of types 5-7, indicating that there may be incomplete processing of the capsid precursor during growth in cell culture.

Molecular epidemiology of human astroviruses.

Human astroviruses (HAstVs) are associated with 5-9 percent of cases of gastroenteritis in young children. Seven serotypes (HAstV-1 to -7), which correlate with genotypes, have been defined by using immune typing methods. We have used partial nucleotide sequence information from the capsid protein gene for molecular typing of 29 unique human astrovirus strains obtained from prospective studies of children with gastroenteritis in Egypt and Malawi. HAstV-1 was the most commonly detected strain, consistent with previous studies, but a surprising variety of strains were identified in both collections. An eighth astrovirus type, HAstV-8, has been defined on the basis of the complete capsid protein gene sequence and was detected in both collections analysed in this study. Although HAstV-8 and HAstV-4 strains segregate into well resolved clades by analysis of sequences from the region encoding protein P2 (VP32), the pair-wise distances between these types are less than those between strains of the other serotypes. In contrast, analysis of sequences from the region encoding protein P3 unambiguously resolve HAstV-4 and HAstV-8 strains, consistent with their classification as distinct serotypes. Overall, strains representing six of the eight serotypes were detected in two collections of samples from prospective studies of gastroenteritis in young children indicating that multiple astrovirus types are frequently co-circulating within communities.

RT-PCR identification and typing of astroviruses and Norwalk-like viruses in hospitalized patients with gastroenteritis: evidence of nosocomial infections.

BACKGROUND: Astroviruses (HAstVs) and 'Norwalk-like viruses' (NLV) are frequent causes of gastroenteritis worldwide, though no data on the strains in circulation or their prevalence is available for France. OBJECTIVES: We applied molecular methods to detect HAstVs and NLVs by reverse transcription-polymerase chain reaction (RT-PCR) in fecal samples collected during a 2-year period from children and adults hospitalized with gastroenteritis. STUDY DESIGN: All samples negative for rotavirus and adenovirus by latex agglutination which contained small (25-40 nm) viral particles observed by electron microscopy (EM) were examined by RT-PCR. RT-PCR products were sequenced to characterize the HAstV and NLV strains present. RESULTS: A total of 75 samples were analyzed by RT-PCR, of which 15 were positive for HAstV and 24 for NLV. Several distinct strains of serotype 1 HAstV, the predominant serotype, circulated during the period. Nineteen of the 24 NLVs were of the G2 genogroup including Mexico-like (n=10), Bristol-like (n=8), and Hawaii-like viruses (n=1); two were genogroup 1. Overall, seven (47%) of the 15 HAstV infections and nine (37.5%) of the 24 NLV infections appeared to be nosocomially acquired based on the date of admission in hospital and the date of illness. CONCLUSION: This study provides additional evidence of the importance of nosocomial infections caused by NLV and HAstV.

Localization of neurotrophin-3-like immunoreactivity in the rat cochlear nucleus.

Immunohistochemistry as well as immunohistofluorescence were used to investigate the distribution of the neurotrophin-3 (NT3) in the adult rat cochlear nucleus. We found a widespread distribution of NT3 immunolabeled neurons throughout the three divisions of this nucleus. NT3-like immunoreactivity was clearly population-specific, with some cell groups heavily (various small neurons and granule cells) or moderately (large neurons of the ventral cochlear nucleus) stained, while others remained negative (a major fraction of medium and large neurons of the dorsal cochlear nucleus). Double-labeling experiments were performed using antibody against the glial fibrillary acid protein, a classic marker for mature astrocytes. This colocalization study revealed that NT3 immunoreactivity was also present in a subpopulation of astrocytes, particularly in the glia limitans and their projections. Numerous small cells also colocalized NT3 together with the glial marker in the granule cell domain and in the molecular cell layer of the dorsal cochlear nucleus. These results suggest that NT3 may exist in widespread populations of adult cochlear nucleus neurons as well as in glial cells. This abundant distribution of NT3-like immunoreactivity implies that this neurotrophin may have an important role in the continued maintenance of mature cochlear nucleus and makes it an attractive candidate for playing a role in regulation or stabilization of neuronal circuits in this nucleus.

Characterization of "Norwalk-like viruses" and astroviruses by liquid hybridization assay.

"Norwalk-like viruses" (NLVs) and human astroviruses are causative agents of gastroenteritis in all age-groups. The typing of these agents is generally done by nucleotide sequencing, blot hybridization, or enzyme immunoassay. These techniques are expensive, time-consuming, and sometimes require scarce reagents, which limits the typing of NLVs and astroviruses to a few reference laboratories. This report describes a liquid hybridization assay that uses broadly reactive probes whose sequences are based on data from specimens in collections available at CDC and GenBank. Two astrovirus genogroup-specific probes were designed and tested successfully on 26 wild strains from all serotypes. Fourteen GII and 16 GI representative NLV strains were typed without cross-hybridization by using P1B- and P2A-specific probes, described previously, and new P2B- and P1A-specific probes. Analysis of the specificity of the probes, the effect of the mismatches during hybridization, and the sensitivity of hybridization assay demonstrates this method to be a rapid and simple technique for molecular typing of NLVs and preliminary characterization of astroviruses.

The burden of norovirus gastroenteritis: an important foodborne and healthcare-related infection.

