SARS-CoV-2 antibodies, serum inflammatory biomarkers and clinical severity of hospitalized COVID-19 patients

Background The involvement of SARS-CoV-2 antibodies in mediating immunopathogenetic events in COVID-19 patients has been suggested. By using several experimental approaches, we investigated the potential association between SARS-CoV-2 IgGs recognizing the spike (S) protein receptor-binding domain (RBD), neutralizing antibodies (NtAb) targeting S, and COVID-19 severity. Patients and methods This unicenter, retrospective, observational study included 51 hospitalized patients (24 at the intensive care unit; ICU). A total of 93 sera from these patients collected at different time points from the onset of symptoms were analyzed. SARS-CoV-2 RBD IgGs were quantitated by ELISA and NtAb50 titers wer…

The G428A Nonsense Mutation in FUT2 Provides Strong but Not Absolute Protection against Symptomatic GII.4 Norovirus Infection

In November 2004, 116 individuals in an elderly nursing home in El Grao de Castellón, Spain were symptomatically infected with genogroup II.4 (GII.4) norovirus. The global attack rate was 54.2%. Genotyping of 34 symptomatic individuals regarding the FUT2 gene revealed that one patient was, surprisingly, a non-secretor, hence indicating secretor-independent infection. Lewis genotyping revealed that Lewis-positive and negative individuals were susceptible to symptomatic norovirus infection indicating that Lewis status did not predict susceptibility. Saliva based ELISA assays were used to determine binding of the outbreak virus to saliva samples. Saliva from a secretor-negative individual boun…

Human milk and mucosal lacto- and galacto-N-biose synthesis by transgalactosylation and their prebiotic potential in Lactobacillus species.

Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho-β-galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 69 ± 4 and 71 ± 1 % of o-nitrophenyl-β-d-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a s…

Unraveling the role of the secretor antigen in human rotavirus attachment to histo-blood group antigens

25 Páginas, 7 figuras, 2 tablas

SARS‐CoV‐2 N‐antigenemia in critically ill adult COVID‐19 patients: Frequency and association with inflammatory and tissue‐damage biomarkers

Abstract The current study aimed at characterizing the dynamics of SARS‐CoV‐2 nucleocapsid (N) antigenemia in a cohort of critically ill adult COVID‐19 patients and assessing its potential association with plasma levels of biomarkers of clinical severity and mortality. Seventy‐three consecutive critically ill COVID‐19 patients (median age, 65 years) were recruited. Serial plasma (n = 340) specimens were collected. A lateral flow immunochromatography assay and reverse‐transcription polymerase chain reaction (RT‐PCR) were used for SARS‐CoV‐2 N protein detection and RNA quantitation and in plasma, respectively. Serum levels of inflammatory and tissue‐damage biomarkers in paired specimens were …

Different transmission patterns of uncommon group A human rotavirus genotypes

The interactions between host glycobiology, bacterial microbiota, and viruses in the gut

Rotavirus (RV) and norovirus (NoV) are the major etiological agents of viral acute gastroenteritis worldwide. Host genetic factors, the histo-blood group antigens (HBGA), are associated with RV and NoV susceptibility and recent findings additionally point to HBGA as a factor modulating the intestinal microbial composition. In vitro and in vivo experiments in animal models established that the microbiota enhances RV and NoV infection, uncovering a triangular interplay between RV and NoV, host glycobiology, and the intestinal microbiota that ultimately influences viral infectivity. Studies on the microbiota composition in individuals displaying different RV and NoV susceptibilities allowed th…

Precise mapping of the Goodpasture epitope(s) using phage display, site-directed mutagenesis, and surface plasmon resonance.

Goodpasture disease is an autoimmune disorder mediated by circulating autoantibodies against the noncollagenous-1 (NC1) domain of the alpha 3 chain of type IV collagen (alpha 3(IV)NC1). The structure of Goodpasture epitope(s) has been previously mapped into two main binding regions (E-A and E-B) of the alpha 3(IV)NC1 domain using a residue mutation approach on the highly related alpha 1(IV)NC1 domain. Here we combined phage display and surface plasmon resonance technology to more precisely localize the pathogenic binding sites. Peptides mimicking the Goodpasture epitope(s) were used to identify residues involved in autoantibody binding and found involvement of eight residues previously unre…

Human norovirus hyper-mutation revealed by ultra-deep sequencing

Human noroviruses (NoVs) are a major cause of gastroenteritis worldwide. It is thought that, similar to other RNA viruses, high mutation rates allow NoVs to evolve fast and to undergo rapid immune escape at the population level. However, the rate and spectrum of spontaneous mutations of human NoVs have not been quantified previously. Here, we analyzed the intra-patient diversity of the NoV capsid by carrying out RT-PCR and ultra-deep sequencing with 100,000-fold coverage of 16 stool samples from symptomatic patients. This revealed the presence of low-frequency sequences carrying large numbers of U-to-C or A-to-G base transitions, suggesting a role for hyper-mutation in NoV diversity. To mor…

Recombinant Noroviruses Circulating in Spain from 2016 to 2020 and Proposal of Two Novel Genotypes within Genogroup I.

Noroviruses are the leading cause of sporadic cases and outbreaks of viral gastroenteritis. For more than 20 years, most norovirus infections have been caused by the pandemic genotype GII.4, yet recent studies have reported the emergence of recombinant strains in many countries. In the present study, 4,950 stool samples collected between January 2016 and April 2020 in Valencia, Spain, from patients with acute gastroenteritis were analyzed to investigate the etiological agent. Norovirus was the most frequently detected enteric virus, with a positivity rate of 9.5% (471/4,950). Among 224 norovirus strains characterized, 175 belonged to genogroup II (GII) and 49 belonged to GI. Using dual geno…

Seroepidemiology of porcine enteric sapovirus in pig farms in Venezuela

Porcine enteric sapovirus (PES) has been shown to cause diarrhea under experimental conditions in gnotobiotic piglets. However, the role of PES as enteric pathogens in porcine farms remains unclear. To further understand the PES-host interactions under field conditions, a serological survey was carried out. To this end the capsid gene of a PES isolate was cloned in the baculovirus expression system and an ELISA was developed based on virus-like particles from the baculovirus-expressed PES capsid protein. A total of 85 serum samples collected from pigs ranging from 8 weeks to over 54 weeks of age were analyzed. An overall seroprevalence to PESs of 62% was found, with significant differences …

Unique Microbial Catabolic Pathway for the Human Core N-Glycan Constituent Fucosyl-α-1,6-N-Acetylglucosamine-Asparagine

The survival of commensal bacteria in the human gut partially depends on their ability to metabolize host-derived molecules. The use of the glycosidic moiety of N-glycoproteins by bacteria has been reported, but the role of N-glycopeptides or glycoamino acids as the substrates for bacterial growth has not been evaluated. We have identified in Lactobacillus casei strain BL23 a gene cluster (alf-2) involved in the catabolism of the glycoamino acid fucosyl-α-1,6-N-GlcNAc-Asn (6′FN-Asn), a constituent of the core-fucosylated structures of mammalian N-glycoproteins. The cluster consists of the genes alfHC, encoding a major facilitator superfamily (MFS) permease and the α-l-fucosidase AlfC, and t…

Selection of single-chain antibodies against the VP8* subunit of rotavirus VP4 outer capsid protein and their expression in Lactobacillus casei.

ABSTRACTSingle-chain antibodies (scFv) recognizing the VP8* fraction of rotavirus outer capsid and blocking rotavirus infection in vitro were isolated by phage display. Vectors for the extracellular expression inLactobacillus caseiof one of the scFv were constructed.L. caseiwas able to secrete active scFv to the growth medium, showing the potential of probiotic bacteria to be engineered to express molecules suitable for in vivo antirotavirus therapies.

Structures of collagen IV globular domains: insight into associated pathologies, folding and network assembly

15 páginas, 6 figuras, 1 tabla.

Therapeutic Opportunities in Intestinal Microbiota–Virus Interactions

The host microbiota has emerged a third player in interactions between hosts and viral pathogens. This opens new possibilities to use different tools to modulate the intestinal microbial composition, aimed at reducing the risk of or treating viral enteric infections.