Human norovirus (NoV) is now recognized as one of the most important causative agents of gastroenteritis in all age groups worldwide. During the course of NoV infection, symptoms are usually mild and disappear within 48 h after onset. The incidence of NoV infection is high, with hundreds of cases per 10 000 of the population, although the number of infections is still underestimated. Epidemiological surveys conducted in Europe and North America have shown that NoV infections constitute a major disease burden, especially for young children and the elderly, in whom NoV infection leads to high rates of hospitalization and mortality. NoV infections are also of concern in hospitals, where viral infections can be persistent in immunocompromised patients. Although the cost of NoV infection in the hospital community has not yet been clearly established, it appears that NoV infections could cost hundreds of thousands of euros in terms of unit closure, and NoV-related sickness in patients and health workers. Besides their clinical burden, NoVs, as foodborne pathogens, also cause to millions of dollars of losses for the healthcare system and the food industry. Recent estimates in the USA showed that, annually, NoV illness cost $2 billion and led to a loss of approximately 5000 quality-adjusted life-years, making NoV one of the top five pathogens causing enteric illnesses. The highest cost among 14 foodborne pathogens is also attributed to human NoV in The Netherlands. This accumulation of evidence underlines the enormous impact of NoV on populations.

Presence of enteric hepatitis viruses in the sewage and population of Greater Cairo.

In Egypt, the disease burden of viral hepatitis is one of the heaviest worldwide. We conducted a survey of hepatitis A virus (HAV) and hepatitis E virus (HEV) in patients and sewage in Cairo. Our data showed that HAV (genotype IB) was predominant over HEV (genotype 3) and was circulating in the population and the environment.

Outbreak of gastroenteritis in military recruits associated with serotype 3 astrovirus infection.

A serotype 3 astrovirus was identified in stool samples from an outbreak of acute gastroenteritis that occurred among military recruits in France. Sixteen stools samples and eight pairs of acute- and convalescent-phase serum were collected from affected individuals. Astrovirus was detected in two stool samples by electron microscopy and in four stool samples by reverse transcriptase-polymerase chain reaction (RT-PCR). Seroconversion to the astrovirus present in one stool was detected in seven patients by using solid-phase immune electron microscopy (SPIEM) and dot blot. For three patients, the serological results were consistent with the PCR results, indicating that astrovirus was a cause of gastroenteritis in these young adults. This study describes the characterization of the serotype 3 astrovirus associated with this outbreak. The virus has a buoyant density in cesium chloride of 1.365 gm/ml and contains two proteins immuno-precipitated with rabbit serum. Only the larger protein was recognized by immunoblotting using a convalescent-phase human serum. The protein composition of this virus differs from that reported for serotype 1 astrovirus, indicating heterogeneity in the capsid composition among astrovirus serotypes.

Detection and genetic differentiation of human astroviruses: phylogenetic grouping varies by coding region.

Astroviruses are single-stranded, positive-sense RNA viruses that are associated with gastroenteritis in humans and animals. We describe a reverse transcription-polymerase chain reaction (RT-PCR) assay using primers targeted to a nonstructural protein coding region that allowed sensitive detection and genetic typing of representative strains of seven astrovirus serotypes. Phylogenetic analysis of the nucleotide sequences of PCR products from the reference strains and several wild isolates indicated two distinct genogroups of sequences in open reading frame 1a (ORF 1a). These genogroups correlated with serotype: genogroup A included strains of types 1 to 5, while genogroup B included strains of types 6 and 7. This phylogenetic arrangement differs from the nearly equidistant clustering of serotypes seen when comparing nucleotide sequences from either ORF 1b or ORF 2. It is possible that recombination was responsible for the observed difference in genetic relationships.

The epidemiology of enteric caliciviruses from humans: a reassessment using new diagnostics.

In the United States, acute gastroenteritis is one of the most commonly noted illnesses on hospital discharge records and death certificates, yet few of these cases have an etiologic diagnosis. The application of new molecular diagnostic methods has shown caliciviruses (previously referred to as the Norwalk family of viruses or small round structured viruses) to be the most common cause of acute gastroenteritis (AGE) outbreaks in the United States, and they may emerge as a common cause of sporadic cases of AGE among both children and adults. Novel molecular methods have permitted outbreak strains to be traced back to their common source and have led to the first identification of virus in implicated vehicles of infection-water, shellfish, and foods contaminated both at their source and by food handlers. The broad application of these methods to routine diagnosis in hospitals and public health laboratories is advancing our appreciation of the full burden of calicivirus-associated diarrhea, and it is opening new avenues for its prevention and control.

Characterization of capsid genes, expressed in the baculovirus system, of three new genetically distinct strains of "Norwalk-like viruses".

"Norwalk-like viruses" (NLVs), members of a newly defined genus of the family Caliciviridae, are the most common agents of outbreaks of gastroenteritis in the United States. Two features of NLVs have hindered the development of simple methods for detection and determination of serotype: their genetic diversity and their inability to grow in cell culture. To assess the immune responses of patients involved in outbreaks of gastroenteritis resulting from infection with NLVs, we previously used recombinant-expressed capsid antigens representing four different genetic clusters, but this panel proved insufficient for detection of an immune response in many patients. To extend and further refine this panel, we expressed in baculovirus the capsid genes of three additional genetically distinct viruses, Burwash Landing virus (BLV), White River virus (WRV), and Florida virus. All three expressed proteins assembled into virus-like particles (VLPs) that contained a full-length 64-kDa protein, but both the BLV and WRV VLPs also contained a 58-kDa protein that resulted from deletion of 39 amino acids at the amino terminus. The purified VLPs were used to measure the immune responses in 403 patients involved in 37 outbreaks of acute gastroenteritis. A majority of patients demonstrated a fourfold rise in the titer of immunoglobulin G to the antigen homologous to the outbreak strain, but most seroconverted in response to other genetically distinct antigens as well, suggesting no clear pattern of type-specific immune response. Further study of the antigenicity of the NLVs by use of VLPs should allow us to design new detection systems with either broader reactivity or better specificity and to define the optimum panel of antigens required for routine screening of patient sera.