Nearly Complete Genome Sequence of a Human Norovirus GII.P17-GII.17 Strain Isolated from Brazil in 2015

Human noroviruses are the most common cause of nonbacterial acute gastroenteritis worldwide. We report here the nearly complete genome sequence (7,551 nucleotides) of a human norovirus GII.P17-GII.17 strain detected in July 2015 in the stool sample from an adult with acute gastroenteritis in Brazil.

Inference of SARS-CoV-2 spike-binding neutralizing antibody titers in sera from hospitalized COVID-19 patients by using commercial enzyme and chemiluminescent immunoassays

medRxiv: https://doi.org/10.1101/2020.09.07.20188151

Infant gut microbiota modulation by human milk disaccharides in humanized microbiome mice

Human milk glycans present a unique diversity of structures that suggest different mechanisms by which they may affect the infant microbiome development. A humanized mouse model generated by infant fecal transplantation was utilized here to evaluate the impact of fucosyl-α1,3-GlcNAc (3FN), fucosyl-α1,6-GlcNAc, lacto-N-biose (LNB) and galacto-N-biose on the fecal microbiota and host–microbiota interactions. 16S rRNA amplicon sequencing showed that certain bacterial genera significantly increased (Ruminococcus and Oscillospira) or decreased (Eubacterium and Clostridium) in all disaccharide-supplemented groups. Interestingly, cluster analysis differentiates the consumption of fucosyl-oligosacc…

Microbiota Depletion Promotes Human Rotavirus Replication in an Adult Mouse Model

Intestinal microbiota-virus-host interaction has emerged as a key factor in mediating enteric virus pathogenicity. With the aim of analyzing whether human gut bacteria improve the inefficient replication of human rotavirus in mice, we performed fecal microbiota transplant (FMT) with healthy infants as donors in antibiotic-treated mice. We showed that a simple antibiotic treatment, irrespective of FMT, resulted in viral shedding for 6 days after challenge with the human rotavirus G1P[8] genotype Wa strain (RVwa). Rotavirus titers in feces were also significantly higher in antibiotic-treated animals with or without FMT but they were decreased in animals subject to self-FMT, where a partial re…

The extracellular wall-bound β-N-acetylglucosaminidase from Lactobacillus casei is involved in the metabolism of the human milk oligosaccharide lacto-N-triose

Human milk oligosaccharides (HMOs) are considered to play a key role in establishing and maintaining the infant gut microbiota. Lacto-N-triose forms part of both type 1 and type 2 HMOs and also of the glycan moieties of glycoproteins. Upstream of the previously characterized gene cluster involved in lacto-N-biose and galacto-N-biose metabolism from Lactobacillus casei BL23, there are two genes, bnaG and manA, encoding a β-N-acetylglucosaminidase precursor and a mannose-6-phosphate isomerase, respectively. In this work, we show that L. casei is able to grow in the presence of lacto-N-triose as a carbon source. Inactivation of bnaG abolished the growth of L. casei on this oligosaccharide, dem…

Nearly Complete Genome Sequences of Human Norovirus Belonging to Several Genotypes from Valencia, Spain

Human noroviruses are responsible for most nonbacterial acute gastroenteritis cases. The GII.2, GII.4, and GII.17 genotypes of human noroviruses have recently arisen as the most frequent genotypes found in humans worldwide. We report here seven nearly complete genomes of these genotypes from patients with acute gastroenteritis in Valencia, Spain.

Antibodies against Lewis antigens inhibit the binding of human norovirus GII.4 virus-like particles to saliva but not to intestinal Caco-2 cells.

BACKGROUND: Human noroviruses (NoVs) are the main cause of gastroenteritis worldwide. The most commonly detected NoV strains belong to the genetically diverse GII.4 genotype, with new pandemic variants emerging periodically. Despite extensive efforts, NoV investigation has been hampered by the lack of an effective in vitro cell culture system. However, NoV-derived recombinant virus-like particles (VLPs) resembling empty capsids are good surrogates for analysing NoV antigenicity and virus-ligand interactions. NoV VLPs have been reported to bind to histo-blood group antigens (HBGAs). We have analysed the ability of NoV VLPs derived from GI.1 genotype and from three GII.4 genotype variants, GI…

Histo-blood group antigens in children with symptomatic rotavirus infection

Group A rotaviruses are a major cause of acute gastroenteritis in children. The diversity and unequal geographical prevalence of rotavirus genotypes have been linked to histo-blood group antigens (HBGAs) in different human populations. In order to evaluate the role of HBGAs in rotavirus infections in our population, secretor status (FUT2+), ABO blood group, and Lewis antigens were determined in children attended for rotavirus gastroenteritis in Valencia, Spain. During three consecutive years (2013&ndash

Norovirus infection: why are the genogroup II genotype 4 strains so persistent in the population?

Intestinal Microbiota and Susceptibility to Viral Infections

During pathogenesis, viruses come in contact with the microbiota that colonizes the mucosal sites they infect. The intestinal microbiota has emerged as a critical factor in intestinal viral susceptibility. While the interaction of virus-intestinal commensal bacteria can lead to enhanced or decreased viral infection capacity, several scientific studies support the use of probiotics as antiviral therapies. Thus, probiotics and the modulation of the intestinal microbiota are envisaged as therapeutic strategies in the prevention and treatment of viral infection.

Replication of Human Norovirus in Mice after Antibiotic-Mediated Intestinal Bacteria Depletion

Human noroviruses (HuNoVs) are the main cause of acute gastroenteritis causing more than 50,000 deaths per year. Recent evidence shows that the gut microbiota plays a key role in enteric virus infectivity. In this context, we tested whether microbiota depletion or microbiota replacement with that of human individuals susceptible to HuNoVs infection could favor viral replication in mice. Four groups of mice (n = 5) were used, including a control group and three groups that were treated with antibiotics to eliminate the autochthonous intestinal microbiota. Two of the antibiotic-treated groups received fecal microbiota transplantation from a pool of feces from infants (age 1-3 months) or an au…

Expression and purification of polyhistidine-tagged rotavirus NSP4 proteins in insect cells

The rotavirus nonstructural NSP4 protein, a transmembrane endoplasmic reticulum-specific glycoprotein, has been described as the first viral enterotoxin. Purified NSP4 or a peptide corresponding to NSP4 residues 114-135 induces diarrhea in young mice. NSP4 has a membrane-destabilizing activity and causes an increase in intracellular calcium levels and chloride secretion by a calcium-dependent signalling pathway in eucaryotic cells. In this study, four recombinant baculoviruses were generated expressing the rotavirus NSP4 glycoprotein from the human strains Wa and Ito, the porcine strain OSU, and the simian strain SA11, which belong to two different NSP4 genotypes, A and B. The recombinant g…

Molecular Virology of Enteric Viruses (with Emphasis on Caliciviruses)

Human caliciviruses are members of the family Caliciviridae and are responsible for a majority of the outbreaks of acute nonbacterial gastroenteritis. In fact, they now are considered a common cause of sporadic cases of diarrhea in the community (Glass et al., 2000b; Koopmans et al., 2002; Lopman et al., 2002; Hutson et al., 2004). These viruses were implicated in as many as 95% of the reported viral gastroenteritis outbreaks examined over a 4.5-year period in the United States (Fankhauser et al., 2002), and similar high incidence rates have been found in other studies (Maguire et al., 1999; Glass et al., 2000b; Koopmans et al., 2000; Lopman et al., 2003). Common features of the Calicivirid…

Combined kinetic analysis of SARS-CoV-2 RNAemia, N-antigenemia and virus-specific antibodies in critically ill adult COVID-19 patients

Combined kinetic analysis of plasma SARS-CoV-2 RNAemia, Nucleocapsid (N)-antigenemia and virus-specific antibodies may help ascertain the role of antibodies in preventing virus dissemination in COVID-19 patients. We performed this analysis in a cohort of 71 consecutive critically ill COVID-19 patients (49 male; median age, 65 years) using RT-PCR assay, lateral flow immunochromatography method and receptor binding domain (RBD) and N-based immunoassays. A total of 338 plasma specimens collected at a median of 12 days after symptoms onset were available for analyses. SARS-CoV-2 RNAemia and N-antigenemia were detected in 37 and 43 specimens from 26 (36.5%) and 30 (42.2%) patients, respectively.…

Human milk and mucosa-associated disaccharides impact on cultured infant fecal microbiota

Human milk oligosaccharides (HMOs) are a mixture of structurally diverse carbohydrates that contribute to shape a healthy gut microbiota composition. The great diversity of the HMOs structures does not allow the attribution of specific prebiotic characteristics to single milk oligosaccharides. We analyze here the utilization of four disaccharides, lacto-N-biose (LNB), galacto-N-biose (GNB), fucosyl-α1,3-GlcNAc (3FN) and fucosyl-α1,6-GlcNAc (6FN), that form part of HMOs and glycoprotein structures, by the infant fecal microbiota. LNB significantly increased the total levels of bifidobacteria and the species Bifidobacterium breve and Bifidobacterium bifidum. The Lactobacillus genus levels wer…

Relevance of secretor status genotype and microbiota composition in susceptibility to rotavirus and norovirus infections in humans

Host genetic factors, such as histo-blood group antigens (HBGAs), are associated with susceptibility to norovirus (NoV) and rotavirus (RV) infections. Recent advances point to the gut microbiome as a key player necessary for a viral pathogen to cause infection. In vitro NoV attachment to host cells and resulting infections have been linked to interactions with certain bacterial types in the gut microbiota. We investigated the relationship between host genotype, gut microbiota, and viral infections. Saliva and fecal samples from 35 adult volunteers were analysed for secretor status genotype, the gut microbiota composition by 16S rRNA gene sequencing, and salivary IgA titers to NoV and RV. Hi…

Rotavirus stimulates release of serotonin (5-HT) from human enterochromaffin cells and activates brain structures involved in nausea and vomiting

Rotavirus (RV) is the major cause of severe gastroenteritis in young children. A virus-encoded enterotoxin, NSP4 is proposed to play a major role in causing RV diarrhoea but how RV can induce emesis, a hallmark of the illness, remains unresolved. In this study we have addressed the hypothesis that RV-induced secretion of serotonin (5-hydroxytryptamine, 5-HT) by enterochromaffin (EC) cells plays a key role in the emetic reflex during RV infection resulting in activation of vagal afferent nerves connected to nucleus of the solitary tract (NTS) and area postrema in the brain stem, structures associated with nausea and vomiting. Our experiments revealed that RV can infect and replicate in human…

Infant Gut Microbial Metagenome Mining of α- l -Fucosidases with Activity on Fucosylated Human Milk Oligosaccharides and Glycoconjugates

The gastrointestinal microbiota members produce α-l-fucosidases that play key roles in mucosal, human milk, and dietary oligosaccharide assimilation. Here, 36 open reading frames (ORFs) coding for putative α-l-fucosidases belonging to glycosyl hydrolase family 29 (GH29) were identified through metagenome analysis of breast-fed infant fecal microbiome. Twenty-two of those ORFs showed a complete coding sequence with deduced amino acid sequences displaying the highest degree of identity with α-l-fucosidases from Bacteroides thetaiotaomicron, Bacteroides caccae, Phocaeicola vulgatus, Phocaeicola dorei, Ruminococcus gnavus, and Streptococcus parasanguinis. Based on sequence homology, 10 α-l-fuco…

Characterization of a Novel Conformational GII.4 Norovirus Epitope: Implications for Norovirus-Host Interactions

ABSTRACT Human noroviruses (NoVs) are the main etiological agents of acute gastroenteritis worldwide. While NoVs are highly diverse (more than 30 genotypes have been detected in humans), during the last 40 years most outbreaks and epidemics have been caused by GII.4 genotype strains, raising questions about their persistence in the population. Among other potential explanations, immune evasion is considered to be a main driver of their success. In order to study antibody recognition and evasion in detail, we analyzed a conformational epitope recognized by a monoclonal antibody (3C3G3) by phage display, site-directed mutagenesis, and surface plasmon resonance. Our results show that the predi…

Quasispecies dynamics and molecular evolution of human norovirus capsid P region during chronic infection.

In this novel study, we have for the first time identified evolutionarily conserved capsid residues in an individual chronically infected with norovirus (GGII.3). From 2000 to 2003, a total of 147 P1-1 and P2 capsid sequences were sequenced and investigated for evolutionarily conserved and functionally important residues by the evolutionary trace (ET) algorithm. The ET algorithm revealed more absolutely conserved residues (ACR) in the P1-1 domain (47/53, 88 %) as compared with the P2 domain (86/133, 64 %). The capsid P1-1 and P2 domains evolved in time-dependent manner, with a distinct break point observed between autumn/winter of year 2000 (isolates P1, P3 and P5) and spring to autumn of y…

Epidemiological and Genetic Characterization of Sapovirus in Patients with Acute Gastroenteritis in Valencia (Spain)

Sapovirus is a common cause of acute gastroenteritis in all age groups. Sapovirus infections are seldom investigated in Spain, and its epidemiology in the country is not well known. The use of molecular diagnostic procedures has allowed a more frequent detection of sapoviruses in patients with diarrhea. A total of 2545 stool samples from patients with acute gastroenteritis attended from June 2018 to February 2020 at the Clinic University Hospital in Valencia, Spain, were analyzed by reverse transcription (RT) and real-time multiplex PCR (RT-PCR) to investigate the etiology of enteric infections. Sapovirus was the second enteric virus detected with a positive rate of 8%, behind norovirus (12…

Phylogenetic analysis of G12 group A rotavirus circulating in Spain during 2012–2015: Detection of different clusters with distinct evolutionary origins

Single-chain variable fragment (scFv) antibodies against rotavirus NSP4 enterotoxin generated by phage display.

The rotavirus non-structural NSP4 protein causes membrane destabilization as well as an increase in intracellular calcium levels in eukaryotic cells and induces diarrhea in young mice, acting as a viral enterotoxin. In this study the phage display technique was used to generate a panel of single-chain variable fragment (scFv) antibodies specific for the NSP4 protein of the human rotavirus strain Wa from a human semi-synthetic scFv library. After several rounds of panning and selection on NSP4 adsorbed to polystyrene tubes, individual scFv were isolated and characterised by fingerprinting and by sequencing the VH and VL genes. The isolated scFv antibodies specifically recognize NSP4 in enzym…

Adaptive immune responses to SARS-CoV-2 in recovered severe COVID-19 patients

ABSTRACTObjectivesThere is an imperative need to determine the durability of adaptive immunity to SARS-CoV-2. We enumerated SARS-CoV-2-reactive CD4+ and CD8+ T cells targeting S1 and M proteins and measured RBD-specific serum IgG over a period of 2-6 months after symptoms onset in a cohort of subjects who had recovered from severe clinical forms of COVID-19.MethodsWe recruited 58 patients (38 males and 20 females; median age, 62.5 years), who had been hospitalized with bilateral pneumonia, 60% with one or more comorbidities. IgG antibodies binding to SARS-CoV-2 RBD were measured by ELISA. SARS-CoV-2-reactive CD69+-expressing-IFNγ-producing-CD4+ and CD8+ T cells were enumerated in heparinize…

Suitability of two rapid lateral flow immunochromatographic assays for predicting SARS‐CoV‐2 neutralizing activity of sera

Purpose: Assessment of commercial SARS-CoV-2 immunoassays for their capacity to provide reliable information on sera neutralizing activity is an emerging need. We evaluated the performance of two commercially-available lateral flow immunochromatographic assays (LFIC) (Wondfo SARS-CoV-2 Antibody test and the INNOVITA 2019-nCoV Ab test) in comparison with a SARS-CoV-2 neutralization pseudotyped assay for COVID-19 diagnosis in hospitalized patients, and investigate whether the intensity of the test band in LFIC associates with neutralizing antibody (NtAb) titers. Patients and Methods: Ninety sera were included from 51 patients with moderate to severe COVID-19. A green fluorescent protein (GFP)…

Noroviral P-Particles as an In Vitro Model to Assess the Interactions of Noroviruses with Probiotics

Noroviruses (NoVs) are the main etiologic agents of acute epidemic gastroenteritis and probiotic bacteria have been reported to exert a positive effect on viral diarrhea. The protruding (P) domain from NoVs VP1 capsid protein has the ability to assemble into the so-called P-particles, which retain the binding ability to host receptors. We purified the P-domains from NoVs genotypes GI.1 and GII.4 as 6X(His)-tagged proteins and determined that, similar to native domains, they were structured into P-particles that were functional in the recognition of the specific glycoconjugated receptors, as established by surface plasmon resonance experiments. We showed that several lactic acid bacteria (pr…

Norovirus infections and seroprevalence of genotype gii.4-specific antibodies in a spanish population

Genotype II.4 noroviruses (NoVs) are a leading cause of epidemic acute gastroenteritis in children and adults worldwide. The prevalence of different NoV genotypes causing outbreaks and sporadic cases of acute gastroenteritis in the region of Valencia, Spain, during a 4-year period (2008–11) was investigated. NoVs were detected in 42 out of 55 (76.3%) outbreaks and in 26 out of 332 (7.8%) sporadic cases of acute gastroenteritis. Genogroup GII strains were predominant in outbreaks and sporadic cases. Different genotype GII.4 variants were found (Yerseke_2006a, Den Haag_2006b, Apeldoorn_2007, and New Orleans_2009), with the latter variant detected most frequently (35.3%). A recombinant P domai…

Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples

Shellfish contamination by human noroviruses (HuNoVs) is a serious health and economic problem. Recently an ISO procedure based on RT-qPCR for the quantitative detection of HuNoVs in shellfish has been issued, but these procedures cannot discriminate between inactivated and potentially infectious viruses. The aim of the present study was to optimize a pretreatment using PMAxx to better discriminate between intact and heat-treated HuNoVs in shellfish and sewage. To this end, the optimal conditions (30 min incubation with 100 μM of PMAxx and 0.5% of Triton, and double photoactivation) were applied to mussels, oysters and cockles artificially inoculated with thermally-inactivated (99 °C for 5 …

Antiviral activity of aged green tea extract in model food systems and under gastric conditions.

Aged-green tea extract (GTE) is known to reduce the infectivity of hepatitis A virus (HAV) and murine norovirus (MNV), a human norovirus surrogate, in vitro and in washing solutions. Initially, the effect of aged-GTE was evaluated on virus like particles (VLPs) of human norovirus (HuNoV) genogroup I (GI) by a porcine gastric mucine (PGM)-enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM), and on HuNoV GI suspensions by an in situ capture-RT-qPCR method, suggesting that HuNoVs are very sensitive to aged-GTE treatment at 37 °C. Moreover, the potential application of aged-GTE was evaluated using model foods and simulated gastric conditions. Then, aged-GTE samp…

Structures of collagen IV globular domains: insight into associated pathologies, folding and network assembly. Corrigendum

The article by Casino et al. [IUCrJ (2018). 5, 765–779] is corrected.

Role of nitric oxide during rotavirus infection.

The pathophysiological mechanisms behind rotavirus-induced diarrhoea still remain incomplete. Current views suggest that the non-structural protein 4 (NSP4) of rotavirus and the enteric nervous system (ENS) participate in water secretion and diarrhoea. In the present work the role of nitric oxide (NO) in rotavirus infection and disease has been studied in vitro, mice and humans. Incubation of human intestinal epithelial cells (HT-29) with purified NSP4 but not with infectious virus produced NO2/NO3 accumulation in the incubation media. The NSP4-induced release of NO metabolites occurred within the first minutes after the addition of the toxin. Mice infected with murine rotavirus (strain EDI…

Humoral immune response to rotavirus NSP4 enterotoxin in Spanish children.

The rotavirus non-structural protein 4 (NSP4) has been shown to play a crucial role in rotavirus-induced diarrhea, acting as a viral enterotoxin. It has also been demonstrated that antibody to NSP4 can reduce the severity of rotavirus-induced diarrhea in newborn mice. Two recombinant baculoviruses, expressing the NSP4 protein from the SA11 and Wa rotavirus strains, genotypes A and B, respectively, were used to produce and purify these glycoproteins, which were applied as antigen in an enzyme-linked immunosorbent assay (ELISA) to test the specific antibody response to NSP4 in human sera. Serum samples from 30 children convalescing from a rotavirus infection, from 54 healthy children under 5-…

Improving efficiency of viability-qPCR for selective detection of infectious HAV in food and water samples.

Aim: To improve the efficacy of intercalating dyes to distinguishing between infectious and inactivated hepatitis A virus (HAV) in food. Methods and Results: Different intercalating dyes were evaluated for the discrimination between infectious and thermally inactivated HAV suspensions combining with the RT‐qPCR proposed in the ISO 15216. Among them, PMAxx was the best dye in removing the RT‐qPCR signal from inactivated HAV. Applied to lettuce and spinach, PMAxx–Triton pretreatment resulted in complete removal of the RT‐qPCR signal from inactivated HAV. Likewise, this study demonstrates that this pretreatment is suitable for the discrimination of inactivated HAV in shellfish without further …

The Rotavirus Vaccine Landscape, an Update

Rotavirus is the leading cause of severe acute childhood gastroenteritis, responsible for more than 128,500 deaths per year, mainly in low-income countries. Although the mortality rate has dropped significantly since the introduction of the first vaccines around 2006, an estimated 83,158 deaths are still preventable. The two main vaccines currently deployed, Rotarix and RotaTeq, both live oral vaccines, have been shown to be less effective in developing countries. In addition, they have been associated with a slight risk of intussusception, and the need for cold chain maintenance limits the accessibility of these vaccines to certain areas, leaving 65% of children worldwide unvaccinated and …

The lactose operon from Lactobacillus casei is involved in the transport and metabolism of the human milk oligosaccharide core-2 N-acetyllactosamine

The lactose operon (lacTEGF) from Lactobacillus casei strain BL23 has been previously studied. The lacT gene codes for a transcriptional antiterminator, lacE and lacF for the lactose-specific phosphoenolpyruvate: phosphotransferase system (PTS) EIICB and EIIA domains, respectively, and lacG for the phospho-β-galactosidase. In this work, we have shown that L. casei is able to metabolize N-acetyllactosamine (LacNAc), a disaccharide present at human milk and intestinal mucosa. The mutant strains BL153 (lacE) and BL155 (lacF) were defective in LacNAc utilization, indicating that the EIICB and EIIA of the PTS are involved in the uptake of LacNAc in addition to lactose. Inactivation of lacG aboli…

Characterisation of a household norovirus outbreak occurred in Valencia (Spain)

Background Human noroviruses (NoVs) are the main cause of non-bacterial gastroenteritis worldwide. Several studies have linked human susceptibility to NoVs with the expression of histo-blood group antigens (HBGAs). In January 2012, a NoV gastroenteritis outbreak affected a household in Valencia, Spain, and the personal susceptibility to NoV was investigated. Methods To reach this aim 8 members of the affected household were recruited for this study and their secretor status, ABO and Lewis antigens were determined. NoV-specific saliva IgA and serum IgG antibody titers were analyzed. Their capacity to block viral binding to saliva receptors was analyzed, using virus-like particles (VLPs) of t…

Interaction of Intestinal Bacteria with Human Rotavirus during Infection in Children

The gut microbiota has emerged as a key factor in the pathogenesis of intestinal viruses, including enteroviruses, noroviruses and rotaviruses (RVs), where stimulatory and inhibitory effects on infectivity have been reported. With the aim of determining whether members of the microbiota interact with RVs during infection, a combination of anti-RV antibody labeling, fluorescence-activated cell sorting and 16S rRNA amplicon sequencing was used to characterize the interaction between specific bacteria and RV in stool samples of children suffering from diarrhea produced by G1P[8] RV. The genera Ruminococcus and Oxalobacter were identified as RV binders in stools, displaying enrichments between …